Sorting and Recycling Endosomes

Methods in Cell Biology Sorting and Recycling Endosomes Volume 130

Series Editors Leslie Wilson Department of Molecular, Cellular and Developmental Biology University of California Santa Barbara, California

Phong Tran University of Pennsylvania Philadelphia, USA & Institut Curie, Paris, France

Methods in Cell Biology Sorting and Recycling Endosomes Volume 130

Edited by

Wei Guo University of Pennsylvania, Biology Department, Philadelphia, PA, USA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

Academic Press is an imprint of Elsevier

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Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-12-802829-2 ISSN: 0091-679X For information on all Academic Press publications visit our website at http://store.elsevier.com

Contents Contributors.......................................................................................... xiii Preface ................................................................................................ xix

CHAPTER 1 Dynamic Imaging of the Recycling Endosomal Network in Macrophages......................................... 1 Adam A. Wall, Nicholas D. Condon, Jeremy C. Yeo, Nicholas A. Hamilton, Jennifer L. Stow Introduction.......................................................................2 1. Methods ...........................................................................4 2. Experimental Strategies.......................................................5 Summary......................................................................... 16 References....................................................................... 17

CHAPTER 2 Analyzing the Functions of Rab11-effector Proteins During Cell Division ..................................19 Rytis Prekeris Introduction..................................................................... 20 1. Methods for Analyzing Proteins Regulating Cytokinesis ......... 22 2. Time-Lapse Analysis of Endosome and Actin Dynamics during Cytokinesis............................................................ 27 Summary......................................................................... 33 Acknowledgments............................................................. 33 References....................................................................... 33

CHAPTER 3 Expression and Localization of Exocytic and Recycling Rabs from Magnaporthe Oryzae in Mammalian Cells ...................................................35 Yaoyao Qi, M. Caleb Marlin, Zhimin Liang, Dongmei Zhang, Jie Zhou, Zonghua Wang, Guodong Lu, Guangpu Li Introduction..................................................................... 36 1. Materials and Equipment................................................... 37 2. Methods ......................................................................... 38 Summary......................................................................... 42 Acknowledgments............................................................. 44 References....................................................................... 44

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CHAPTER 4 Methods to Analyze Novel Liaisons between Endosomes and Centrosomes..................................47 Hui-Fang Hung, Heidi Hehnly, Stephen Doxsey Introduction: The Relationship between the Recycling Endosome and Centrosome ................................................ 48 1. Live Cell Imaging of REs throughout the Cell Cycle.............. 50 2. RE’s Centrosome Localization by Electron Microscopy.......... 51 3. Centrosome Isolation and Identification of RE Components..... 53 Acknowledgments............................................................. 56 References....................................................................... 56

CHAPTER 5 Biochemical Analysis of Rabin8, the Guanine Nucleotide Exchange Factor for Rab8 .....................59 Bin Wu, Juanfei Wang, Yuting Zhao, Wei Guo Introduction..................................................................... 60 1. Method........................................................................... 61 Acknowledgments............................................................. 66 References....................................................................... 67

CHAPTER 6 Simple In Vitro Assay of Arf GAPs and Preparation of Arf Proteins as Substrates ..................................69 Pei-Wen Chen, Xiaoying Jian, Ruibai Luo, Paul A. Randazzo Introduction..................................................................... 70 1. Methods ......................................................................... 71 Summary......................................................................... 78 Acknowledgment.............................................................. 78 References....................................................................... 78

CHAPTER 7 An ACAP1 Coat Complex Acting in Endocytic Recycling ..............................................................81 Jian Li, Victor W. Hsu Introduction..................................................................... 82 1. Materials ........................................................................ 83 2. Methods ......................................................................... 85 Discussion and Perspective................................................. 96 Acknowledgments............................................................. 97 References....................................................................... 97

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CHAPTER 8 Biochemical Methods for Studying Kinetic Regulation of Arf1 Activation by Sec7................... 101 1. 2. 3. 4. 5. 6.

Brian C. Richardson, J. Christopher Fromme Introduction................................................................... 103 Materials ...................................................................... 104 Purification of Required Proteins ...................................... 106 Liposomes .................................................................... 115 Measuring GEF Activity.................................................. 117 Identifying ProteineArf1 Interactions................................ 121 Supporting Protocol........................................................ 123 Acknowledgments........................................................... 125 References..................................................................... 125

CHAPTER 9 Rab and Arf G Proteins in Endosomal Trafficking ... 127 Dipannita Dutta, Julie G. Donaldson Introduction................................................................... 128 1. Objectives and Rationale ................................................. 130 2. Materials and Instruments................................................ 131 3. Methods ....................................................................... 132 Summary....................................................................... 137 References..................................................................... 137

CHAPTER 10 Qualitative and Quantitative Analysis of Endocytic Recycling............................................. 139

1. 2. 3. 4.

James B. Reineke, Shuwei Xie, Naava Naslavsky, Steve Caplan Introduction................................................................... 140 Materials ...................................................................... 142 Qualitative Assessment of Endocytic Recycling................... 143 Quantitative Assessment of Endocytic Recycling................. 149 Quantification of MHC I Antibody-Receptor Complex Recycling Using Immunofluorescence Microscopy and ImageJ.................................................................... 152 Acknowledgments........................................................... 154 References..................................................................... 154

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CHAPTER 11 Methods for Analyzing the Role of Phospholipase A2 Enzymes in Endosome Membrane Tubule Formation................................. 157 Danielle N. Kalkofen, Paul de Figueiredo, William J. Brown Introduction................................................................... 158 1. In Vitro Endosome Membrane Tubule Formation................. 160 2. In Vivo Endosome Membrane Tubule Formation ................. 167 3. PLA2 Enzymes in Endosome Membrane Tubule Formation... 174 Acknowledgments........................................................... 178 References..................................................................... 178

CHAPTER 12 In Vivo Analysis of Recycling Endosomes in Caenorhabditis elegans .................................... 181 Anbing Shi, Barth D. Grant Introduction................................................................... 182 1. Objectives and Rationale ................................................. 183 2. Materials and Instruments................................................ 184 3. Methods ....................................................................... 184 Summary....................................................................... 195 References..................................................................... 195

CHAPTER 13 Monitoring Receptor Trafficking Following Retromer and WASH Deregulation......................... 199

1. 2. 3. 4. 5. 6. 7. 8.

Douglas G. Osborne, Christine A. Phillips-Krawczak, Daniel D. Billadeau Introduction................................................................... 200 Analysis of Retrograde Trafficking: Objectives and Rationale ...................................................................... 201 Experimental Strategy..................................................... 203 Methods ....................................................................... 204 Experimental Analysis .................................................... 206 Analysis of Integrin Recycling: Objectives and Rationale...... 208 Experimental Strategy..................................................... 208 Methods ....................................................................... 209 Experimental Analysis .................................................... 210 Summary....................................................................... 212 References..................................................................... 212

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CHAPTER 14 Quantitative Analysis of Endosome Tubulation and Microdomain Organization Mediated by the WASH Complex .............................................. 215 Emmanuel Derivery, Alexis Gautreau Introduction................................................................... 216 1. Imaging of Endosome Tubulation ..................................... 218 2. Live Imaging of WASH Microdomains .............................. 222 3. Quantitative 3D Analysis of WASH Domain Localization in Fixed Cells................................................................ 225 Conclusion .................................................................... 231 Acknowledgments........................................................... 232 References..................................................................... 232

CHAPTER 15 Rab GTPases and Kinesin Motors in Endosomal Trafficking ........................................................... 235 1. 2. 3. 4.

Ce´dric Delevoye, Bruno Goud Introduction................................................................... 236 Screen for Rab GTPaseseKinesin Interactions .................... 236 Spatiotemporal Distribution of Kinesins and Rab GTPases.... 237 Role of the Microtubule Cytoskeleton in the Dynamics of Kinesin and Rab GTPases............................................ 239 Functional Assays .......................................................... 239 Conclusions................................................................... 245 References..................................................................... 246

CHAPTER 16 Measuring Receptor Recycling in Polarized MDCK Cells.......................................................... 247 Luciana Gallo, Gerard Apodaca Introduction................................................................... 248 1. Objectives and Rationale ................................................. 250 2. Materials and Instruments................................................ 253 3. Methods ....................................................................... 257 Summary....................................................................... 266 References..................................................................... 267

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CHAPTER 17 Structural and Functional Analysis of Endosomal Compartments in Epithelial Cells ......... 271 Andres E. Perez Bay, Ryan Schreiner, Enrique Rodriguez-Boulan Introduction................................................................... 272 1. Objectives and Rationale ................................................. 275 2. Experimental Protocols ................................................... 275 References..................................................................... 285

CHAPTER 18 Analyzing the Role of AP-1B in Polarized Sorting from Recycling Endosomes in Epithelial Cells.................................................................... 289 Heike Fo¨lsch Introduction................................................................... 290 1. Objectives and Rationale ................................................. 291 2. Materials and Instruments................................................ 292 3. Methods ....................................................................... 293 Summary....................................................................... 303 Acknowledgments........................................................... 303 References..................................................................... 303

CHAPTER 19 Recycling of the Insulin-responsive Glucose Transporter Glut4 Regulated by the Small GTPase RalA and the Exocyst Complex.................. 307 Chao Nie, Xiao-Wei Chen Introduction................................................................... 308 1. Objective and Rationale .................................................. 309 2. Materials and Instruments................................................ 310 3. Methods and Results....................................................... 311 Summary....................................................................... 317 References..................................................................... 317

CHAPTER 20 GPCR Sorting at Multivesicular Endosomes ........... 319 Michael Robert Dores, JoAnn Trejo Introduction................................................................... 320 1. Objectives and Rationale ................................................. 321 2. Materials and Instruments................................................ 322 3. Methods ....................................................................... 323 Summary....................................................................... 330 References..................................................................... 330

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CHAPTER 21 Visualizing and Quantitating Sequence-dependent GPCR Recycling ................................................... 333 Shanna L. Bowman, Amanda L. Soohoo, Manojkumar A. Puthenveedu Introduction................................................................... 334 1. Objectives and Rationale ................................................. 335 2. Materials and Instruments................................................ 335 3. Methods ....................................................................... 336 Summary....................................................................... 344 References..................................................................... 344

CHAPTER 22 Methods to Study Endocytic Trafficking of the EGF Receptor ....................................................... 347 1. 2. 3. 4.

Itziar Pinilla-Macua, Alexander Sorkin Introduction................................................................... 348 Objectives and Rationale ................................................. 349 Materials ...................................................................... 350 Basic Techniques ........................................................... 351 Experimental Strategies................................................... 352 Summary....................................................................... 366 References..................................................................... 366

Volumes in Series .................................................................................369 Index ..................................................................................................383

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Contributors Gerard Apodaca Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA Daniel D. Billadeau Department of Biochemistry and Molecular Biology, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA Shanna L. Bowman Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA William J. Brown Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Steve Caplan Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Pei-Wen Chen Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Xiao-Wei Chen Institute of Molecular Medicine, Peking University, Beijing, China; PKU-THU Center for Life Sciences, Peking University, Beijing, China Nicholas D. Condon Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia Paul de Figueiredo Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA; Department of Microbial Pathogenesis and Immunology, Norman Borlaug Center, Texas A&M University, College Station, TX, USA Ce´dric Delevoye Institut Curie, PSL Research University, Paris, France; CNRS UMR 144, Structure and Membrane Compartments, Paris, France Emmanuel Derivery Department of Biochemistry, Sciences II, University of Geneva, Geneva, Switzerland

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Julie G. Donaldson Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Michael Robert Dores Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA, USA Stephen Doxsey Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA Dipannita Dutta Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA Heike Fo¨lsch Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA J. Christopher Fromme Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Luciana Gallo Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA Alexis Gautreau Laboratoire de Biochimie, Unite´ Mixte de Recherche 7654, Centre National de la Recherche Scientifique, Ecole Polytechnique, Palaiseau Cedex, France Bruno Goud Institut Curie, PSL Research University, Paris, France; CNRS UMR 144, Molecular Mechanisms of Intracellular Transport, Paris, France Barth D. Grant Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ, USA Wei Guo Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Nicholas A. Hamilton Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia; Research Computing Centre, The University of Queensland, Brisbane, QLD, Australia

Contributors

Heidi Hehnly Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA; Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY, USA; Department of Pharmacology, University of Washington, Seattle, WA, USA Victor W. Hsu Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Boston, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA Hui-Fang Hung Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA Xiaoying Jian Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA Danielle N. Kalkofen Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA Guangpu Li Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Jian Li Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Boston, MA, USA; Department of Medicine, Harvard Medical School, Boston, MA, USA Zhimin Liang Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Guodong Lu Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China Ruibai Luo Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

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M. Caleb Marlin Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Naava Naslavsky Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Chao Nie Institute of Molecular Medicine, Peking University, Beijing, China Douglas G. Osborne Department of Biochemistry and Molecular Biology, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA Andres E. Perez Bay Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY, USA Christine A. Phillips-Krawczak Department of Biochemistry and Molecular Biology, Division of Oncology Research, Mayo Clinic, Rochester, MN, USA Itziar Pinilla-Macua Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Rytis Prekeris Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Manojkumar A. Puthenveedu Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA Yaoyao Qi Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China; Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Paul A. Randazzo Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA James B. Reineke Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA

Contributors

Brian C. Richardson Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA Enrique Rodriguez-Boulan Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY, USA Ryan Schreiner Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY, USA Anbing Shi Department of Medical Genetics, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; Institute for Brain Research, Huazhong University of Science and Technology, Wuhan, Hubei, China Amanda L. Soohoo Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA Alexander Sorkin Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Jennifer L. Stow Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia JoAnn Trejo Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA, USA Adam A. Wall Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia Juanfei Wang Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Zonghua Wang Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China Bin Wu Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

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Shuwei Xie Department of Biochemistry and Molecular Biology and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA Jeremy C. Yeo Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia Dongmei Zhang Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China Yuting Zhao Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Jie Zhou Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China

Preface Endosomes not only serve as a receiving compartment for proteins endocytosed from cell surface, but also function as a donor compartment and sorting station for cargos designated to lysosomes, Golgi, and plasma membrane. In recent years, the importance of endosomes as sorting and recycling compartments has become increasingly appreciated. However, the complexity of the endosome system also divulges the gap in our knowledge of this fascinating system. Like their sophisticated cousin Golgi, endosomes are dynamic and functionally versatile, yet they are even more elusive due to their highly elaborate and heterogeneous structures. Scientists from various fields of cell biology, membrane traffic, and beyond, see the need to communicate the methods used to study the dynamics and functions of this endomembrane system. The aim of this volume is to bring together specialists from the field to contribute their expertise to a broad range of biomedical researchers interested in endosomal sorting and recycling. The first part of the volume consists of chapters focusing on the Rab family of small GTPases, which are not only key regulators of different trafficking steps, but also landmarks of various endosomal compartments. Methods for biochemical, imaging, and functional studies of Rab proteins and their regulators are included (Chapters 1e5). These chapters are followed by approaches studying the Arf GTPases as they control the generation of tubular vesicular carriers from the endosomes (Chapters 6e9). In addition, methods to study the proteins and lipids that control membrane morphology and dynamics are presented (Chapters 10e12). As in most cases, vesicle budding and transport from endosomes involve the action of, and coordination with, cytoskeleton. Several chapters present methods to study the actin polymerization machinery and microtubule-based transport during endosomal trafficking (Chapters 13e15). This volume also includes studies of epithelial cells, which are characterized by their complicated sorting machinery that ensures the correct targeting of proteins to distinct apical and basolateral domains at the plasma membrane. Experimental protocols are presented to investigate the various routes of protein transport from endosomes in this polarized system (Chapters 16e18). The last part of the volume presents studies that link endosomal trafficking and signaling (Chapters 19e22). Elucidating the mechanisms of receptor recycling and degradation is essential for the understanding of the endosome function as well as signal transduction. Biochemical and microscopic imaging approaches are presented for quantitative studies of receptor internalization, recycling, and degradation. Clearly, due to the complexity of the system and rapid progress of the field, these chapters cannot cover all aspects of endosomal sorting and recycling. It is my hope that the expertise gathered in this volume will serve as a useful resource and platform for future investigation of this fascinating membrane system. Wei Guo

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Dynamic imaging of the recycling endosomal network in macrophages

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Adam A. Wall*, Nicholas D. Condon*, Jeremy C. Yeo*, Nicholas A. Hamilton*, x, Jennifer L. Stow*, 1 *Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia x Research Computing Centre, The University of Queensland, Brisbane, QLD, Australia 1

Corresponding author: E-mail: [email protected]

CHAPTER OUTLINE Introduction ................................................................................................................ 2 1. Methods ................................................................................................................ 4 1.1 Cell Culture and Transfection ................................................................... 4 1.2 Materials and Instruments ....................................................................... 4 1.2.1 Reagents.............................................................................................. 4 1.2.2 Instruments.......................................................................................... 5 1.3 Live Cell Imaging .................................................................................... 5 2. Experimental Strategies.......................................................................................... 5 2.1 Strategy 1: RE Subcompartments Defined by Rabs .................................... 5 2.1.1 Background and objective .................................................................... 5 2.1.2 Flow of the experiment ......................................................................... 6 2.1.3 Results................................................................................................. 6 2.2 Strategy 2: Cargo Movement through RE ................................................... 8 2.2.1 Background and Objective.................................................................... 8 2.2.2 Flow of the experiments........................................................................ 9 2.2.3 Experimental considerations ............................................................... 11 2.2.4 Results............................................................................................... 11 2.3 Strategy 3: Delivery of RE to Phagosomes ............................................... 14 2.3.1 Background and objective .................................................................. 14 2.3.2 Flow of the experiment ....................................................................... 15 2.3.3 Experimental considerations ............................................................... 15 2.3.4 Results............................................................................................... 16 Summary .................................................................................................................. 16 References ............................................................................................................... 17

Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.04.007 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 1 Dynamic imaging of the recycling endosomal network

Abstract Recycling endosomes (REs) form an extensive and complex network of subcompartmentalized vesicular and tubular elements that connect with the cell surface and other endosomes in macrophages. As surveillance and defense cells of the innate immune system, macrophages are highly dependent on REs for their active and voluminous cell surface turnover and endocytic, exocytic, and recycling of membrane and cargo. Here we set out three approaches for imaging and analyzing REs in macrophages, based on the expression of fluorescently labeled RE-associated proteins and the uptake of fluorescent cargo. Subcompartments of the REs are identified by co-expression and co-localization analysis of RE associated Rab GTPases. Transferrin is a well-known cargo marker as it recycles through REs and it is compared here to other cargo, revealing how different endocytic routes intersect with REs. We show how the movement of transferrin through REs can be modeled and quantified in live cells. Finally, since phagosomes are a signature organelle for macrophages, and REs fuse with the maturing phagosome, we show imaging of REs with phagosomes using a genetically encoded pH-sensitive SNARE-based probe. Together these approaches provide multiple ways to comprehensively analyze REs and the important roles they play in these immune cells and more broadly in other cell types.

INTRODUCTION In most eukaryotic cells, recycling endosomes (REs) form a central hub interconnecting endocytic and exocytic trafficking (Hsu & Prekeris, 2010; Maxfield & McGraw, 2004). In macrophages, the REs form a vast, interconnected network of vesicular and tubular elements that are collectively distributed from the perinuclear region to the subplasma membrane zone (Lacy & Stow, 2011; Manderson, Kay, Hammond, Brown, & Stow, 2007). This highly dynamic organellar system is crucial for the immune surveillance roles of macrophages, which include the ingestion and sampling of fluid, particulate matter and pathogens, the destruction of pathogens and cell debris, and the secretion of critical mediators like chemokines and cytokines (Epelman, Lavine, & Randolph, 2014). REs are in intimate contact with the plasma membrane, providing a reservoir of extra membrane for the formation of cell surface projections, like filopodia, lamellipodia, dorsal ruffles, and phagocytic cups (Huynh, Kay, Stow, & Grinstein, 2007). Cell surface turnover in these cells is rapid and voluminous and so REs also receive membrane from the cell surface through constant macropinocytic and, sometimes, phagocytic pathways, which feed membrane back into the intracellular recycling pool (Hsu & Prekeris, 2010). Finally, REs are also secretory compartments, acting as a sorting substation for many recycling or newly synthesized cargo proteins and then fusing with the plasma membrane to deliver proteins to the cell surface or releasing secreted proteins (Lacy & Stow, 2011; Murray, Kay, Sangermani, & Stow, 2005). Thus, REs traffic a wide variety of cargo and large expanses of membrane into and out of the cell. They also participate in receptor signaling pathways for cell activation via pathogen-associated and danger signals, and opsins such as immunoglobulin and complement (Husebye et al., 2010; NairGupta et al., 2014). Growth factor receptors and G protein-coupled receptors often

Introduction

traverse the RE network in routes that help to determine the fate of signaling through receptor recycling or degradation (Marchese, 2014). The highly dynamic nature of REs in macrophages means that this compartment is optimally visualized and its functions best appreciated through fluorescence imaging in live cells. Labeled REs can be readily imaged in fixed macrophages as a series of small puncta, contrasting with imaging in live cells where the dynamic connections and tubules that interconnect the RE network and provide connections to other parts of the cell, can be resolved (Manderson et al., 2007). The expression of fluorescently tagged proteins in primary macrophages and macrophage cell lines has revealed many of the unique structure/function aspects of REs in these cells. The combined use of fluorescently tagged resident RE cellular proteins and labeled cargo, allows for visualization and quantification of endocytic and exocytic traffic through REs. Coexpression of multiple, tagged RE-associated proteins shows the complex subcompartmentalized nature of the RE network itself. Members of the Rab family of small GTPases have proven to be invaluable experimental markers for REs and other organelles (Chavrier, Parton, Hauri, Simons, & Zerial, 1990; Hutagalung & Novick, 2011). In their active, GTP-bound form, Rabs bind to specific organelles or membrane carriers to participate in membrane trafficking, fusion, budding, and in signalling (Das & Guo, 2011; Hutagalung & Novick, 2011). The quintessential RE marker across many cell types is Rab11a and, in macrophages too, this protein defines a major subpopulation of REs, particularly those containing cargo destined for the cell surface (Das & Guo, 2011; Murray et al., 2005; Stow & Murray, 2013). Members of the SNARE protein family are also commonly used for demarking membrane compartments in cells (Stow, Manderson, & Murray, 2006). SNAREs function as part of large multisubunit membrane fusion complexes (Stenmark, 2009; Su¨dhof & Rothman, 2009; Wickner, 2010) and as such, the majority of SNAREs are transmembrane (TM) proteins with long cytoplasmic tails containing a coiled-coil SNARE domain and short lumenal or extracellular domain. As “resident” proteins, SNAREs can be used as markers for endosomes, either by immunolabeling or through expression of GFP-tagged SNAREs. In macrophages, VAMP3 is well-established as an RE marker, where it functions by interacting with the SNAREs syntaxin4 and SNAP-23 on the plasma membrane or on phagosomes to fuse REs and their cargo to these sites (Nair-Gupta et al., 2014; Stow et al., 2006). Basic methods for visualizing RE-associated proteins and lipids in live macrophages form the basis for studying a host of receptors, secreted and endocytosed products that traverse REs. Understanding RE behavior and connections to the cell surface and to other organellesdparticularly phagosomes, autophagosomes, lysosomes, Golgi, endoplasmic reticulum, and the cell surfacedis essential for elucidating macrophage immune responses in homeostasis and in a variety of infections and inflammatory and developmental diseases. Macrophages provide perhaps one of the most extreme examples of REsdin terms of the volume, dynamism, and variety of functionsdof any cell type. For the same reasons, macrophages are ideal subjects for imaging and analyzing REs.

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1. METHODS 1.1 CELL CULTURE AND TRANSFECTION Macrophages of the macrophage-like line RAW264.7 are maintained in RPMI supplemented with 10% (v/v) fetal bovine serum (pre-screened for non-activation of cells) and 1% (v/v) L-glutamine at 37 C, 5% CO2 incubator. Macrophages are passaged in 10 cm non-tissue culture treated plates and split thrice weekly for experimental use. Fixed cell imaging requires macrophages to be seeded on #1, 11 mm glass coverslips within a 24-well tissue culture plate at a density of 0.1 106 cells/mL for next day transfection or 0.2 106 cells/mL for next day fixation. Macrophages to be imaged live are seeded onto 35 mm MatTek glass bottom dishes at the same cell densities. Transient transfection of fluorescent DNA constructs by lipofection is performed approximately 16 h after plating when cells are in exponential growth phase, yielding better expression in the often difficult-to-transfect macrophages. Cells are incubated prior to transfection with reduced serum medium (OptiMEM, Gibco). For transfection, we use 2 mg plasmid DNA per MatTek or 0.5 mg per coverslips in a 24-well tissue culture plate following the recommended protocol for Lipofectamine 2000 (Life-Technologies). Cells are incubated with the DNA/Lipofectamine for 2 h at 37 C, 5% CO2, before being replaced with regular growth medium. Macrophages can be imaged from 6 to 16 h post transfection. This methodology yields a typical transfection efficiency of 10e30% that is more than sufficient for imaging experiments. Primary mouse macrophages are transfected for imaging using the Amaxa nucleofection method according to manufacturer’s protocol. In brief, cells are washed with OptiMEM and 1 106 cells are used per reaction in a total of 100 mL volume. Cells are then resuspended in OptiMEM and mixed with 10 mg of DNA per Amaxa electroporation cuvette. Electroporation is carried out using the D-032 setting as per manufacturer’s protocol. Cells are then immediately transferred to prewarmed complete RPMI medium for recovery.

1.2 MATERIALS AND INSTRUMENTS 1.2.1 Reagents RPMI culture medium Fetal bovine serum L-Glutamine Lipofectamine2000 OptiMEM 30 mm Latex Beads Lipopolysaccaharide, Salmonella minnesota R595 (LPS) Fluorescently tagged cargo: Alexa647 transferrin, Alexa488-dextran (10,000 mw), and Alexa594-low density lipoprotein (LDL)

2. Experimental strategies

1.2.2 Instruments Applied precision personal DeltaVision microscope Based on Olympus IX71 inverted wide-field epi-fluorescence and brightfield microscope fitted with a Roper Coolsnap HQ2 monochrome camera with a 120 W xenon lamp coupled with an incubation chamber and CO2 control.

1.3 LIVE CELL IMAGING RAW264.7 macrophages are seeded onto a 35 mm MatTek glass bottom dishes. For next day imaging, a total of 0.8 106 cells are plated or for next day transfection we use a total of 0.4 106 cells to allow for cell growth and to maintain optimal imaging conditions. Where needed, macrophages are activated by adding LPS at 100 ng/mL to the medium from 30 min before imaging. Activated macrophages have more active trafficking and more dynamic REs than preactivated cells. The Personal DeltaVision deconvolution microscope system is used for timelapse imaging for rapid, wide-field illumination and acquisition of images. The microscope is equipped with a chamber for maintaining live cells at 37 C and 5% CO2. The chamber should be temperature equilibrated 37 C for at least 30 min prior to use for maximum stabilization of the imaging system. If CO2 infusion is not available; CO2-independent medium can be used. The 60 objective with objective oil of 1.20 refractive index is typically used to image single cells. In our live cell examples, we utilize the DeltaVisions optical axis integration (OAI) scan function to limit the time required to take a small optical z-scan. This helps to speed up image acquisition and decrease photobleaching and photo-toxicity. Imaging conditions are carefully selected to minimize photobleaching and pixel saturation during image acquisition.

2. EXPERIMENTAL STRATEGIES 2.1 STRATEGY 1: RE SUBCOMPARTMENTS DEFINED BY RABS 2.1.1 Background and objective The RE network is highly compartmentalized with subcompartments that collect and sort exocytic, recycling, and endocytic cargos (Manderson et al., 2007; Marchese, 2014). Membranes that pinch off the RE network are also carriers that can fuse with the plasma membrane to deliver exocytic or secretory cargo. As in epithelial cells, REs in macrophages organize “polarized” trafficking to different cell surface projections or to the front and back of migrating cells (Das & Guo, 2011; Hsu & Prekeris, 2010). The multiple members of the Rab family associate with and demark different subcompartments of REs. Comparing two or more tagged Rabs in cotransfected cells for instance highlights the segregation of Rabs often on

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overlapping but also distinct RE membranes. Rabs11a and 8a are sequentially acting Rabs on REs in exocytic pathways (Das & Guo, 2011), while they often exhibit coordinated actions through their effectors and accessory proteins, they typically occupy nonidentical membrane domains in the RE network. An important point for further investigation is how guanine nucleotide exchange factors and other cellular machinery target and retain Rabs on these very specific membrane domains. Since specific Rabs are perhaps the most recognizable markers for REs, expressing and localizing fluorescently tagged Rabs is a mainstay of RE studies. To highlight RE subcompartments in live cells, and to assign individual Rabs to different structural and functional aspects of the RE network in macrophages, we describe the coexpression and imaging of fluorescently tagged RE-associated GTPases, Rab8a, and Rab11a. Imaging in fixed and live cells highlights the segregation of these two Rabs on different membrane domains within the RE network.

2.1.2 Flow of the experiment 1. Transfect macrophages plated on coverslips for fixed cells or MatTek dishes (for live imaging) with a 1:1 mixture of GFP-Rabb1a and td-tomato-Rab8a plasmids. 2. Wash cells and replace with RPMI medium for 16 h. 3. Cells can be image fixed or live on a fluorescence microscope of choice (e.g., Personal DeltaVision deconvolution microscope). 4. Prior to experiments, cells are stimulated with 100 ng/mL of LPS for 30 min to activate them. 5. For fixed cell examples, images are merged using ImageJ to see overlap and colocalization. 6. For live cell examples, images are collected using time-lapse microscopy that is defined by the fluorescent intensity of the transfected proteins. Using the DeltaVision microscope, we utilize the OAI scan function and keep the lamp power at 30% or bellow to limit photo-toxicity. For fixed cell examples, a higher power can be utilized. 7. Aquired OAI scans are deconvolved as projections using Applied Precision’s online microscope software and visualized using ImageJ.

2.1.3 Results In fixed macrophages, REs appear as puncta throughout the cytoplasm and in concentrations in the perinuclear area (Figure 1(A)). GFP-Rab11a is on many puncta, consistent with its widespread distribution on REs. Rab8a is much less abundant on internal membranes. It is found on some RE puncta but also on surface ruffles and some larger, circular macropinosomes or short tubular elements. The overlap of Rabs 8a and 11a can be seen on specific RE puncta (Figure 1(A), box) where examples demonstrate the range of colocalization scenarios for these two RE Rabs. Rabs 8a and 11a can be on separate RE puncta, they can be seen as adjacent labeling on the same puncta and on a few REs these is complete overlap of the two labeled

RAW264.7 macrophages coexpressing GFP-Rab11a and td-Tomato-Rab8a. (A) Fixed cells show perinculear concentrations of active Rabs11a and 8a on REs and scattered RE puncta throughout the cells. Rab8a is additionally found on larger peripheral macropinosomes. The inset shows a selection of punctate REs highlighting examples of complete overlap of both Rabs, their side-by-side juxtaposition on some REs and separately labeled REs, together representing the dynamic flux of membranes containing these Rabs on intersecting subcompartments. (B) Live cell imaging of cotransfected macrophages. The REs in live cells appear much more tubular, often ringlike and their dynamic nature can be appreciated in movies. Frames from a movie show Rab8a positive tubular membranes forming macropinosomes from surface ruffles and coursing back into the REs from the surface. These tubules skirt around a vacuole and intersect with Rab11a REs (arrowheads). Scale bar ¼ 10 mm. (See color plate)


FIGURE 1 Localization of recycling endosome-associated Rab GTPases in macrophages.

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Rabs. These distributions reflect the presence of Rabs11a and 8a on highly dynamic, intersecting membranes within the RE network. Live imaging provides a clearer picture of the dynamic nature of membranes labeled for the two Rabs. In the cell depicted (Figure 1(B)), GFP-Rab11a appears on punctate membranes scattered throughout the RE network. In movies, these Rab11a puncta can be seen moving around in the cell, often moving toward the cell surface or moving back and forth between the cytoplasm and cell surface. In contrast, td-tomatoRab8a is found on surface ruffles in macrophages (Luo et al., 2014) and on very tubular membranes that typically form from the cell surface and bring membrane back into the REs. Live cell imaging in macrophages highlights the prominent and perhaps preferential association of Rab11a with exocytic components of REs and of Rab8a with membrane being retrieved from the cell surface back into REs.

2.2 STRATEGY 2: CARGO MOVEMENT THROUGH RE 2.2.1 Background and Objective Interconnected endosomal networks within macrophages handle many different types of cargo molecules, with varied destinations (Maxfield & McGraw, 2004). Different cytokines traverse the REs during movement from the trans-Golgi network towards the cell surface for secretion (Manderson et al., 2007; Stow et al., 2013; Beaumont et al., 2011). Receptors can be internalized into REs and then targeted for degradation or recycling (Marchese, 2014). The RE is also important for handling receptors during pathogen contact (Husebye et al., 2010) and for bringing nutrients into the cell on recycling surface receptors (Maxfield & McGraw, 2004). Transferrin receptors in complex with the iron chelating protein transferrin traverse this network, bringing iron into the cell and then returning to the surface for new rounds of uptake. Both transferrin receptors and labeled transferrin are quintessential markers of REs in most cell types (Maxfield & McGraw, 2004). For studies on REs, fluorescently labeled cargo can be used to demark the compartment itself and to track movement of the cargo and the REs through endocytic, recycling, or exocytic routes. Tracking multiple cargos, such as different cytokines, can also demonstrate how cargo is sorted and compartmentalized within the RE network (Manderson et al., 2007). Tracking and measuring the rates at which fluorescently tagged cargo moves throughout the RE network can provide insights into the functions of RE-associated proteins and it can be used to measure disease-related cell dysfunction. Here we describe two experiments that can be used to study the function of RE associated proteins. The first uses three different endocytic markers to identify three trafficking routes that all intersect with the RE cargo transferrin at some point during recycling. Fluorescent dextran labels all fluid phase entry into the cell such as macropinocytosis, while LDL represents receptor-mediated entry. Both that bth ultimately lead to a degradative fate in the lysosomes. The second experiment measures the half-life of transferrin in the RE by measuring the transferrin total fluorescence over-time. These two experiments can be used to study the function of RE resident proteins in both knockdown or over-expression studies.


2.2.2 Flow of the experiments Experiment 1: Recycling endosome component trafficking pathways Stage #1 Experimental Setup and Acquisition 1. RAW264.7 macrophages are seeded onto #1.5 glass coverslips the day prior to experimentation. 2. The following day, cells are washed three times with serum-free medium and serum starved at 37 for 30 min. 3. Coverslips are then inverted onto 50 mL drops of serum free medium containing fluorescently labeled transferrin and incubated for 10 min at 37 (pulse). 4. Coverslips are washed three times with cold complete medium to remove any excess fluorescently labeled transferrin. 5. Coverslips are then inverted onto 50 mL drops of pre-warmed complete medium containing fluorescently labeled Alexa488-dextran (200 mg/mL) and Alexa594-LDL (5 mg/mL) and incubated for 37 (chase). 6. Coverslips are washed three times with cold PBS at chosen time-points, and fixed with 4% paraformaldehyde for 30 min. 7. Individual cells are imaged at 60 magnification using a DeltaVision deconvolution microscope. Fluorescent transferrin, dextran and LDL are captured in their respective channels and are deconvolved using Applied Precisions online microscope software. Bright field is also captured for cell outline. Stage #2 Analysis of Images 1. Cell outlines are manually traced using the bright-field image and added to ROI manager. 2. Images are split into their individual channels for each of the fluorescently labeled endosomal cargo and are cropped based off the ROI. 3. Calculating colocalization of RE constituentsdFluorescent measurements of colocalization are determined for each marker pair (dextran/LDL, transferrin/LDL, transferrin/dextran) using the default colocalization tool within FIJI, Colocalization Threshold, which implements the Manders overlap coefficient. Relative levels of fluorescent colocalization are compared over multiple time-points to determine the changes in cargo coincidence throughout the endosomal network.

Experiment 2: Rates of Transferrin Trafficking from the Cell Surface Stage #1 Experimental Setup and Acquisition 1. RAW264.7 macrophages were seeded on MatTek dishes. 2. The following day, cells are washed three times with serum-free medium and serum starved at 37 for 30 min prior to experimentation. Excess medium is then removed from the outer part of the MatTek dish, which is then wiped dry, leaving a small volume of medium over center coverslip in order to minimize incubation volumes for labeled transferrin.

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3. Fluorescently labeled Alexa647-transferrin diluted in medium at a final concentration of 10 mg/mL is added directly to the cells for 10 min (pulse) at 37 C. 4. Excess transferrin, after the pulse, is washed away rapidly, the MatTek dish is placed on ice for transport to the imaging chamber, where it is washed with 37 C pre-warmed complete medium before imaging. 5. A single field of view would be captured per/dish at 40 magnification to increase the number of cells that can be imaged simultaneously. 6. Fluorescent channels captured included the tagged transferrin, fluorescently transfected marker (usually eGFP) and bright field to capture cell outline for later analysis. OAI scans were acquired every 20 s for a period of 20 min as the transferrin exited the cell (chase). 7. Live cell image files are deconvolved as projections using Applied Precision’s online microscope software. Stage #2 Analysis of Images - Calculating the Half-Life of Transferrin 1. Image analysis is performed in FIJI open access image software. 2. Images undergo a background subtraction, using the default plug-in with a rolling ball radius of 50 pixels. Previous experimentation has suggested that minimal bleaching occurs with 20 s time-lapses of fluorescently labeled Alexa-dyes. 3. For live cells, image sequences are split into three different color channels (transferrin-cargo, eGFP (e.g., for an over-expressed Rab) and bright field (cell outline). 4. Cell outline images are generated from the first frame of the bright-field image sequence. The assumption is that cells do not move significantly during the acquisition so individual segmentation at each time point is not necessary. Cell outlines are individually traced and selected as their own region of interest (ROI). 5. ROIs are then overlaid with the fluorescent transferrin channel and individual fluorescent measurements are taken at each time point. To correct background intensity variation, a background region was selected and the median intensity in this region was subtracted from the mean cell region reading, for each slice. 6. Intensity values are plotted against time, and multiple cells measurements are grouped for statistical analysis. 7. For the determination of the transferrin half-life we need to solve for the exit rate constant (Kexit). We have modeled the change in amount of transferrin in a cell as follows: the rate of change in the amount of the transferrin in the cell over time is proportional to the amount of transferrin in the cell, that is represented by the equation dXin/dt=KexitXin. Therefor; Xin(t) ¼ Xin(0)eKexit(t) where we measure Xin(t) (total Tfn fluorescence at any timepoint (t)), Xin(0) (Tfn fluorescence at time 0) and use Eulers constant (e) to solve for Kexit, using Matlab or similar software.


2.2.3 Experimental considerations This method can be applied to a variety of experimental questions; however, there are a number of experimental considerations: •

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Cells should be grown to a semi-confluent state to allow for increased cell numbers from each single acquisition, however, overcrowding can influence the ability to individually segment cells to single ROIs. Cells can be treated with a number of pharmacological inhibitors to test their effects on recycling in the cells, or secretion of cargo via the REs. Cells can be labeled with more than one cargo and dual pathways can be explored, fluorescently labeled dextran or LDL can be added in conjunction with transferrin to look at maturation of early endosomes (EEs) to lysosomes, and or the effects on sorting of cargo as it passes through REs. Manipulation of host proteins with the use of dominant negative Rab constructs, or siRNA knockdown of host trafficking Rabs can also be used to explore potential defects in cell recycling pathways using either fixed RE constituents or live cell transferrin exit quantification. Data can be collected using these approaches to explore more complex trafficking events of different cargo components throughout the entire endosomal network. The development of this quantification strategy is described elsewhere in more detail (Lamberton, Condon, Stow, & Hamilton, 2014).

2.2.4 Results Alexa647-transferrin internalized for 10 min labels an extensive series of punctate or vesicular structures extending from near the cell surface (labeled for F-actin) to the perinuclear area of fixed macrophages (Figure 2(A)). Uptake of Alexa647transferrin in cells transfected with GFP-Rab11a shows the extensive, but not complete, colocalization of the two markers in RE puncta throughout the cytoplasm (Figure 2(B)). This shows that internalized transferrin is largely in REs but that, as a recycling cargo, it does not extend to all parts of the RE network, particularly the juxtanuclear RE where GFP-Rab11a is more concentrated. Transferrin can be compared to other cargo moving through or intersecting with the RE network in cells harboring multiple labelled proteins. The fluid phase marker, Alexa488-10,000 MW-dextran is taken up by and labels macropinosomes. Alexa594eLDL is internalized by either macropinocytosis or by receptor-mediated endocytosis in clathrin-coated vesicles that merge with early/sorting endosomes. The resulting fixed cells show a series of puncta with one, two, or three labeled cargo. Widespread puncta labeled for LDL and/or transferrin (Figure 3(C)) show the extensive reach of the combined early/sorting and RE networks. The larger puncta labeled with dextran indicate the presence of macropinosomes. The puncta magnified in the inset demonstrate how all three cargos intersect in a proportion of endosomes. Performing imaging at different time points of uptake or in pulse-chase regimes, can be used to chart and quantify the dynamic course of cargo moving through EEs

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FIGURE 2 Recycling endosome cargo and trafficking in macrophages. RAW264.7 macrophages trafficking the well-known recycling endosome cargo Transferrin. (A) Macrophages pulsed with Alexa647-transferrin (pseudo colored green) for 10 min and chased into REs for 20 min were fixed and F-actin labeled with Alexa594 phalloidin (red), the nuclei are labeled with DAPI (blue). The presence of internalized transferrin in REs throughout the cytoplasm is evident. (B) Macrophages transiently transfected with GFPRab11a (green) and pulsed with Alexa647-transferrin (red) both label REs, the plasma membrane is labeled with Alexa350-wheat germ agglutinin (blue). Rab11a and transferrin overlap in some REs but are not identical, highlighting the dynamic and differential distribution of elements within this network. (C) Macrophages pulsed with Alexa647transferrin (pseudo-colored blue) to label REs, Alexa488-dextran labeling macropinosomes (pseudo-colored red) and fluorescently tagged Alexa594-LDL (pseudo-colored green) showing receptor-mediated uptake over 20 min chase. Inset shows tri-labeled RE (arrows) containing transferrin, dextran, and LDL. (D) Quantification of colocalization between fluorescently labeled transferrin (Tfn), dextran (dex), and LDL over time. Manders overlap coefficient is represented as % colocalization. (E) Live cell image acquisition of macrophages pulsed with fluorescently labeled Alexa647-transferrin, with frames showing exit of transferrin over acquisition period. (F) Model for exit quantification of transferrin. Xin (total intracellular transferrin), Xout (extracellular transferrin), Kexit (arrow, rate of loss of transferrin). (G) Graphical comparison of experimental data (blue) of transferrin exit over time, and mathematically modeled decay (red) of transferrin exit, with a predicted rate constant of 0.0023116, which equates to a transferrin half-life of 300 s. (See color plate)

FIGURE 3 Recruitment of VAMP3 during early stage phagocytosis.


(A) Schematic diagram of VAMP3 and C-terminal position of fluorescent tags. VAMP3 contains a cytoplasmic helical coiled-coiled domain that interacts with other SNARES, a TM domain. (Right panel) RAW264.7 macrophages transiently expressing GFP-VAMP3 phagocytosing an IgGopsonized bead. Box indicates GFP-VAMP3 on surface membrane ruffles. Inset shows the localization of GFP-VAMP3 to the nascent phagocytic cup and on the phagosome after internalization (arrows). Another internalized IgG-bead is seen in the vicinity with GFP-VAMP3 enrichment on the phagosome which gradually dissociates from the maturing phagosome (asterisks). (B) Schematic diagram of fluorescence dynamics of SEPVAMP3 showing quenching of SEP-VAMP3 in acidic endosomal compartments. SEP-VAMP3 fluoresces brightly upon exposure to the external environment (pH 7.4) when recycling endosomes fuse at the plasma membrane. (Right panel) RAW264.7 macrophages transiently expressing SEP-VAMP3 show SEP-VAMP3 on peripheral compartments, on surface ruffles (arrow) and on early phagosomes (box). The phagocytic membrane around an IgG-opsonized bead shows fluorescence “bursts” of SEP-VAMP3 denoting RE fusion at the phagocytic cup (arrowhead). More mature phagosomes in the inner part of the cytoplasm are not fluorescing, having lost VAMP3 or having SEP-VAMP3 quenched by the acidic lumens. Scale bar ¼ 10 mm. (See color plate)

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and onto REs and Lysosomes. The results depicted in Figure 2(D) record the fates of transferrin, dextran, and LDL internalized simultaneously in macrophages. Cells were fixed at intervals over 30 min to quantify the fluorescence for each cargo, and colocalization of pairs of cargo proteins are demonstrated. Over time, dextran and LDL, which pass through REs but are largely destined for lysosomes, have increasing colocalization with each other. Transferrin initially and transiently increases in colocalization with LDL, and to a lesser extent with dextran, as these cargo proteins traverse before being sorted to REs and lysosomes, moving away from the transferrin in the recycling system. This demonstrates the concept of tracking cargo moving through the endocytic system and pinpoints key steps that are required for sorting to REs. Live imaging of internalized fluorescent transferrin is used to chart its uptake from the surface as a bolus, its recycling course and exit from the cell over time, which results in a progressive diminution of total intracellular transferrin (Figure 2(E)). The loss of transferrin fluorescence can be modeled (Figure 2(F) and (G)). Actual fluorescence measurements (blue line, Figure 2(G)) show the results concur with the model of predicted exit (red line, Figure 2(G)) of transferrin from REs. By our measurements, transferrin typically exits macrophages with a half-life of approximately 250e300 s. In conclusion, tracking cargo is a powerful strategy for studying the nature and functions of REs in this highly dynamic network in macrophages.

2.3 STRATEGY 3: DELIVERY OF RE TO PHAGOSOMES 2.3.1 Background and objective REs connect with the cell surface and with membrane protrusions and invaginations forming directly from the plasma membrane. The process of phagocytosis is used by macrophages for internalizing and destroying pathogens, dead cells, or particles (Botelho & Grinstein, 2011). The plasma membrane and a variety of endosomes, including REs, contribute membrane for the extension of phagocytic cups and then endosomes participate in the two-way exchange of membrane during maturation of the phagosome (Huynh et al., 2007). Different membrane domains labeled with Rab11a or Rab8a associate with forming phagosomes or their precursor ruffles for signaling from Toll-like receptors to activate macrophages during pathogen responses (Husebye et al., 2010; Luo et al., 2014). Thus fusion of REs with early stage phagosomes is critical for multiple facets of the innate immune response. The membrane fusion protein, VAMP3da member of the SNARE protein familydis a well-established resident of REs in macrophages (Stow et al., 2006). VAMP3 coordinates fusion of the REs with membranes at the base of nascent phagocytic cups in macrophage (Bajno et al., 2000; Murray et al., 2005). Transmembrane VAMP3 can be labeled with GFP attached to its short lumenal domain (Figure 3(A)) and this allows localization of GFP-VAMP3 throughout the cell. Alternatively, VAMP3 can be tagged with a pH-sensitive variant of GFPdsuper-ecliptic pHluorin (SEP)dwhich fluoresces at neutral pH but not in the acidic lumens of REs or other endosomes (Sankaranarayanan, De Angelis, Rothman, & Ryan, 2000). Using this


construct, the actual fusion of REs with phagosomes will be registered by the sudden fluorescing of the SEP-VAMP3 when the probe is exposed to the neutral pH of the external medium or in the very early stage phagosome. The goal of this experiment was to image the behavior of GFP-VAMP3-labeled REs in the vicinity of nascent phagosomes and second to use SEP-VAMP3 to image the fusion of REs with early stage phagosomes.

2.3.2 Flow of the experiment 1. Transfect RAW264.7 macrophages plated on MatTek dishes with GFP- or SEPVAMP3. Maintain cells at 37 C for 24 h. Cells should be 80e90% confluent upon imaging. 2. Activate cells with 100 ng/mL LPS for 30 min before imaging to enhance the phagocytic capacity of the macrophages. 3. Prepare twice the concentration of IgG beads in RPMI 1640 medium (4 mL of 10% IgG bead suspension in 1 mL of medium). 4. Equilibrate MatTek dishes and IgG bead containing culture medium in heating chamber for 10 min. 5. Withdraw 1 mL of culture medium from the MaTtek dish. 6. Choose an optimally spaced and spread out cell expressing SEP-VAMP3. The cells should not be very bright as SEP-VAMP3 fluorescence is significantly quenched at 5.5e6.5 low intracellular pH. 7. Depending on the fluorescence intensity, the emission power and excitation time can be ideally reduced as SEP-VAMP3 fluoresces brightly at the plasma membrane upon fusion of SEP-VAMP3 positive REs. 8. Set desired top and bottom Z-slice at 0.2e0.4 mm/step for OAI scan of 3e4 mm slice. 9. Set up image capturing parameters at one frame/s for 10 min. 10. Add 1 mL of IgG bead culture medium. 11. Allow IgG beads to settle down onto cells. This usually takes 2e4 min. The IgG beads can be observed using DIC to track their movement on cells. 12. Upon cell contact, imaging capturing can be initiated.

2.3.3 Experimental considerations The focal depth of phagocytosis might change during the process as the cell draws the IgG bead into the cell (from top to bottom). Thus manual operation of the finetuning knob on the microscope can be used to accommodate this. Latex beads usually exhibit an optical fluorescence aberration (spherical aberration) around the circumference of the bead, however the bright VAMP3-SEP fluorescence “bursts” at the cell surface are obvious even superimposed on this optical effect. To avoid the optical aberration obtained with beads, phagocytosis experiments can be done using IgG-opsonized sheep red blood cells which do not exhibit this aberration. These image-based methods can be used for rapid, quantitative screening of

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phagocytosis in macrophages under drug-treatment regimes or to assess loss of protein function (Yeo, Wall, Stow, & Hamilton, 2013).

2.3.4 Results Expression of GFP-VAMP3 can be used to localize REs and to track their movement in live cells (Figure 3(A)). In macrophages, the distribution of GFP-VAMP3 mirrors the trafficking cycle of VAMP3. GFP-VAMP3 resides on the perinuclear and punctate REs through the cell from where it functions to fuse REs with the cell surface or phagosomes. GFP-VAMP3 appears transiently on these destination membranes before it is reinternalized and returned to the REs for subsequent rounds of fusion. Accordingly, GFP-VAMP3 is concentrated (transiently) on surface ruffles where membrane fusion is prominent (Figure 3(A)). When cells begin to phagocytose surface-attached opsonized beads, GFP-VAMP3 appears early on at the base of the bead and GFP-VAMP3-labeled membranes can be seen gradually encircling the beads (Figure 3(A), arrow). During internalization of the phagocytic cup, there is significant remodeling of the membranes and GFP-VAMP3 is on ruffles, tubules and vesicular structures as membrane is exchanged between the phagosome and endosome and the cell surface. At later stages of phagocytosis, GFP-VAMP3 is lost from the mature phagosome (Figure 3(A), asterisk). VAMP3 with a C-terminal SEP tag can be used to track the exocytosis of REs, fluorescing as the REs fuse with the plasma membrane or with the still neutral early phagosomal membranes before phagosomes mature and acquire an acidic lumen (Figure 3(B)). Newly synthesized SEP-VAMP3 fluoresces moderately in the near neutral pH of the Golgi complex as it makes its way out to the REs, but then its fluorescence is suppressed in the slightly acidic environs of the trans-Golgi network, post-Golgi vesicles, and REs. Some puncta near the cell surface fluoresce perhaps as surface-connected REs or newly internalized endosomes prior to acidification. Strongly fluorescent SEP-VAMP3 is present on surface ruffles (Figure 3(B), arrow) as the probe is exposed to the neutral pH of the external milieu. During phagocytosis (Figure 3(B)), SEP-VAMP3 fluoresces brightly on early stage phagosomes either prior to closure, or immediately after closure. However, as the phagosome matures and becomes acidified, SEP-VAMP3 no longer fluoresces. The point of fusion of a SEP-VAMP3-labeled RE with the phagosome is seen as a bright burst or spot of fluorescence (Figure 3(B), arrowhead). This approach provides direct evidence that REs fuse with early stage phagosomes. Thus, this construct is especially useful for observing fusion of REs at the cell surface in the context of exocytosis and secretion.

SUMMARY In this chapter, we have described several approaches for the imaged-based analysis of the RE network in macrophages. The detailed methodology presented will aid replication of the RE labeling and analysis approaches we have optimized for use in macrophages as an example of a highly differentiated, “professional trafficking”

References

cell type. The approaches and results can be extrapolated to other cell types and indeed it would be interesting to do so as a means of comparing the relative contributions of REs to cell type specific functions. Live cell imaging using deconvolution, confocal, or spinning disc microscopes provides a wealth of dynamic, structural, and functional information about the REs and RE-associated molecules in macrophages. We have provided snapshots of three approaches for labeling and imaging REs, each of which can be extended to perform complex analyses or modified for rapid screening purposes. Comparing sets of Rabs by coexpression and colocalization is a key means for revealing the diverse subcompartments of REs and cells will tolerate for a short time, transient coexpression of up to five or six labeled Rabs to extend this type of analysis. An endless array of labeled cargo can be employed for tracking exocytic and endocytic routes through the cell, many of which involve REs. We have provided one set of algorithms for quantifying cargo movement in live cells, focused here on the transit and exit of a bolus of transferrin. In addition to Rabs, SNAREs are also useful experimentally as organelle markers and here we demonstrate the use of labelled VAMP3 to show fusion of REs with phagosomes. Moreover the use of the pH-sensitive label is ideal for imaging exocytosis and stage-specific phagosome delivery. The use and extension of all these imaging approaches will continue to be a mainstay for exploring REs as a multifunctional cell organelle and for understanding complex RE roles in innate immune responses.

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Huynh, K. K., Kay, J. G., Stow, J. L., & Grinstein, S. (2007). Fusion, fission, and secretion during phagocytosis. Physiology (Bethesda), 22, 366e372. Lacy, P., & Stow, J. L. (2011). Cytokine release from innate immune cells: association with diverse membrane trafficking pathways. Blood, 118, 9e18. Lamberton, T. O., Condon, N. D., Stow, J. L., & Hamilton, N. A. (2014). On linear models and parameter identifiability in experimental biological systems. Journal of Theoretical Biology, 358, 102e121. Luo, L., Wall, A. A., Yeo, J. C., Condon, N. D., Norwood, S. J., Schoenwaelder, S., et al. (2014). Rab8a interacts directly with PI3Kg to modulate TLR4-driven PI3K and mTOR signalling. Nature Communication, 15(5), 4407. http://dx.doi.org/10.1038/ncomms5407. Manderson, A. P., Kay, J. G., Hammond, L. A., Brown, D. L., & Stow, J. L. (2007). Subcompartments of the macrophage recycling endosome direct the differential secretion of IL-6 and TNF{alpha}. The Journal of Cell Biology, 178, 57e69. Marchese, A. (2014). Endocytic trafficking of chemokine receptors. Current Opinion in Cell Biology, 27, 72e77. Maxfield, F. R., & McGraw, T. E. (2004). Endocytic recycling. Nature Reviews Molecular Cell Biology, 5, 121e132. Murray, R. Z., Kay, J. G., Sangermani, D. G., & Stow, J. L. (2005). A role for the phagosome in cytokine secretion. Science, 310, 1492e1495. Nair-Gupta, P., Baccarini, A., Tung, N., Seyffer, F., Florey, O., Huang, Y., et al. (2014). TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation. Cell, 158, 506e512. Sankaranarayanan, S., De Angelis, D., Rothman, J. E., & Ryan, T. A. (2000). The use of pHluorins for optical measurements of presynaptic activity. Biophysics Journal, 79, 2199e2208. Stenmark, H. (2009). Rab GTPases as coordinators of vesicle traffic. Nature Reviews Molecular Cell Biology, 10, 513e525. Stow, J. L., Manderson, A. P., & Murray, R. Z. (2006). SNAREing immunity: the role of SNAREs in the immune system. Nature Reviews Immunology, 6, 919e929. Stow, J. L., & Murray, R. Z. (2013). Intracellular trafficking and secretion of inflammatory cytokines. Cytokine Growth Factor Reviews, 24, 227e239. Su¨dhof, T. C., & Rothman, J. E. (2009). Membrane fusion: Grappling with SNARE and SM proteins. Science, 323, 474e477. Wickner, W. (2010). Membrane fusion: five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles. Annual Reviews Cell and Developmental Biology, 26, 115e136. Yeo, J. C., Wall, A. A., Stow, J. L., & Hamilton, N. A. (2013). High-throughput quantification of early stages of phagocytosis. Biotechniques, 55, 115e124.

CHAPTER

Analyzing the functions of Rab11-effector proteins during cell division

2 Rytis Prekeris

Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA E-mail: [email protected]

CHAPTER OUTLINE Introduction .............................................................................................................. 20 1. Methods for Analyzing Proteins Regulating Cytokinesis .......................................... 22 1.1 Multinucleation Analysis ........................................................................ 22 1.1.1 Procedure .......................................................................................... 23 1.1.2 Data interpretation.............................................................................. 23 1.2 Measuring Delay in Abscission ............................................................... 24 1.2.1 Procedure .......................................................................................... 24 1.2.2 Data interpretation.............................................................................. 24 1.3 Analyzing the Establishment of the Abscission Site .................................. 25 1.3.1 Procedure .......................................................................................... 26 1.3.2 Data interpretation.............................................................................. 26 2. Time-Lapse Analysis of Endosome and Actin Dynamics during Cytokinesis............... 27 2.1 Analyzing Rab11/FIP3-Endosome Transport during Cytokinesis................. 27 2.1.1 Procedure .......................................................................................... 28 2.1.2 Data interpretation.............................................................................. 29 2.2 Analyzing Actin or Microtubule Dynamics during Cytokinesis .................... 29 2.3 Cytokinesis and Apical Lumen Formation in 3D Cultures .......................... 30 2.3.1 Procedure .......................................................................................... 30 Summary .................................................................................................................. 33 Acknowledgments ..................................................................................................... 33 References ............................................................................................................... 33

Abstract Recycling endosomes recently have emerged as major regulators of cytokinesis and abscission steps of cell division. Rab11-endosomes in particular were shown to transport proteins to the mitotic ingression furrow and play a key role in establishing the abscission Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.05.001 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 2 Analyzing the functions of Rab11-effector proteins

site. Rab11 GTPase functions by binding and activating various effector proteins, such as Rab11 family interacting proteins (FIPs). FIPs appear to be at the core of many Rab11 functions, with FIP3 playing a role in targeting of the Rab11-endosomes during mitosis. Here we summarize the newest finding regarding the roles and regulation of FIP3 and Rab11 complex, as well as describe the methods developed to analyze membrane and cytoskeleton dynamics during abscission step of cytokinesis.

INTRODUCTION The last step of cell division is a physical separation of two daughter cells via a process known as cytokinesis (Barr & Gruneberg, 2007; Pollard, 2010). After replication of the genetic material, the mother cell divides by the formation of the cleavage furrow that constricts cytoplasm leaving two daughter cells connected by a thin intracellular bridge (ICB). The resolution of this bridge (abscission) results in separation of two daughter cells. Although the mechanisms that govern abscission are not fully understood, recent evidence suggests that actin cytoskeleton, endosomes, and the ESCRT-III protein complex play a critical role in this process. ESCRT complexes (complexes 0, I, II, and III) were originally described as regulators of multivesicular body formation (Babst, Katzmann et al., 2002). Since then several ESCRT proteins, namely Tsg101, Alix, and ESCRT-III complex proteins, were demonstrated to be required for cytokinesis (Carlton, Agromayor et al., 2008; Carlton & Martin-Serrano, 2007). The model of ESCRT recruitment to the ICB is as follows: Alix and/or Tsg101 are recruited to the midbody by binding to the midbody protein CEP55. These components then recruit various ESCRT-III complex members to the midbody. The ESCRT-III complex has the ability to form w5 nm filaments that are proposed to mediate abscission (Elia, Sougrat et al., 2011; Guizetti, Schermelleh et al., 2011). How ESCRT-III complex proteins move from the midbody to the abscission site remains unclear, but we have shown that localized actin depolymerization and narrowing of the ICB (secondary ingression) are required to establish the abscission site and recruit the ESCRT-III complex (Figure 1). Recycling endosomes (RE) have emerged as important players in mediating abscission (Fielding, Schonteich et al., 2005; Schiel, Childs et al., 2013; Wilson, Fielding et al., 2004). Several reports demonstrated that pronounced changes occur in endocytic recycling during mitosis, and that these changes are required for successful completion of cytokinesis. Originally, it was proposed that REs initiate abscission by fusing with each other and the plasma membrane, thus building a separating membrane in a manner similar to a formation of the phragmoplasts in plant cells. However, recent data from our laboratory (Schiel, Park et al., 2011; Schiel, Simon et al., 2012) have shown that fusion of REs mediates the formation of a “secondary ingression,” thus initiating ESCRT-III recruitment to the abscission site (Figure 1).

FIGURE 1 Mechanisms mediating abscission. In early telophase, as midbody is formed from central spindle microtubules, ESCRT-III complex is recruited to the midbody. In late telophase, Rab11/FIP3 and Rab35 endosomes (likely different endosomal populations) are delivered and fuse with cleavage furrow plasma membrane. Among other factors, these organelles deliver OCRL and p50RhoGAP leading to the localized disassembly of actin cytoskeleton and severing of central spindle microtubules. Actin depolymerization induces formation of the secondary ingression and ESCRT-III translocation from the midbody to the abscission site. Delivery of the ESCRT-III to the secondary ingression leads to a final scission event and separation of daughter cells.

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Rab11, a small GTPase that functions in RE-mediated trafficking of plasma membrane receptors, has recently emerged as a key regulator of RE transport to the ICB during abscission (Fielding et al., 2005; Wilson et al., 2004). All Rab GTPases function by binding and recruiting various effector proteins. While several Rab11-effector proteins have been identified, Rab11 regulates RE delivery to the ICB predominantly via binding to its FIP3-effector protein (Fielding et al., 2005; Wilson et al., 2004). The FIP3/Rab11 complex accumulates at the ICB during mitosis, and depletion of FIP3 by siRNA arrests cells in late telophase, while having no effect on anaphase/early telophase (Simon, Schonteich et al., 2008; Wilson et al., 2004). Interestingly, recent data show that FIP3-endosomes deliver p50RhoGAP (known RhoA GAP) to the ingression furrow during late telophase. Upon delivery of p50RhoGAP to the ICB, it mediates nested depolymerization of the actin cytoskeleton, leading to the formation of the “secondary ingression” and abscission (Schiel et al., 2012) (Figure 1). The past few years have seen a dramatic increase in our understanding of abscission. It is clear that highly dynamic regulation of endosomes, cytoskeleton, and ESCRT complexes is the key to the successful completion of cytokinesis (Schiel et al., 2013). Here, we describe a set of novel techniques/approaches that will allow the further dissection of the machinery governing cytokinesis and abscission.

1. METHODS FOR ANALYZING PROTEINS REGULATING CYTOKINESIS The studies from many research groups led to development of several abscission models. These studies are based on the use of various techniques. The temporary nature of the abscission event makes it difficult to study. Furthermore, the abscission site is very small and difficult to image using standard light microscopy techniques. Finally, there are currently no good markers to clearly identify the abscission site. Consequently, most of the techniques used to detect the abscission defects usually measure the failure of cells to separate rather than the actual abscission event. Unfortunately, the separation of daughter cells is a very complex event that can be affected by many factors. Accordingly, each of the techniques (described bellow) has its shortcomings and a combination of multiple approaches should be used to test the involvement of the candidate protein in the abscission event.

1.1 MULTINUCLEATION ANALYSIS One of the most commonly used and easiest techniques for investigating proteins that regulate cytokinesis is to measure the multinucleation of cells. The idea behind this approach is that the inhibition of abscission results in the regression of the cleavage furrow, thus leading to bi- or multinucleated cells. This technique has been commonly used to detect defects in the formation and ingression of the cleavage furrow. One drawback of using this approach to analyze the abscission event is

1. Methods for analyzing proteins regulating cytokinesis

the failure of cleavage furrow to regress in cells arrested at late telophase. Instead, these cells stay connected with the ICB, often long enough to start a second round of division. Therefore, to better analyze the effect on cell abscission, cells are usually scored for the number of bi- and multinucleated cells along with the number of cells connected by ICBs.

1.1.1 Procedure 1. Coat 22 mm glass coverslips with collagen (placed in 6-well dish). Make sure to clean the glass coverslips with 3% acetic acid, followed by wash with water, beforehand to ensure an even coating of collagen. Alternatively, poly-L-lysine can also be used as a substrate. 2. Under a cell culture hood, plate HeLa cells on collagen-coated glass coverslips. Make sure that cells are no more than 30% confluent. Plating cells too densely will affect their division rates, thus directly affecting the levels of multinucleation. It is paramount to always plate equal number of cells, since the cell density will directly affect cell division rates. 3. Incubate cells in serum-supplemented media for 24 h. 4. Rinse cells with phosphate-buffered saline (PBS) and fix for 15 min with 4% paraformaldehyde (in PBS). 5. Permeabilize cells with 2 mL of blocking solution (PBS, 2% fetal bovine serum, 1% albumin, 2% saponin) by incubating for 20 min. 6. Incubate cells with primary anti-acetylated tubulin antibody (SigmaeAldrich, cat#T7451; dilution 1:100) for 1 h by overlaying 100 mL of blocking solution with primary antibody. Make sure to place a small piece of parafilm on top of the coverslip and wrap 6-well dish in moist paper towel to minimize evaporation. 7. Wash cells three times (5 min each) with 2 mL of blocking solution. Overlay cells with 100 mL of blocking solution with secondary antibody, DAPI, and rhodamine-phalloidin (Invitrogen, cat#R415; dilution 1:50). We typically use anti-mouse IgG antibody conjugated to Alexa488. Incubate for 30 min. 8. Wash cells three times (5 min each) with 2 mL of blocking solution. 9. Mount cells in Vectashield (Vector Laboratories, cat#H-1000). Seal edges with nail polish. 10. Image cells using florescent micropscopy. Rhodamine-phalloidin will allow you to visualize the edges of the cells to count the number of nuclei within each cell. Similarly, anti-acetylated tubulin antibodies will stain the central spindle, allowing visualization of the ICBs.

1.1.2 Data interpretation Typically, untreated HeLa cells have around 2e5% of multinucleated cells. Similarly, 3e6% of cells are still connected with the ICB and therefore in the late telophase stage. Knockdown of known key regulators of abscission, such as FIP3 or CHMP4B (ESCRT-III subunit) usually results in about 20% multinucleation and about 25% cells that remain connected with ICBs (Carlton et al., 2008; Schiel et al., 2013).

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This is presumably due to the fact that some cells that are arrested in late telophase will eventually undergo apoptosis. Indeed, FIP3 knockdown leads to a significant decrease in cell survival (Schiel et al., 2012). Interestingly, a large portion of the telophase-arrested cells will resolve their ICBs using traction-dependent cytokinesis, generating forceful breakage of the extended ICBs and the eventual separation of daughter cells. Thus, if the candidate protein is involved in abscission, one should not expect to see more than 20e25% multinucleated cells.

1.2 MEASURING DELAY IN ABSCISSION Due to the redundancy in cytokinesis machinery knockdown of any single protein involved in mediating abscission may not completely block cell division. A more direct method of evaluating the involvement of candidate proteins in mediating the abscission event is to measure the time required for daughter cells to complete cytokinesis. Indeed, many well-established abscission regulators dramatically increase division time while having a very moderate effect on multinucleation (Dambournet, Machicoane et al., 2011; Schiel et al., 2012).

1.2.1 Procedure 1. Plate Hela cells on collagen-coated glass bottom Grid-50 dishes (Ibidi, cat#81148). Make sure that cells are plated at 20e30% confluency since cells will be imaged for 24 h, during which time the majority of cells will divide at least once. Plating cells too densely will make it difficult to visualize the timing of abscission. 2. Let cells attach for 3e4 h in serum-supplemented media. Replace media to remove floating cells. 3. Set up cells for imaging on an inverted microscope equipped with an X-Y-Z motorized stage and an environmental control system. 4. Image cells with a 20X objective. Pick at least five to six imaging fields (with 10e15 cells each) and image by field-contrast at 30 min time-lapses for 24 h. 5. During cell division the ICB and midbody are quite dynamic and often can leave the field of focus. To ensure visualization of each abscission event at every time point a mini-Z-stack should be imaged. Typically taking 10 images separated by 1 mm Z-step is sufficient to ensure that ICB and midbody can be evaluated at every time point.

1.2.2 Data interpretation Once imaging is complete, the time required for cytokinesis is measured for every cell in each field. Typically, the metaphase is considered to be time point 0, while the resolution of the ICB is marked as the last step in daughter cell separation. The time required for cells to complete mitosis is quite variable and can range from 60 min to 3e4 h. As a result, a large sample of cells is needed (80e100 cells for every condition) to derive a meaningful and statistically sound data about the timing of cytokinesis.

1. Methods for analyzing proteins regulating cytokinesis

1.3 ANALYZING THE ESTABLISHMENT OF THE ABSCISSION SITE Measuring the formation of abscission site is the most direct way of testing the effect of candidate proteins in regulating abscission. Endosomes initiate the abscission site by regulating localized depolymerization of actin cytoskeleton by either delivering p50RhoGAP (Rab11/FIP3-endosomes) or by modulating PI(4,5)P2 levels (Rab35endosomes) (Dambournet et al., 2011; Schiel et al., 2012). Actin depolymerization then leads to the formation of the secondary ingression, leading to the formation of the abscission site (Schiel et al., 2011). The abscission site can be identified by several methods. First, central spindle microtubules are heavily acetylated and are clearly identifiable following staining with anti-acetylated tubulin antibody. Since abscission involves localized spastin-dependent cutting of microtubules (Connell, Lindon et al., 2009) the abscission site can be identified as a gap in the central spindle (Elia et al., 2011; Schiel et al., 2011). Note that the central spindle stained with anti-acetylated tubulin antibodies always has a gap in the middle that is caused by limited access of antibodies to a dense midbody structure. The abscission site usually forms on one or both sides of this midbody-gap and can be identified as secondary gaps in anti-acetylated tubulin staining (Figure 2). Alternatively, the ESCRT-III

FIGURE 2 Models of asymmetric and symmetric abscission. In many dividing cells abscission sites are established bilaterally on both sides of the midbody. That leads to a shedding of the midbody to the extracellular space (left panel). However, abscission site can also be formed only on one side of the midbody, leading to asymmetric abscission (right panel). This type of asymmetric abscission results in midbody inheritance by one of the daughter cells.

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complex can also be used as an abscission site marker. Typically, CHMP4B-GFP is used in these types of studies, since good anti-CHMP4B antibodies are not available. Along with other ESCRT complex members, CHMP4B-GFP is first recruited to the midbody (Figure 1) (Elia et al., 2011; Schiel et al., 2012). After formation of the secondary ingression, part of CHMP4B-GFP translocates to the abscission site and can be identified as a puncta on one or both sides of the midbody. If cells are costained with anti-acetylated tubulin antibodies, these CHMP4B puncta will be present in the secondary gaps within central spindle microtubules (Elia et al., 2011; Schiel et al., 2012).

1.3.1 Procedure 1. Under a cell culture hood, plate HeLa cells on collagen or polycoated glass coverslips. Make sure that cells are no more than 30% confluent. Plating cells too densely will make it difficult to visualize central spindle and the midbody. 2. Incubate cells in serum-supplemented media for 24 h. 3. If using CHMP4B-GFP as an abscission marker, transfect cells and incubate for another 28 h. 4. Rinse cells with phosphate-buffered saline (PBS) and fix for 15 min with 4% paraformaldehyde (in PBS). 5. Permeabilize cells with 2 mL of blocking solution (PBS, 2% fetal bovine serum, 1% albumin, 2% saponin) by incubating for 20 min. 6. Incubate cells with primary anti-acetylated tubulin antibody (SigmaeAldrich, cat#T7451; dilution 1:100) for 1 h by overlaying 100 mL of blocking solution with primary antibody. Make sure to place a small peace of parafilm on top of the coverslip and wrap 6-well dish in a moist paper towel to minimize evaporation. 7. Wash cells three times (5 min each) with 2 mL of blocking solution. Overlay cells with 100 mL of blocking solution with secondary antibody and DAPI. We typically use anti-mouse IgG antibody conjugated to Alexa594 to allow coimaging of acetylated tubulin with CHMP4B-GFP. Incubate for 30 min. 8. Wash cells three times (5 min each) with 2 mL of blocking solution. 9. Mount cells in Vectashield (Vector Laboratories, cat#H-1000). Seal edges with nail polish. 10. Image cells by fluorescent microscopy. Anti-acetylated tubulin antibodies will stain the central spindle. If cells were transfected with CHMP4B-GFP, it can also be used to identify abscission sites.

1.3.2 Data interpretation The abscission rates can be evaluated by counting the percentage of telophase cells that have clearly established an abscission site, as determined by the presence of a gap in the central spindle. Care must be taken not to count a midbody-associated gap. Similarly, abscission sites can be counted by determining the percentage of the cells that contain CHMP4B-GFP puncta within the ICB, but outside the midbody. In addition to counting cells that have an established abscission site, one

2. Time-Lapse analysis of endosome and actin dynamics

can also analyze the effect of experimental treatment on the rates of asymmetric abscission. In about 40% of HeLa cells abscission usually occurs on both sides of the midbody leading to the release of the midbody into the media (Figure 2). Interestingly, in a majority of the cell divisions the abscission site is only established on one side of the midbody (Kuo, Chen et al., 2011). As a result of this asymmetric abscission, one daughter cell will inherit the postmitotic midbody (Figure 2). Importantly, midbody inheritance has been proposed as a mechanism for regulating cell fate and differentiation in daughter cells (Chen, Ettinger et al., 2012; Ettinger, Wilsch-Brauninger et al., 2011; Kuo et al., 2011). Thus, it will be important to identify the factors that mediate the establishment of these asymmetric abscissions. One of the potential drawbacks of using CHMP4B-GFP overexpression as an abscission site marker is the fact that high levels of CHMP-GFP can inhibit abscission presumably by interfering with ESCRT-III function. Thus, care must be taken to analyze only cells expressing low to moderate levels of CHMP4B-GFP.

2. TIME-LAPSE ANALYSIS OF ENDOSOME AND ACTIN DYNAMICS DURING CYTOKINESIS Microscopy analysis of fixed cells remains a powerful tool for the initial characterization of candidate proteins that regulate abscission. However, static image analysis will not uncover the properties and regulation of dynamic changes in action cytoskeleton and membrane dynamics during cell division. Thus, time-lapse analysis of live dividing cells is needed to investigate rapid and localized changes in cytoskeleton and endosomes during abscission. The approaches listed below will allow researchers to investigate the changes in Rab11/FIP3-endosomes (A) or actin and microtubules (B).

2.1 ANALYZING Rab11/FIP3-ENDOSOME TRANSPORT DURING CYTOKINESIS Rab11/FIP3-endosomes have recently emerged as key mediators of abscission (Wilson et al., 2004; Schiel et al., 2012). Importantly, the dynamic movement and localization of Rab11/FIP3-endosomes change dramatically as cells proceed from metaphase to telophase (Figure 3). During metaphase Rab11/FIP3-endosomes accumulate around centrosomes, where they stay until telophase (Figure 3) (Schiel et al., 2011). After cells form the midbody and start progressing to late telophase, Rab11/ FIP3-endosomes move along the central spindle microtubules to the cleavage furrow (Figure 3) (Schiel et al., 2011; Simon et al., 2008). This leads to the translocation of all Rab11/FIP3-endosomes from centrosomes to the endocytic pools situated on each side of the midbody (Figure 3). The translocation of the Rab11/FIP3-endosomes is usually nonsymmetric, with endosomes from one centrosome arriving at the midbody slightly ahead of the endosomes from the other side (Schiel et al., 2011). Since

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FIGURE 3 Rab11/FIP3-endosome dynamics during cytokinesis. During metaphase and anaphase Rab11/FIP3-endosomes associate with centrosomes. As dividing cell progresses to telophase and midbody is formed, Rab11/FIP3-endosomes start translocated along central spindle microtubules to the close proximity of the midbody. By late telophase almost all Rab11/FIP3-endosomes are localized at and around the midbody. This translocation precedes and is required for the final abscission event.

Rab11/FIP3-endosomes initiate the formation of the secondary ingression and the establishment of the abscission site, potentially this asynchronous arrival at the cleavage furrow determines asymmetric abscission and midbody inheritance by one daughter cell. Despite the potential impact on cell fate and differentiation, the machinery mediating asynchronous translocation of Rab11/FIP3-endosomes to the cleavage furrow remains essentially unknown. Thus, time-lapse analysis of FIP3GFP dynamics during cell division is perfectly suited to decipher the mechanisms governing Rab11/FIP3-endosome dynamics and midbody inheritance.

2.1.1 Procedure 1. Transfect HeLa cells with FIP3-GFP and plate cells on collagen-coated glass bottom Grid-50 dishes (Ibidi, cat#81148). Make sure that cells are plated at 30e 50% confluency to allow clear visualization of FIP3-GFP dynamics. Plating cells too dense will result in the formation of short intercellular bridges, making visualization more difficult. 2. Set up cells for imaging on an inverted microscope equipped with an environmental control system. Cells need to be imaged at 37 C to ensure proper endosome dynamics and cytokinesis. Imaging cells at room temperature often leads to either mitotic arrest or dramatically increase the time required for abscission. 3. Image cells with a 63X oil-objective. Pick cells at metaphase and image it every 10e15 min. Since during metaphase cells round up, small Z-stack with 500 nm Z-step. About 30 stack images will be needed to image the entire cell. 4. Imaging cells every 10e15 min will uncover the steady-state changes in FIP3GFP localization but will not allow tracking of individual cells. To visualize and analyze the speed and dynamics of individual FIP3-GFP-endosomes 300 ms time-lapses will be needed. Unfortunately, imaging dividing metaphase cells using such short time-lapses will lead to phototoxicity and can dramatically affect the cell’s ability to divide. Therefore, to analyze the dynamics of

2. Time-Lapse analysis of endosome and actin dynamics

individual FIP3-endosomes we usually pick cells at early telophase that contain a formed midbody which substantially reduces the number of time points needed to visualize FIP3-GFP dynamics through the abscission event. Generally, no more than 50e80 time points should be taken for each cell. Furthermore, picking cells at telophase results in much flatter cells compared to metaphase cells, thus eliminating the need to do z-stacks and further reducing imaging-induced photodamage.

2.1.2 Data interpretation Time-lapse analysis is a powerful tool for investigating many different aspects of Rab11/FIP3-endosome dynamics. Using 10e15 min time-lapse windows, any changes in the steady-state Rab11/FIP3-endosome distribution at different mitotic stages can be captured. Using 300 ms time-lapse windows will allow for measurement of the directionality, processivity, and speed of individual Rab11/FIP3endosomes.

2.2 ANALYZING ACTIN OR MICROTUBULE DYNAMICS DURING CYTOKINESIS In addition to changes in Rab11/FIP3-endosome dynamics, the actin and microtubule cytoskeleton also undergo dramatic and very dynamic changes. As described earlier, during telophase central spindle microtubules are compacted together to form a very dense cellular structure known as the midbody. The spastin-dependent localized severing of these central spindle microtubules is a key step in determining the location and timing of the abscission. Although the machinery determining the site of spastin-dependent microtubule severing remains unclear the location of “cut” appears to depend on central spindle bending during late telophase (Simon et al., 2008). Actin filaments also dynamically change during cytokinesis. During early anaphase actin forms a filamentous actomyosin contractile “ring” at the midzone of the dividing cell. The contraction of this actomyosin ring leads to the formation and ingression of the cleavage furrow. As ingression progresses, the actomyosin ring gets compacted leading to the dramatic increase in filamentous actin amount at the plasma membrane of the cleavage furrow. However, once the ingression is complete and the midbody has formed, the acto-myosin network undergoes a very rapid and localized disassembly, the step that is controlled by endocytic transport and is required for the abscission (Dambournet et al., 2011; Schiel et al., 2012). These findings clearly demonstrate the dynamic nature of cytoskeleton and support the ides that to fully understand the regulation of cytokinesis it is imperative to perform time-lapse microscopy to analyze filamentous actin and microtubule dynamics. Microtubules are typically analyzed using GFP or mCherry-tagged tubulin. This type of analysis usually works better in cells stably expressing tagged-tubulin. Similarly, actin dynamics can be analyzed in cells transfected with GFP-actin. However, some published reports have suggested that GFP-tagging can affect filamentous

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actin dynamics. Therefore, alternatives for imaging actin have recently been developed, including LifeAct and utrophin. LifeAct is a recombinant reporter utilizing a yeast-derived peptide that binds specifically to filamentous actin (Riedl, Crevenna et al., 2008). Tagging LifeAct with either GFP or mCherry enables visualization of the formation and dynamics of actin filaments. Alternatively, actin-binding peptide derived from utrophin can also be tagged with GFP and used to visualize actin filaments. Although LifeAct is more commonly used, we found that both these actin biosensors work very similarly (Schiel et al., 2012). Once transfected with tubulinGFP, actin-GFP, LifeAct-GFP, or utrophin-GFP cells are then analyzed by time-lapse microscopy as described in part (A).

2.3 CYTOKINESIS AND APICAL LUMEN FORMATION IN 3D CULTURES While the main function of cytokinesis is to physically separate a mitotic cell into two daughter cells, several noncanonical functions for cytokinesis have been recently proposed. For example, asymmetric abscission leads to midbody inheritance by one of the daughter cells and has been implicated in regulating cell fate (Chen et al., 2012). Additionally, we recently proposed that cytokinesis and midbody formation is the first symmetry-breaking event that determines the timing and location of apical lumen during epithelial tissue morphogenesis (Li, Mangan et al., 2014) (Figure 4). Based on these findings, several approaches have been developed to image cytokinesis during the process of epithelia polarization and apical lumen formation. One of them is time-lapse analysis of mitosis and epithelia polarization in 3D Matrigel cultures (Li et al., 2014).

2.3.1 Procedure 1. Plate MDCK cells on a 100 mm tissue culture plate in 10 mL MDCK media and let it grow for 24 h at 37 C. 2. One to two hours before plating cells take an aliquot of Matrigel and set it on ice to thaw out. It is very important to keep Matrigel cold even while thawing, since it solidifies rapidly at room temperature. 3. Aspirate media and rinse cells with 10 mL PBS. This step is essential. Leaving some of the serum-supplemented media will inhibit trypsin and will make it very difficult to lift individual MDCK cells. 4. Add 2 mL 0.25% Trypsin-EDTA and let set at 37 C for 10e15 min. MDCK cells are usually difficult to dislodge. Thus, if needed, they can be incubated for 20e25 min. 5. Dislodge cells by gently tapping at the side of the 100 mm dish. Harvest cells by adding 8 mL MDCK media to plate and transferring cells to a 15 mL tube. Sediment cells by centrifugation at 1000x rpm for 3 min. 6. Aspirate media and resuspend cells in 1 mL of MDCK media by pipetting up and down using 1 mL blue pipette tip. Pipetting up and down many times helps to separate cells. It is crucial for this assay to embed individual cells in Matrigel. Embedding cell clumps will lead to formation of cell aggregates

FIGURE 4 Model depicting the role of midbody-associated apical membrane initiation site (AMIS) formation and endosome transport during lumenogenesis in epithelial cells. Upon division of nonpolarized epithelial cell, AMIS forms abound midbody in late telophase. Rab11/FIP3-endosomes containing apical cargo, such as Crumbs3 and gp135, are then delivered along central spindle microtubules to the AMIS, where they fuse and form nascent apical lumen. As polarized epithelial cells divide these nascent lumens mature to become a function apical lumens.

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12.

with multiple lumens. We found that pipetting cells 50 times with a 1 mL pipette usually gives a maximum number of individual cells; however, this number may vary. Thus, it is advisable to initially pipette cells varying number of times, while monitoring the efficiency of cell separation. Count cells to determine number of cells/mL and add 20,000 cells to 25 mL MDCK media in 1.5 mL Eppendorf tube. Make a 75% Matrigel solution by adding 75 mL Matrigel to the 25 mL cell solution from step 7. Gently mix by pipetting up and down. Plate the cell/ Matrigel mix immediately, by placing a drop onto the center of a 5 cm gridded glass-bottom dish. This step needs to be done quickly, since the Matrigel-cell mixture solidifies readily at room temperature. Place cells in the incubator and let it solidify in the tissue culture incubator at 37 C for 30 min. The percentage of Matrigel may also vary between 25 and 80%. The volume of media to suspend cells in and volume of Matrigel added can be adjusted to determine ideal conditions. In lower concentrations of Matrigel, cells may sink through the Matrigel matrix (during process of solidifying the Matrigel) and stick to the bottom of the plate, counteracting the function of the Matrigel to observe the cells suspended in the matrix. If the Matrigel concentration is too high, the cells might not be able to suspend in the matrix and instead would just sit on top, which is again not ideal for 3D imaging. Add 5 mL of MDCK media to the dish and let cells acclimate for 6e12 h at 37 C. Typically cells will start dividing within 12 h after embedding. Imaging them within 6e12 h time period will ensure that at least some cells will be undergoing first cell division. Mount dish on fluorescent microscope and use a gridded chart to label the location of several individual cells, especially those that may be in metaphase (starting division). Adjust focus to set top and bottom of each cell and take initial 0.2 mm-step Z-stack images. Repeat the imaging according to the time frame required for desired observation. This will result in taking a mini-Z-stack for every time point. Generally, avoid taking more than 50e80 time-lapse Z-stacks. To visualize apical lumen formation during cell division, we typically use a 10 min time-lapse. To visualize lumen formation and expansion, we use 30 min time-lapses. Finally, if the motility of individual Rab11/FIP3-endosomes needs to be observed, we use 200e500 ms time-lapse. Use grid etched in glass to locate various cells for imaging at different time points. Taking mini-Z-stacks at every time point allows the use of postacquisition image analysis to generate 3D images of MDCK cells at each time point during lumen formation. Alternatively, individual images that best represent the lumen formation dynamics can be selected and displayed/analyzed for every time point.

References

SUMMARY While the importance of polarized endosomal transport during cytokinesis is clearly established, the function and regulation of this transport is only beginning to emerge. Similarly, we still do not fully understand the machinery mediating localized reorganization of actin and microtubule cytoskeleton. This is in part due to the technical limitations in studying abscission, the very transient cellular event that is hard to image and quantify. Here, we described the newest techniques currently available to study membrane and cytoskeleton dynamics during abscission. It is important to note that while the emergence of live imaging techniques provided significant insights in understanding abscission, many of these techniques still have significant limitations in spatial and temporal resolution to directly analyze the cytokinesis and abscission events. Thus, the use of multiple approaches rather then reliance on a singe technique will be the key in understanding Rab11-endosome and cytoskeleton dynamics during cytokinesis.

ACKNOWLEDGMENTS I am grateful to Abitha Jacob and Dr Alexander Blasky for critical reading of the manuscript. I apologize to all colleagues whose work could not be cited due to space limitations. I also acknowledge the financial support byNIH (DK064380), Cancer League of Colorado, andSusan G. Komen for the Cure foundation (BCTR0706749).

REFERENCES Babst, M., Katzmann, D. J., et al. (2002). Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting. Development Cell, 3(2), 271e282. Barr, F. A., & Gruneberg, U. (2007). Cytokinesis: placing and making the final cut. Cell, 131(5), 847e860. Carlton, J. G., Agromayor, M., et al. (2008). Differential requirements for Alix and ESCRT-III in cytokinesis and HIV-1 release. Proceedings of the National Academy of Sciences of the United States of America, 105(30), 10541e10546. Carlton, J. G., & Martin-Serrano, J. (2007). Parallels between cytokinesis and retroviral budding: a role for the ESCRT machinery. Science, 316(5833), 1908e1912. Chen, C. T., Ettinger, A. W., et al. (2012). Resurrecting remnants: the lives of post-mitotic midbodies. Trends in Cell Biology, 23(3), 118e120. Connell, J. W., Lindon, C., et al. (2009). Spastin couples microtubule severing to membrane traffic in completion of cytokinesis and secretion. Traffic, 10(1), 42e56. Dambournet, D., Machicoane, M., et al. (2011). Rab35 GTPase and OCRL phosphatase remodel lipids and F-actin for successful cytokinesis. Nature Cell Biology, 13(8), 981e988. Elia, N., Sougrat, R., et al. (2011). Dynamics of endosomal sorting complex required for transport (ESCRT) machinery during cytokinesis and its role in abscission. Proceedings of the National Academy of Sciences of the United States of America, 108(12), 4846e4851.

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Ettinger, A. W., Wilsch-Brauninger, M., et al. (2011). Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour. Nature Communications, 2, 503. Fielding, A. B., Schonteich, E., et al. (2005). Rab11-FIP3 and FIP4 interact with Arf6 and the exocyst to control membrane traffic in cytokinesis. The EMBO Journal, 24(19), 3389e3399. Guizetti, J., Schermelleh, L., et al. (2011). Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. Science, 331(6024), 1616e1620. Kuo, T. C., Chen, C. T., et al. (2011). Midbody accumulation through evasion of autophagy contributes to cellular reprogramming and tumorigenicity. Nature Cell Biology, 13(10), 1214e1223. Li, D., Mangan, A., et al. (2014). FIP5 phosphorylation during mitosis regulates apical trafficking and lumenogenesis. EMBO Reports, 15(4), 428e437. Pollard, T. D. (2010). Mechanics of cytokinesis in eukaryotes. Current Opinion in Cell Biology, 22(1), 50e56. Riedl, J., Crevenna, A. H., et al. (2008). Lifeact: a versatile marker to visualize F-actin. Nature Methods, 5(7), 605e607. Schiel, J. A., Childs, C., et al. (2013). Endocytic transport and cytokinesis: from regulation of the cytoskeleton to midbody inheritance. Trends in Cell Biology, 23(7), 319e327. Schiel, J. A., Park, K., et al. (2011). Endocytic membrane fusion and buckling-induced microtubule severing mediate cell abscission. Journal of Cell Science, 124(Pt 9), 1411e1424. Schiel, J. A., Simon, G. C., et al. (2012). FIP3-endosome-dependent formation of the secondary ingression mediates ESCRT-III recruitment during cytokinesis. Nature Cell Biology, 14(10), 1068e1078. Simon, G. C., Schonteich, E., et al. (2008). Sequential Cyk-4 binding to ECT2 and FIP3 regulates cleavage furrow ingression and abscission during cytokinesis. The EMBO Journal, 27(13), 1791e1803. Wilson, G. M., Fielding, A. B., et al. (2004). The FIP3-Rab11 protein complex regulates recycling endosome targeting to the cleavage furrow during late cytokinesis. Molecular Biology of the Cell, 16(2), 849e860.

CHAPTER

Expression and localization of exocytic and recycling Rabs from Magnaporthe oryzae in mammalian cells

3

Yaoyao Qi*, x, M. Caleb Marlinx, Zhimin Liangx, Dongmei Zhang*, Jie Zhou*, Zonghua Wang*, Guodong Lu*, Guangpu Li*, x, 1 *Key Laboratory of Biopesticide and Chemical Biology, Fujian Agriculture and Forestry University, Fuzhou, China x Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA 1


CHAPTER OUTLINE Introduction .............................................................................................................. 36 1. Materials and Equipment ...................................................................................... 37 1.1 Reagents for Cloning and Plasmid Construction ....................................... 37 1.2 Reagents for Protein Expression and Analysis .......................................... 37 1.3 Materials and Reagents for Confocal Fluorescence Microscopy .................. 38 1.4 Equipment ........................................................................................... 38 2. Methods .............................................................................................................. 38 2.1 Cloning of MoRab11, MoRab8, and MoRab1 ........................................... 38 2.1.1 Extraction of total RNA from the M. oryzae strain Guy11...................... 38 2.1.2 Amplifying the cDNAs of MoRab11, MoRab8, and MoRab1 by RT-PCR and cloning into the bidirectional expression vector pBI-Tet with human Rab counterparts ................................................ 39 2.2 Expression and Intracellular Localization of MoRab11, MoRab8, and MoRab1 ......................................................................................... 40 2.2.1 Transfection of BHK-21 cells .............................................................. 40 2.2.2 Immunoblot analysis of protein expression .......................................... 40 2.2.3 Localization by confocal fluorescence microscopy ............................... 42 Summary .................................................................................................................. 42 Acknowledgments ..................................................................................................... 44 References ............................................................................................................... 44 Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.05.002 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 3 Fungal recycling Rabs

Abstract Rab GTPases are master regulators of intracellular membrane trafficking along endocytic and exocytic pathways. In this chapter, we began to characterize the exocytic and recycling Rabs from the filamentous fungus Magnaporthe oryzae (M. oryzae) that causes the rice blast disease. Among the 11 putative Rabs identified from the M. oryzae genome database (MoRabs), MoRab1, MoRab8, and MoRab11 appear orthologs of mammalian Rab1, Rab8, and Rab11 and likely function in exocytosis and endosomal recycling. To test this contention, we cloned, expressed, and determined intracellular localization of the three MoRabs in mammalian cells, in comparison to their human counterparts (hRabs). The MoRabs were well expressed as GFP fusion proteins and colocalized with the tdTomatolabeled hRabs on exocytic and recycling organelles, as determined by immunoblot analysis and confocal fluorescence microscopy. The colocalization supports the contention that the MoRabs are indeed Rab orthologs and may play important roles in the development and pathogenicity of M. oryzae.

INTRODUCTION Rab GTPases are molecular switches that regulate intracellular membrane trafficking on endocytic, exocytic, and recycling pathways (Hutagalung & Novick, 2011; Li & Segev, 2012; Pfeffer, 2013). Each Rab targets to a specific organelle and controls multiple steps of vesicular transport by alternating between GTP-bound “on” conformation and GDP-bound “off” conformation with the assistance of upstream regulators and downstream effectors (Stenmark, 2009). Rab11, Rab8, and Rab1 in particular are involved in endosomal recycling and exocytosis, respectively. Rab11 is localized to so-called slow recycling endosomes mediating transport of internalized receptors via clathrin-dependent endocytosis, e.g., transferrin receptor, back to the plasma membrane (Ullrich, Reinsch, Urbe, Zerial, & Parton, 1996). In polarized cells, Rab11 specifically targets to apical recycling endosomes destined to the apical plasma membrane and plays an important role in the transcytosis of polymeric IgA receptor from basolateral to apical cell surface (Goldenring, Roland, & Lapierre, 2012). Rab8 is involved in recycling different populations of cargoes including those internalized via clathrin-independent endocytosis such as MHC I. Interestingly, Rab11 and Rab8 form a Rab-activation cascade in recycling cargoes to specialized cell surface structures such as the primary cilia (Knodler et al., 2010). This recycling Rab cascade is mediated by Rabin8, which is a guanine nucleotide exchange factor (GEF) for Rab8 but also an effector of Rab11 and can be recruited to the recycling endosomes by Rab11 for activation of Rab8 (Knodler et al., 2010). Rab8 is also suggested to deliver newly synthesized membrane cargoes directly to the cell surface via the exocytic pathway, especially in polarized cells (Goldenring et al., 2012). Along the exocytic pathway, there is another Rab, Rab1, which is localized at the endoplasmic reticulum (ER) exit sites and the preGolgi intermediate compartment (IC) to mediate ER to Golgi transport (Stenmark, 2009; Taussig, Chen, & Segev, 2012). In addition, Rab1 is also involved in the initiation of autophagy (Lynch-Day et al., 2010; Taussig et al., 2012).

1. Materials and equipment

These three recycling and exocytic Rabs are conserved in evolution from the last eukaryotic common ancestor (LECA) to humans (Diekmann & Pereira-Leal, 2013; Elias, Brighouse, Gabernet-Castello, Field, & Dacks, 2012; Klopper, Kienle, Fasshauer, & Munro, 2012), suggesting functions fundamental to eukaryotic cells. Here we present methods for cloning and initial localization study of Rab11, Rab8, and Rab1 homologs from M. oryzae, a pathogenic filamentous fungus in plants that causes rice blast disease (Ebbole, 2007). Although the three Rabs have multiple isoforms in mammalian cells, they have only one isoform each in M. oryzae and are termed here as MoRab11, MoRab8, and MoRab1 among a total of 11 MoRabs identified from the M. oryzae genome database. The MoRabs and their human counterparts are coexpressed in mammalian cells as eGFP and tdTomato fusion proteins, respectively, via a bidirectional expression vector, and are found to colocalize to recycling endosomes and exocytic structures. The results suggest that MoRab11, MoRab8, and MoRab1 are authentic orthologs of mammalian counterparts and should help understand the function of endosomal recycling and exocytosis in the development and pathogenicity of M. oryzae.

1. MATERIALS AND EQUIPMENT 1.1 REAGENTS FOR CLONING AND PLASMID CONSTRUCTION Plasmids: pGEM-T Easy vector (Promega), pBI-Tet (Clontech), pTet-Off (Clontech) Bacterial strains: E. coli DH5a and MC1061 Fungal strain: Guy11 Growth media: For bacteria: LB liquid (Difco) and LB Agar (Difco). For M. oryzae: Complete medium plates containing 0.6% yeast extract, 0.6% casein hydrolysate, 1% sucrose, and 1.5% agarose Ampicillin: 1000x stock solution (100 mg/mL in ddH20 sterilized by filtration, aliquoted and stored at 20 C) Diethylpyrocarbonate (DEPC) (TIANGEN Biotech) RNAiso and SYBR PrimeScriptÔ RT-PCR Kit (Takara) ImProm-IIÔ Reverse Transcription System (Promega)

1.2 REAGENTS FOR PROTEIN EXPRESSION AND ANALYSIS Tissue culture: Baby hamster kidney (BHK) cells (BHK-21 cell line from ATCC) Growth media: a-minimal essential medium (MEM) (Invitrogen) containing 5% fetal bovine serum (FBS) (Invitrogen), glutamine, penicillin/streptomycin Lipofectamine 2000 transfection reagent (Invitrogen) Phosphate-buffered saline (PBS) (Sigma) 1X SDS-loading buffer: 50 mM Tris-HCl (pH6.8), 2% (W/V) SDS, 0.1% (W/V) bromophenol blue, 10% (V/V) glycerol, 1% b-mercaptoethanol (add before use)

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Reagents for SDS-PAGE (12% separating gel): Separating gel 40% Acrylamide:Bis 1.5M Tris-HCl pH8.8 10% SDS Distilled H2O Mix well, then add 10% Ammonium persulfate TEMED

3 mL 2.5 mL 100 mL 4.5 mL 100 mL 10 mL

Stacking gel 40% Acrylamide:Bis 0.5M Tris-HCl pH 6.8 10% SDS Distilled H2O Mix well, then add 10% Ammonium persulfate TEMED

0.5 mL 1.25 mL 50 mL 3.25 mL 50 mL 5 mL

Immobilon-P PVDF membrane (Millipore) Antibodies: anti-GFP monoclonal antibody (mAb) (BD Biosciences), antiDsRed mAb (Sigma), anti-actin mAb (Sigma), IRDye 800CW goat anti-mouse IgG (LI-COR Biosciences)

1.3 MATERIALS AND REAGENTS FOR CONFOCAL FLUORESCENCE MICROSCOPY Coverslips: 12 Circular-1 (Fisher Scientific) Glass Slides: VWR Micro Slides, 25 75 mm 1.0 mm thick ProLong Gold Antifade Reagent with DAPI (Invitrogen) 16% paraformaldehyde (PFA) (Electron Microscopy Sciences)

1.4 EQUIPMENT UV spectrophotometer (GeneQuant, GE Healthcare) Centrifuge (5415R, Eppendorf) CO2 tissue culture incubator (Fisher Scientific) Platform shaker (New Brunswick Scientific) Bio-Rad Semi-Dry Transfer Apparatus Odyssey Infrared Imaging System (LI-COR Biosciences) Leica SP2 MP confocal laser scanning microscope

2. METHODS 2.1 CLONING OF MoRab11, MoRab8, AND MoRab1 2.1.1 Extraction of total RNA from the M. oryzae strain Guy11 1. Guy11 strain was grown in 100 mL liquid complete medium (CM) for 3 days, and the mycelium body was harvested with a filter paper, frozen in liquid nitrogen, and grounded into powder.

2. Methods

2. The powder was transferred to a 1.5-mL Eppendorf centrifuge tube (DEPCtreated) to which 1 mL RNAiso reagent was added and mixed by vortexing for 30 sec, followed by incubation at room temperature for 5 min. 3. Add 200 mL of chloroform to the tube and mix well by vortexing. 4. Centrifuge at 12,000 rpm for 15 min at 4 C. 5. Transfer the supernatant to a new 1.5 mL tube (DEPC-treated), and add 0.6 volume of isopropanol. Mix well and incubate at room temperature for 20 min. 6. Centrifuge at 12,000 rpm for 15 min at 4 C. 7. Aspirate the supernatant, wash the pellet twice with 70% ethanol, and air-dry at room temperature for 20 min. 8. Add 200 mL RNase-free H2O to dissolve the pellet, then add 2 mL 5 mg/mL DNase I and incubate at 37 C for 1 h. 9. Inactivate the DNase I by incubation at 65 C for 20 min. 10. Add RNase-free H2O to bring the total volume up to 400 mL, then add an equal volume of H2O-saturated phenol: chloroform: isoamyl alcohol mixture (25:24:1). 11. Centrifuge at 12,000 rpm for 15 min at 4 C. 12. Transfer the supernatant to a new 1.5 mL tube (DEPC-treated), add 2 volume of ethanol and 0.1 volume of 3M NaAc, mix well and incubate at 80 C for 2 h. 13. Centrifuge at 12,000 rpm for 15 min at 4 C. 14. Aspirate the supernatant, wash the pellet twice with 70% ethanol, and air-dry at room temperature for 20 min. 15. Add 50 mL RNase-free H2O to dissolve the pellet and store the RNA preparation at 80 C. 16. Measure the RNA concentration and purity by using a GeneQuant spectrophotometer.

2.1.2 Amplifying the cDNAs of MoRab11, MoRab8, and MoRab1 by RT-PCR and cloning into the bidirectional expression vector pBI-Tet with human Rab counterparts 1. The M. oryzae RNA extract was used as template for the synthesis of cDNAs of MoRab11 (MGG_01079), MoRab8 (MGG_06135), and MoRab1 (MGG_06962) by RT-PCR using the SYBR PrimeScriptÔ RT-PCR Kit. The resulting cDNAs were cloned into the pGEM-T Easy vector and confirmed by direct DNA sequencing. 2. The cDNAs of MoRab11, MoRab8, and MoRab1 were amplified by PCR and cloned into the NotI/SalI sites of the pBI-Tet expression vector, in-frame with the upstream eGFP cDNA previously cloned at the PstI/NotI sites. The three resulting plasmid constructs are named: pBI/eGFP-MoRab11, pBI/eGFPMoRab8, and pBI/eGFP-MoRab1. 3. The pBI-Tet vector is a bidirectional expression vector and can express a second gene cloned at the Mlu/NheI sites. To this end, the cDNAs of human Rab counterparts and Rab5 as control were cloned into the Mlu/NheI sites and the

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tdTomato (tdTom) cDNA was cloned in-frame upstream of the MluI site. As such, eight expression constructs were generated, which are as follows (Figure 1): pBI/eGFP-MoRab11/tdTom-hRab11 pBI/eGFP-MoRab11/tdTom-hRab5 pBI/eGFP-MoRab8/tdTom-hRab8 pBI/eGFP-MoRab8/tdTom-hRab5 pBI/eGFP-MoRab1/tdTom-hRab1 pBI/eGFP-MoRab1/tdTom-hRab5

2.2 EXPRESSION AND INTRACELLULAR LOCALIZATION OF MoRab11, MoRab8, AND MoRab1 2.2.1 Transfection of BHK-21 cells 1. BHK-21 cell monolayers were grown in either 6-well culture plates or 24-well culture plates with coverslips in a-MEM containing 5% heat-inactivated FBS and 20 U/mL penicillin/streptomycin, for immunoblot analysis of protein expression and confocal fluorescence microscopy, respectively. The cells were seeded and then incubated for 24 h in a 37 C tissue culture incubator with 5% CO2 for the cell monolayer to reach w70e80% confluence before transfection. 2. Each aforementioned expression construct (2 mg or 0.4 mg for 6-well and 24-well plates, respectively) was cotransfected with pTet-Off (2 mg or 0.4 mg) into each well of cells using the Lipofectamine 2000-mediated transfection procedure. In the experiments for immunoblot analysis or confocal microscopy, the plasmid DNAs were mixed in 250 mL or 50 mL of a-MEM in a 1.5 mL Eppendorf tube, and 10 mL or 2 mL of Lipofectamine 2000 were mixed in 250 mL or 50 mL of aMEM in a separate tube. Incubate at room temperature for 4 min. Then transfer the DNA mix to the Lipofectamine 2000 mix, and incubate at room temperature for 20 min. Then add the 500 mL or 100 mL of DNA-Lipofectamine 2000 complexes dropwise to the cells in 6-well or 24-well plates. 3. Place the cells back in the 37 C incubator for 5 h. 4. Replace the transfection medium with full growth medium, and continue the incubation for another 24 h.

2.2.2 Immunoblot analysis of protein expression 1. Aspirate the medium and wash the cell monolayer once with 1 mL of PBS. 2. Add 200 mL of 1X SDS-loading buffer directly to each well, grind the cells off the plate with a syringe plug. 3. Transfer the cell lysate to a 1.5 mL tube with a syringe, and pass through a 25G3/ 8 needle 10 times to shear the nuclear DNA and reduce stickiness. 4. Boil the cell lysate for 3 min, then run 20 mL on SDS-PAGE (12% gel).

2. Methods

FIGURE 1 Schematic structure of the pBI-Tet bidirectional expression vector and coexpression of eGFP-labeled MoRabs with tdTomato-labeled hRabs in BHK cells. (A) Schematic diagram of the pBI-Tet constructs with eGFP-MoRabs (Green and light gray) cloned at the NotI/SalI sites in the MCSII region and tdTomato-hRabs (Red and dark gray) cloned at the MluI/NheI sites in the MCSI region. Upon cotransfection with pTet-Off, the pBI-Tet construct may coexpress an eGFP-MoRab and a tdTomato-hRab, via the TRE region and promoter. (B) Immunoblots of eGFP-MoRabs (G-MoRabs) and tdTomato-hRabs (T-hRabs). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with the antibody for eGFP, antibody for DsRed that recognizes tdTomato, and antibody for actin that serves as an internal loading control. Molecular mass standards (in kDa) are indicated on the left side of the panels.

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5. Transfer the proteins from the gel to a PVDF membrane by using the Bio-Rad Semi-Dry Transfer apparatus. 6. Probe the membrane with the primary antibodies for GFP (1:1000 dilution), DsRed (1:1000 dilution), and actin (1:500 dilution), respectively, followed by the secondary antibody (IRDye 800CW goat anti-mouse IgG) at 1:15,000 dilution. 7. Visualize and quantify the labeled proteins on the membrane by using an LI-COR Odyssey Infrared Imaging System, with manufacturer’s analytical software (Figure 1).

2.2.3 Localization by confocal fluorescence microscopy 1. Aspirate the medium and wash the cell monolayer on coverslip once with 0.5 mL of PBS. 2. Add 0.5 mL of freshly made 4% PFA to fix the cells in the 37 C CO2 incubator for 15 min. 3. Aspirate the fixation solution, and wash the cell monolayer on coverslip once with 0.5 mL PBS. 4. Pick up the coverslip with a pair of fine forceps, and mount it onto 10 mL of ProLong Gold Antifade Reagent with DAPI on a glass slide. Make sure that the cell monolayer faces the mounting reagent on the slide. 5. Keep the slide in the dark and at room temperature overnight to dry the residual liquid. 6. Observe and analyze the intracellular localization of fluorescent eGFP-MoRabs and tdTomato-hRabs in the cell by a Leica confocal laser scanning microscope with Ar-488 and Kr-568 laser excitation and associated analytical software (Figure 2).

SUMMARY In this chapter, we have described the experimental protocols for cloning and subcellular localization of recycling and exocytic Rabs from the rice blast fungus M. oryzae, including MoRab11, MoRab8, and MoRab1. Each MoRab is coexpressed with a human counterpart (hRab) in mammalian cells as eGFP- and tdTomatotagged fluorescent proteins, respectively. The bidirectional expression vector pBITet is used to express a MoRab and a hRab simultaneously in the same cell to facilitate the single-cell assay and confocal fluorescence microscopy analysis of their colocalization. Our results show that MoRab11, MoRab8, and MoRab1 colocalizes with hRab11, hRab8, and hRab1, respectively, on recycling endosomes and exocytic organelles, but not with hRab5 that is associated with early endosomes to promote endocytic traffic to late endosomes and lysosomes. The results suggest that MoRab11, MoRab8, and MoRab1 are indeed orthologs of mammalian Rab11, Rab8, and Rab1 and may play the same roles in recycling endocytosed

Summary

FIGURE 2 Colocalization of MoRab1, MoRab8, and MoRab11 with hRab1A/B, hRab8, and hRab11 in BHK cells. (A) Shown are confocal fluorescence images of intracellular structures in BHK cells labeled by eGFP-MoRabs (green) and tdTomato-hRabs (red), as indicated. The early endosomal hRab5 here serves as a negative control versus recycling endosomes. Nuclei are identified with DAPI staining (blue). The results are reproducible in three independent experiments. Bar ¼ 10 mm. (B) Quantification of colocalization between the eGFP-MoRabs and the coexpressed tdTomato-hRabs in the same cells. The graph shows Pearson’s correlation coefficient between each eGFP-MoRab and the corresponding tdTomato-hRabs, which is calculated using the confocal fluorescent images from four cells and the colocalization tool in the Velocity software. Shown are the mean and calculated SEM. (See color plate)

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receptors and cargoes and transporting newly synthesized membrane proteins to the plasma membrane in M. oryzae. The experimental protocols described here may be extended for cloning and validation of additional MoRabs to understand the function of intracellular membrane trafficking in the development and pathogenicity of M. oryzae.

ACKNOWLEDGMENTS This work was supported, in whole or in part, by the NIH grant R01GM074692, the NSFC grants 31328002 and 31070124, and a scholarship from the China Scholarship Council.

REFERENCES Diekmann, Y., & Pereira-Leal, J. B. (2013). Evolution of intracellular compartmentalization. Biochemical Journal, 449, 319e331. http://dx.doi.org/10.1042/Bj20120957. Ebbole, D. J. (2007). Magnaporthe as a model for understanding host-pathogen interactions. Annuual Review of Phytopathology, 45, 437e456. http://dx.doi.org/10.1146/ annurev.phyto.45.062806.094346. Elias, M., Brighouse, A., Gabernet-Castello, C., Field, M. C., & Dacks, J. B. (2012). Sculpting the endomembrane system in deep time: high resolution phylogenetics of Rab GTPases. Journal of Cell Science, 125(Pt 10), 2500e2508. http://dx.doi.org/10.1242/jcs.101378. Goldenring, J. R., Roland, J. T., & Lapierre, L. A. (2012). Rab11a, Rab8a and myosin V: regulators of recycling and beyond. In G. Li, & N. Segev (Eds.), Rab GTPases and membrane trafficking (pp. 123e131). Bentham Science Publishers. Hutagalung, A. H., & Novick, P. J. (2011). Role of Rab GTPases in membrane traffic and cell physiology. Physiological Reviews, 91(1), 119e149. http://dx.doi.org/10.1152/ physrev.00059.2009. Klopper, T. H., Kienle, N., Fasshauer, D., & Munro, S. (2012). Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis. BMC Biology, 10, 71. http://dx.doi.org/10.1186/1741-7007-10-71. Knodler, A., Feng, S., Zhang, J., Zhang, X., Das, A., Peranen, J., et al. (2010). Coordination of Rab8 and Rab11 in primary ciliogenesis. Proceedings of the National Academy of Sciences of the United States of America, 107(14), 6346e6351. http://dx.doi.org/ 10.1073/pnas.1002401107. Li, G., & Segev, N. (2012). Ypt/Rab GTPases and intracellular membrane trafficking: an overview. In G. Li, & N. Segev (Eds.), Rab GTPases and membrane trafficking (pp. 3e17). Bentham Science Publishers. Lynch-Day, M. A., Bhandari, D., Menon, S., Huang, J., Cai, H., Bartholomew, C. R., et al. (2010). Trs85 directs a Ypt1 GEF, TRAPPIII, to the phagophore to promote autophagy. Proceedings of the National Academy of Sciences of the United States of America, 107(17), 7811e7816. http://dx.doi.org/10.1073/pnas.1000063107. Pfeffer, S. R. (2013). Rab GTPase regulation of membrane identity. Current Opinion in Cell Biology, 25(4), 414e419. http://dx.doi.org/10.1016/j.ceb.2013.04.002. Stenmark, H. (2009). Rab GTPases as coordinators of vesicle traffic. Nature Reviews Molecular Cell Biology, 10(8), 513e525. http://dx.doi.org/10.1038/nrm2728.

References

Taussig, D. T., Chen, S. H., & Segev, N. (2012). The golgi gatekeepers: Ypt1-Rab1 and Ypt31/32-Rab11. In G. Li, & N. Segev (Eds.), Rab GTPases and membrane trafficking (pp. 18e33). Bentham Science Publishers. Ullrich, O., Reinsch, S., Urbe, S., Zerial, M., & Parton, R. G. (1996). Rab11 regulates recycling through the pericentriolar recycling endosome. Journal of Cell Biology, 135(4), 913e924.

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CHAPTER

Methods to analyze novel liaisons between endosomes and centrosomes

4

Hui-Fang Hung*, Heidi Hehnly*,y, x,1, Stephen Doxsey*, 1 *Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA y Department of Cell and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY, USA x Department of Pharmacology, University of Washington, Seattle, WA, USA 1

Corresponding authors: E-mail: [email protected]; [email protected]

CHAPTER OUTLINE Introduction: The Relationship between the Recycling Endosome and Centrosome ........ 48 1. Live Cell Imaging of REs throughout the Cell Cycle................................................. 50 1.1 Methods for Live Cell Imaging of REs in Interphase Cells.......................... 50 1.2 Methods for Live Cell Imaging of REs in Mitotic Cells............................... 51 2. RE’s Centrosome Localization by Electron Microscopy............................................ 51 3. Centrosome Isolation and Identification of RE Components...................................... 53 3.1 Methods to Isolate Centrosomes at Interphase ......................................... 53 3.2 Methods for Staining Isolated Centrosomes ............................................. 55 3.3 Methods for Reconstituting GST-Rab11 on Isolated Centrosomes .............. 56 Acknowledgments ..................................................................................................... 56 References ............................................................................................................... 56

Abstract For some time, it has been known that recycling endosomes (REs) are organized in a nebulous “pericentrosomal” region in interphase cells. However, the collective use of previously developed methods, including centrosome isolation, live cell imaging, and electron microscopy, suggested that there is much more going on between the centrosome and the RE than previously imagined. By exploiting these approaches, we uncovered novel roles of the centrosome in RE function and, conversely, novel roles for REs in centrosome function. We first found that REs dynamically localized to the centrosome Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.03.024 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 4 Methods to analyze novel liaisons

throughout the cell cycle. More specifically, we found that REs interacted with appendages of the older centriole in interphase cells to control endosome recycling, and this interaction was governed by RE-machinery including the small GTPase Rab11. We next determined that REs carry centrosome proteins to spindle poles as part of the “centrosome maturation” process. Here we discuss the methods used and materials needed to complete these types of studies.

INTRODUCTION: THE RELATIONSHIP BETWEEN THE RECYCLING ENDOSOME AND CENTROSOME While there is a plethora of studies showing a requirement between microtubules and the endocytic pathway, the relationship between the recycling endosome (RE) and the microtubule organizing center, A.K.A. centrosome, is poorly understood (Das, Hehnly, & Doxsey, 2014). During interphase, extracellular material at the plasma membrane is endocytosed, passed through the early endosome to the RE (Figure 1). At the RE, cargo is stored then recycled back to the plasma membrane (Scott, Vacca, & Gruenberg, 2014). It is known that REs localize to a nebulous “pericentrosomal” compartment during interphase, but its localization during other cell cycle stages was largely ignored. By utilizing simple live cell imaging approaches, we found that REs maintained their organization around the centrosome throughout the cell cycle, from the simple centrosome in interphase to the large, complex mitotic spindle poles in mitotic cells (Hehnly & Doxsey, 2014). This was quite Plasma Membrane Localized

Early Endosome Localized

Recycling Endosome Localized

Addition of Tfn-568 (time point 0 min)

5 min incubation of Tfn-568


Tfn-568 Centrosome Nucleus

FIGURE 1 Loading endosomes with Tfn-568. Hela cells grown on glass coverslips are incubated with Tfn-568 on ice for 5 min to inhibit the endocytic pathway. Tfn will associate with its receptor at the plasma membrane. Cells are washed and placed into 37 C media for 5e30 min. At 5 min, Tfn-568 localizes mostly to peripheral early endosomes. By 30 min, Tfn-568 localizes to the pericentrosomal RE compartment. (See color plate)

Introduction

surprising, since another organelle that is similarly arranged around the centrosome, the Golgi apparatus, is dramatically disassembled from the centrosome area during mitosis so that the mitotic spindle can be established (Shima, Haldar, Pepperkok, Watson, & Warren, 1997). To examine the relationship between the centrosome and RE, time-lapse imaging was used to track REs using the green fluorescent protein (GFP)-tagged RE protein, GFP-Rab11, and the Rab11-effector protein, FIP3GFP. We found that REs were actively maintained and recruited to mitotic spindle poles (Hehnly & Doxsey, 2014). These findings suggested that the REs might have important roles in centrosome function throughout the cell cycle. We unexpectedly identified a more intimate connection between the RE and the centrosome in 2012 using electron microscopy. More specifically, we identified a direct interaction between endosomes (loaded with a marker) and appendages of the older/mother centriole of the centriole pair (Hehnly, Chen, Powers, Liu, & Doxsey, 2012). At the RE, two Rab GTPases, Rab8 and Rab11, regulate cargo recycling into and out of the organelle (Das et al., 2014; Grosshans, Ortiz, & Novick, 2006). More recently, studies showed that these GTPases and other RE components interact directly with mother/older centriole appendage proteins. For example, Rab11 and Rab8 can both interact directly with the appendage protein, Cenexin (Chang et al., 2013; Hehnly et al., 2012). Rab11 also interacts with a vesicle-tethering complex, the exocyst, through the specific exocyst subunit, Sec15 (Gromley et al., 2005; Wu, Mehta, Pichaud, Bellen, & Quiocho, 2005). Interestingly, this same subunit was shown to have a direct interaction with the mother centriole appendage protein, Centriolin (Gromley et al., 2005). Another centrosome-targeted protein, Evi5, is proposed to be a Rab11-specific GTPase activation protein, (Dabbeekeh, Faitar, Dufresne, & Cowell, 2007; Laflamme et al., 2012). Centrosome isolation and immunofluorescence staining demonstrated that all these molecules were preferential localized to the mother centriole and were lost when mother centriole appendages were depleted (Hehnly et al., 2012). Strikingly, isolated centrosomes incubated with bacterially expressed Evi5 mislocalized Rab11 from centrosomes presumably by converting the GTPase to its inactive state. This suggested that molecules of the RE might modulate RE interactions with mother centriole appendages. These findings have important implications in understanding the coordination between REs and the centrosome to regulate key cellular processes in time and space, such as ciliogenesis and spindle formation. For instance, Evi5 is degraded during mitosis causing an increase in Rab11 activity during spindle formation (Eldridge et al., 2006; Hehnly & Doxsey, 2014). This correlates nicely with the increase in RE movement towards mitotic spindle poles that we observed (Hehnly & Doxsey, 2014). Also, the same RE-localized proteins that we identified at mother centriole appendages using isolated centrosome preps (e.g., exocyst, Rab8, and Rab11) are involved in primary cilia formation (Hehnly et al., 2012; Kno¨dler et al., 2010; Westlake et al., 2011; Yoshimura, Egerer, Fuchs, Haas, & Barr, 2007). We propose that using a combination of the techniques discussed below, such as live cell imaging, electron microscopy, and/or centrosome isolation, the behavior of RE proteins with the centrosome can be elucidated at distinct cell cycle stages.

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1. LIVE CELL IMAGING OF REs THROUGHOUT THE CELL CYCLE RE has been characterized and defined by loading it with either iodinated- or fluorescently conjugated cargo proteins that transverse through the RE at a given time after endocytosis (Apodaca, 1994; Sheff, Daro, Hull, & Mellman, 1999). Two commonly used cargos are immunoglobulin receptor’s ligand, IgA (Apodaca, 1994), and transferrin receptor’s ligand, transferrin (Tfn; Sheff et al., 1999). These tagged cargo proteins can be incubated with cells on ice for 5e10 min. After this short incubation, nontagged cargoes are then chased into cells at 37 C for 5, 10, 15, 30, 45, and 60 min. When iodinated cargo is used, the cells are obtained and separated from the surrounding media and the amount of cargo recycled out into the surrounding media is measured using a gamma-counter and compared to the amount of cargo remaining in cells (Sheff et al., 1999). When using fluorescently labeled cargo, studies have commonly fixed cells at these time points to define positions within the endocytic pathway (Apodaca, 1994; Hehnly, Longhini, Chen, & Stamnes, 2009; Lin, Gundersen, & Maxfield, 2002; Sheff et al., 1999, Figure 1). For instance, after 5 min incubation at 37 C, fluorescently conjugated Tfn-488 predominantly localizes at peripheral early endosomes, whereas after 30e40 min Tfn488 will be enriched at the RE (Figure 1). Below we will describe how to utilize this approach for live-cell microscopy of RE dynamics.

1.1 METHODS FOR LIVE CELL IMAGING OF REs IN INTERPHASE CELLS 1. Grow GFP-centrin (HeLa or U2OS) cells to confluence (used in Kuo et al., 2011) in a glass bottom dish (MatTek, catalog# P35G-1.5). Any cell type here is fine, but make sure they are human or stably express the human transferrin receptor. 2. Incubate cells in serum-free media (DMEM plus HEPES, Life Technolgies, catalog# 21063) for 45 min to deplete cells of transferrin. 3. In serum-free media dilute 5 mg/mL Tfn-568 stock to 25 mg/mL (Life Technologies, catalog# T-23365) and add to glass bottom dishes on ice for 5 min. 4. Wash cells with ice-cold movie media (high-glucose DMEM without phenol red and with HEPES (Life Technologies, catalog# 21063-029) plus 100 mg/mL unlabeled Tfn (Sigma, catalog# T8158)) three times, then place cells in warmed movie media. 5. Place dishes on a heated stage (37 C) or in a heated environmental chamber attached to a spinning disc confocal microscope or a wide-field epifluorescence microscope. In our hands, we have had success with visualizing endosomes at the centrosome using a spinning disc over an epifluorescent system. The microscope system we use can be referred to in Hehnly et al. (2012) and Hehnly and Doxsey (2014). 6. After endocytosis, we image approximately four optical sections with 1 mm z-axis increments, where the middle sections (Sections 1e2) are focused on

2. RE’S centrosome localization by electron microscopy

GFP-centrin. A small z-stack is obtained every 2 min for approximately 20e30 min or until the arrival of endosomes to the centrosome is observed.

1.2 METHODS FOR LIVE CELL IMAGING OF REs IN MITOTIC CELLS In contrast to interphase cells, endocytosis occurs less frequently in cells entering mitosis (Fielding & Royle, 2013; Tacheva-Grigorova, Santos, Boucrot, & Kirchhausen, 2013) and are not easily observed using a pulse-chase approach as described above. To overcome this issue, endosomes can be preloaded with Tfn-568 during interphase, then visualized to analyze intracellular dynamics during mitosis. GFP-centrin cells (HeLa cells in this example) should be grown to approximately 70% confluence and serum-starved for 45 min before loading with Tfn-568 (as described above). Proceeding starvation, cells should be incubated in serum-containing movie media (described above) with 25 mg/mL Tfn-568 for 4 h at 37 C. After incubation, wash cells with 37 C movie media minus-Tfn-568 and place on heated stage. Imaging Tfn-568-loaded endosomes is best performed using a spinning disk confocal microscope, as this allows acquisition of clear optical sections above, on, and below the spindle pole over time to optimize visualization of endosome dynamics. Epifluorescence-based microscope systems do not yield the same level of resolution because a single optical section cannot be utilized, causing the spindle pole to become masked by dispersed endosomes or Tfn-568 in the surrounding media. An alternative approach is to use a cell line constitutively expressing FIP3-GFP (HeLa cell; Hehnly & Doxsey, 2014; Wilson et al., 2005). These cells are much easier to engineer than a fluorescently tagged Rab11 cell line because Rab11 over-expression adversely affects cell cycle progression (refer to paper Hehnly & Doxsey, 2014 and below). For individual endosome tracking over short time periods at either an interphase centrosome or mitotic spindle poles, the microscopy layout becomes very important. The microscopy system we use for either transferrin-loaded endosomes or FIP3GFP-decorated endosomes is a Solamere Technology Group CSU10B Spinning Disk Confocal System attached to a Nikon TE2000-E2 motorized inverted fluorescence microscope. To track individual endosomes at the mitotic spindle pole we acquire GFP (either GFP-centrin or FIP3-GFP) and 568 (Tfn-568) every 100 ms using a Rolera MGi EMCCD 14-bit camera (Qimaging). To obtain individual tracks at this time frame, we had to take a single optical section within the z-axis of the spindle. At this point, FIP3-GFP particles could be semiautomatically monitored for 50e100 frames using MetaMorph software (Hehnly & Doxsey, 2014).

2. RE’S CENTROSOME LOCALIZATION BY ELECTRON MICROSCOPY While live cell image acquisition gives impressive spatial resolution in a whole cell volume, morphological interactions between organelles can be limiting. Colin

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Hopkins in 1983 published a method to appreciate the sorting of ligandereceptor complexes within the endocytic pathway and doing so developed a highly useful technique for examining endosome morphology (Hopkins, 1983). We modified this approach using horseradish peroxide (HRP)-conjugated transferrin to image the interaction of RE vesicles/tubules with the centrosome and mother centriole appendages (examples shown in Figure 2). 1. Grow cells on Thermanox TMX Coverslips placed in a 6-well culture plate (we primarily use U2OS or HeLa cells for these studies (Hehnly et al., 2012)). Thermanox plastic coverslips (Electron Microscopy Sciences, catalog# 72273) are used for ease of separation of the sample from the resin. 2. As with live cell imaging studies, cells should be incubated with serum-free media (DMEM no HEPES, Life Technologies, catalog# 11965) for 45 min to deplete cells of transferrin. 3. Dilute 1 mg/mL Tfn-HRP stock (Rockland Immunochemicals, catalog# 009e0334) to 25 mg/mL in serum-free media and add to coverslips on ice for 5 min. 4. Wash cells with serum-free media plus 100 mg/mL unlabeled Tfn three times, then place cells in warmed media plus 10% FCS.

(A)

(B)

(C)

FIGURE 2 Tfn-HRP-loaded endosomes around the centrosome. Shown are three examples of Tfn-HRP-loaded endosomes organized at the centrosome. The centrosome in (A) shows the endosomes organized at mother centriole appendages and was published in Hehnly et al. (2012). Panels (B,C) show unpublished organization of endosomes at the centrosome. We frequently observe centrosomes and endosomes organized to a nuclear envelope pocket as shown in (B).

3. Centrosome isolation and identification of RE components

5. Incubate cells grown on Thermanox coverslips at 37 C for 20e30 min. Some cell types transport Tfn at different rates to the pericentrosome located RE, so we recommend setting up a time series with this experiment ranging from 5e30 min. An additional control for Tfn arrival at the RE is to do a parallel time-course series using cells incubated with fluorescently conjugated Tfn. 6. Incubate coverslips in 2.5% glutaraldehyde diluted in phosphate-buffered saline (PBS) for 30 min, wash with PBS, and incubate in diaminobenzidine (Sigma Aldrich) for 25 min (modified from Ullrich, Reinsch, Urbe´, Zerial, & Parton, 1996). After post-fixation in 1% osmium tetroxide and processing for SPIpon/Araldite embedding, 150e250 nm sections were imaged with an FEI Tecnai Spirit 12 transmission electron microscope (TEM). Conventional protocols for TEM were employed (please refer to Hayat, 1986).

3. CENTROSOME ISOLATION AND IDENTIFICATION OF RE COMPONENTS Using TEM and live cell confocal microscopy our studies demonstrated that the centrosome is localized at and around the centrosome, and that Tfn HRP-loaded vesicles can directly interact with appendages of the mother centriole (Hehnly et al., 2012). However, the nature of this interaction was more elusive. To address this issue and resolve the molecular components of the RE that might directly interact with the centrosome, we manipulated a well-characterized technique of isolating centrosomes or spindle poles (modeled in Figure 3) from interphase (Hehnly et al., 2012; Mitchison & Kirschner, 1986) and mitotic cells (Hehnly & Doxsey, 2014). Using this technique, we identified RE components at the centrosome through both immunoblotting and staining of the isolated centrosome fractions (modeled in Figure 3(B)). A powerful utility of these experiments is the ability to isolate centrosomes from either knock-out (Chen et al., 2014) or small interfering ribonucleic acid (RNA)/small hairpin RNA-depleted cell lines (Hehnly et al., 2012; Hehnly & Doxsey, 2014) and probing for components gained or lost at centrosomes. Another strength of centrosome isolation is the ability to incubate purified proteins with these isolated centrosomes and examine the nature of their interaction. For example, we were able to reconstitute certain RE components (e.g., Evi5 and Rab11) on isolated centrosomes and confirm that GTPRab11 preferentially binds to the mother centriole. Here, we describe the methods to isolate centrosomes and to reconstitute Rab11 components on isolated centrosomes.

3.1 METHODS TO ISOLATE CENTROSOMES AT INTERPHASE 1. Grow cells to 70e100% confluence on two 150-mm plates in DMEM (DMEM, Life Technolgies, catalog# 11995) plus serum. To enrich for cells at G1/S phase, cells are cultured in serum-free media for 48 h. We typically use HeLa or U2OS cells for centrosome preparation. For large-scale centrosome preparation, culture cells in 3 150-mm dishes.

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(A) (1)

(2)

(3)

(4)

(5)

(6)

20% Sucrose 40% Sucrose

20% Ficoll

(B)

20% Ficoll

65% Sucrose

(C) Metal hook to pick up coverslip suuport in the tube

Sucrose Conc. Pericentrrin

Coverslip

Cenexin

Indented slot and hole

Acetylated tubulin Centrosome fraction (~55% sucrose)

Coverslip support

FIGURE 3 Characterization of isolated centrosomes. (A) A workflow of centrosome isolation. (1) Culture cells and arrest cells at interphase. (2) Incubate cells with cytochalasin D and nocodazole to depolymerize actin and microtubules. (3) Wash cells with hypotonic solutions to burst cells, collect centrosome-containing solution, and load onto Ficoll cushion. (4) After centrifugation, the crude centrosome fraction is localized at the Ficoll cushion/lysate interface. (5) Transfer crude centrosome fraction to sucrose gradient. (6) After centrifugation, fractionate gradient. (B) An example immunoblot of centrosome fractions from a sucrose gradient. The centrosome proteins pericentrin, cenexin, and acetylated tubulin localize to a 55% sucrose fraction. (C) The apparatus used to spin down isolated centrosomes on coverslips for immunofluorescence staining. The coverslip support has a slot on the side where the hook can be inserted to take out the coverslip and support from the centrifuge tube.

2. Cells are incubated with 1 mg/mL cytochalasin D (Sigma Aldrich, catalog# C8273) and 10 mg/mL nocodazole (Sigma Aldrich, catalog# M1404) for 2 h in serum-free media at 37 C to depolymerize actin and microtubules. 3. Wash cells consecutively with 1 PBS, 8% w/w sucrose in 0.1 PBS, 8% w/w sucrose in H2O, and finally with lysis buffer (LB, 1 mM TriseHCl at pH 7.4). Note each wash should be performed quickly with solution prechilled on ice. In addition, the wash buffer solutions are carefully removed by aspiration so that the cell monolayer is not disrupted. 4. Cells are incubated with LB buffer plus 0.1% 2-ME and 0.5% NP-40 (200 mL/cm2) and agitated for 5 min at 4 C.

3. Centrosome isolation and identification of RE components

5. The supernatant is then carefully collected without disrupting the cell monolayer. After collection, spin it at 3000 g for 3 min at 4 C to pellet cell debris. 6. Transfer clear supernatant onto a 20% w/w Ficoll (MW 400,000, GE Healthcare, catalog# 17-0300-10) cushion in 10 mM PIPES, 1 mM EDTA, and 8 mM 2-ME. We place the cushion in a 1/2 2 in ultra-clear centrifuge tube (Beckman, catalog# 344057), which fits in a Beckman tabletop rotor TLA 100.3. The supernatant is then centrifuged at 9000 g for 20 min at 4 C. After centrifugation the centrosomes will be localized to the top of the Ficoll cushion (refer to Figure 3(A), step 4). The ultra-clear centrifuge tubes allows for puncturing the side of the tube for easy collection of the cushion interface. The ratio of Ficoll cushion to the lysate in PIPES buffer should be about 1:3. 7. Place centrosome-containing Ficoll/PIPES solution onto sucrose step gradient (20%, 40%, and 65%, 0.5 mL for each step) in ultra-clear centrifuge tubes (same as Step 6). Make sure the centrosome-containing Ficoll/PIPES solution is less than 10% Ficoll before placing it on the sucrose gradient. A stock 65% (w/w) should be made in PE buffer (10 mM PIPES, 1 mM EDTA, 0.1% TritonX-100, and 0.1% 2-ME), and subsequent sucrose solutions (20% and 40%) should be diluted from the 65% stock with PE buffer. 8. The gradient should be centrifuged at 100,000 g for 1 h at 4 C. 9. Pull 100e200 mL fractions from sucrose gradient. Centrosomes normally sediment at approximately 50e55% sucrose. It is a good idea to check fractions for sucrose concentration on a refractometer before proceeding. In addition, a small proportion (10%) of the pulled fraction should be directly added to SDS-loading dye and analyzed by immunoblotting (Figure 3(B)). 10. After confirmation of centrosomes isolation, snap-freeze the centrosome fraction in liquid nitrogen for staining and/or reconstitution experiments.

3.2 METHODS FOR STAINING ISOLATED CENTROSOMES 1. Take 10e20 mL of the centrosome fraction from sucrose gradient described above (confirmed by immunoblotting) diluted with 3e5 mL of PE buffer and added to 15 mL Corex centrifuge tube (Corning, catalog# 8441-15) with a 12 mm coverslip (Thermo Scientific, catalog# 64-0712) and coverslip support (refer to Figure 3(C)). Coverslip support is round at one side to fit the bottom of centrifuge tube and flat at the other side to place a coverslip directly on (Figure 3(C)). 2. Spin at 9000 g in a swinging bucket rotor (Beckman, JS-13.1) for 30 min at 4 C. 3. Carefully take out the coverslip (Figure 3(C)) and rinse with PE buffer. 4. At this point the centrosomes are ready to fix and stain by traditional immunofluorescent method.

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3.3 METHODS FOR RECONSTITUTING GST-Rab11 ON ISOLATED CENTROSOMES 1. The GST-Rab11 bacterial expression vector was obtained from Dr David Lambright (UMMS), expressed, and purified using methods discussed in (Eathiraj, Mishra, Prekeris, & Lambright, 2006; Pan, Eathiraj, Munson, & Lambright, 2006). In brief, 2 L of bacterial expressing GST-Rab11 was cultured. Cells were harvested by centrifugation, lysed (primarily by sonication), and loaded onto a glutathione-sepharose column (GE, 17-0756-01). 2. After eluting GST-Rab11 from a glutathione sepharose 4B column, the elution buffer was exchanged for the reaction buffer containing 20% sucrose with 10 mM PIPES plus protease inhibitors (Thermo Scientific, catalog# 88665). 3. GST-Rab11 was loaded with excess GTPgS or GDP (10 mm). GDP is used as a negative control, since dominant-negative Rab11(S25N) does not colocalize to the centrosome in whole cells. GST-Rab11-GTPgS and GST-Rab11-GDP were then exchanged into buffer lacking either GTPgS or GDP (20% sucrose with 10 mM PIPES plus protease inhibitors). 4. GST-Rab11-GTPgS or GST-Rab11-GDP (10 mg/mL) is incubated with isolated centrosomes, at 37 C for 20 min. The final reaction solution should be 20% sucrose with 10 mM PIPES plus protease inhibitors. 5. Dilute reaction to a final concentration of 5% sucrose with 10 mM PIPES plus protease inhibitors. Centrosomes plus GST-Rab11-GTPgS or GST-Rab11-GDP can be spun onto coverslips and staining performed (refer to Figure 3(C)).

ACKNOWLEDGMENTS We thank Christine Powers (UMMS, TEM), David Lambright (UMMS), and Chun-Ting Chen (UMMS, Doxsey Lab) for contributing to the development of the methods discussed. The following grants supported this work K99GM107355 (to H.H.), GM051994 (to S.D.), and S10RR027897 (TEM, UMMS).

REFERENCES Apodaca, G. (1994). Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes. The Journal of Cell Biology, 125(1), 67e86. http://dx.doi.org/10.1083/ jcb.125.1.67. Chang, J., Seo, S. G., Lee, K. H., Nagashima, K., Bang, J. K., Kim, B. Y., et al. (2013). Essential role of Cenexin1, but not Odf2, in ciliogenesis. Cell Cycle, 12(4), 655e662. http:// dx.doi.org/10.4161/cc.23585. Chen, C.-T., Hehnly, H., Yu, Q., Farkas, D., Zheng, G., Redick, S. D., et al. (2014). A unique set of centrosome proteins requires pericentrin for spindle-pole localization and spindle orientation. Current Biology: CB, 24(19), 2327e2334. http://dx.doi.org/ 10.1016/j.cub.2014.08.029.

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and kinesin. Molecular Biology of the Cell, 13(1), 96e109. http://dx.doi.org/10.1091/ mbc.01-05-0224. Mitchison, T. J., & Kirschner, M. W. (1986). Isolation of mammalian centrosomes. Methods in Enzymology, 134, 261e268. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/ 3821566. Pan, X., Eathiraj, S., Munson, M., & Lambright, D. G. (2006). TBC-domain GAPs for Rab GTPases accelerate GTP hydrolysis by a dual-finger mechanism. Nature, 442(7100), 303e306. http://dx.doi.org/10.1038/nature04847. Scott, C. C., Vacca, F., & Gruenberg, J. (July 2014). Endosome maturation, transport and functions. Seminars in Cell & Developmental Biology, 31, 2e10. http://dx.doi.org/ 10.1016/j.semcdb.2014.03.034. Sheff, D. R., Daro, E. A., Hull, M., & Mellman, I. (1999). The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions. The Journal of Cell Biology, 145(1), 123e139. Retrieved from http://www.pubmedcentral. nih.gov/articlerender.fcgi?artid¼2148223&tool¼pmcentrez&rendertype¼abstract. Shima, D. T., Haldar, K., Pepperkok, R., Watson, R., & Warren, G. (1997). Partitioning of the Golgi apparatus during mitosis in living HeLa cells. The Journal of Cell Biology, 137(6), 1211e1228. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi? artid¼2132532&tool¼pmcentrez&rendertype¼abstract. Tacheva-Grigorova, S. K., Santos, A. J. M., Boucrot, E., & Kirchhausen, T. (2013). Clathrinmediated endocytosis persists during unperturbed mitosis. Cell Reports, 4(4), 659e668. http://dx.doi.org/10.1016/j.celrep.2013.07.017. Ullrich, O., Reinsch, S., Urbe´, S., Zerial, M., & Parton, R. G. (1996). Rab11 regulates recycling through the pericentriolar recycling endosome. The Journal of Cell Biology, 135(4), 913e924. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi? artid¼2133374&tool¼pmcentrez&rendertype¼abstract. Westlake, C. J., Baye, L. M., Nachury, M. V., Wright, K. J., Ervin, K. E., Phu, L., et al. (2011). Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. Proceedings of the National Academy of Sciences of the USA, 108(7), 2759e2764. http://dx.doi.org/ 10.1073/pnas.1018823108. Wilson, G. M., Fielding, A. B., Simon, G. C., Yu, X., Andrews, P. D., Hames, R. S., et al. (2005). The FIP3-Rab11 protein complex regulates recycling endosome targeting to the cleavage furrow during late cytokinesis. Molecular Biology of the Cell, 16(2), 849e860. http://dx.doi.org/10.1091/mbc.E04-10-0927. Wu, S., Mehta, S. Q., Pichaud, F., Bellen, H. J., & Quiocho, F. A. (2005). Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo. Nature Structural & Molecular Biology, 12(10), 879e885. http://dx.doi.org/10.1038/nsmb987. Yoshimura, S.-I., Egerer, J., Fuchs, E., Haas, A. K., & Barr, F. A. (2007). Functional dissection of Rab GTPases involved in primary cilium formation. The Journal of Cell Biology, 178(3), 363e369. http://dx.doi.org/10.1083/jcb.200703047.

CHAPTER

Biochemical analysis of Rabin8, the guanine nucleotide exchange factor for Rab8

5

Bin Wu, Juanfei Wang, Yuting Zhao, Wei Guo1 Department of Biology, University of Pennsylvania, Philadelphia, PA, USA 1


CHAPTER OUTLINE Introduction .............................................................................................................. 60 1. Method................................................................................................................ 61 1.1 Autoinhibitory Conformation of Rabin8.................................................... 61 1.1.1 Background ....................................................................................... 61 1.1.2 Materials ............................................................................................ 62 1.1.3 Expression and purification of NanoLuc-HaloTag fusion proteins.......... 63 1.1.4 BRET assay setup .............................................................................. 64 1.1.5 BRET assay to detect the conformational change of Rabin8 ................ 64 1.2 Using BRET Assay to Analyze the Conformational Change Induced by ERK1/2............................................................................................ 64 1.2.1 Background ....................................................................................... 64 1.2.2 Materials ............................................................................................ 65 1.2.3 Purification of ERK2 ........................................................................... 65 1.2.4 Quantification of Rabin8 conformational change induced by ERK2 phosphorylation .................................................................................. 66 Acknowledgments ..................................................................................................... 66 References ............................................................................................................... 67

Abstract The Rab GTPases are master regulators of endosomal trafficking in eukaryotic cells. Among them, Rab8 plays an important role in tubulovesicular trafficking from the transGolgi network and recycling endosomes to the plasma membrane. Rab8 is activated by its guanine nucleotide exchange factor, Rabin8. In order to understand the molecular mechanisms that control endosomal recycling to the plasma membrane, it is pivotal to understand how Rabin8 is regulated in cells. Recently, biochemical and cell biological Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.06.018 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 5 Biochemical analysis of Rabin8

studies have identified several mechanisms for Rabin8 activation, which involves the relief of the intramolecular autoinhibition of Rabin8. Here we describe biochemical methods that we have used recently to study the activation of Rabin8.

INTRODUCTION The Rab family of small GTPases are master regulators of membrane trafficking in eukaryotic cells (Stenmark, 2009). Rab proteins cycle between their GTP- and GDPbound forms. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins are major regulators of the Rab cycles (Barr & Lambright, 2010; Cherfils & Zeghouf, 2013; Hutagalung & Novick, 2011; Pfeffer, 2013). GEFs stimulate the dissociation of GDP from Rabs, thus switching Rab proteins to their active GTP-bound state (Barr & Lambright, 2010; Cherfils & Zeghouf, 2013). Although the GEFs have been identified for most of the Rab proteins, how these GEFs are regulated remains unclear. Rab8 mediates the trafficking from the trans-Golgi network and recycling endosome to the plasma membrane (Hattula, Furuhjelm, Arffman, & Peranen, 2002; Huber et al., 1993; Peranen, 2011). Rab8 localizes to tubulovesicular carriers and is required for the recycling of a number of proteins such as transferrin receptor, major histocompatibility complex I, and integrins to the plasma membrane (Hattula et al., 2006). Rabin8 (the homolog of yeast Sec2p) is the major GEF of Rab8 (Hattula et al., 2002). Several mechanisms have been speculated for the regulation of its GEF activity toward Rab8. Rab11, which mediates the exit of cargos from the recycling endosomes, interacts with Rabin8 in its GTP-bound form and activates the GEF activity of Rabin8 (Knodler et al., 2010). The Rab11eRabin8eRab8 cascade has been implicated in a number of cellular processes such as apical trafficking in epithelia and ciliogenesis (Bryant et al., 2010; Feng et al., 2012; Knodler et al., 2010; Westlake et al., 2011). In addition, it was recently shown that Rabin8 is phosphorylated by ERK1/2 in response to EGF signaling; the phosphorylation of Rabin8 results in its activation (Wang et al., 2015). A previous study has shown that a Rabin8 mutant (“Rabin8D(300e305)”), in which a highly conserved sequence “SLYNEF” after GEF domain is deleted, has higher binding affinity for Rab8 and stronger GEF activity (Feng et al., 2012). It is thus speculated that Rabin8 adopts an autoinhibitory conformation, with its C-terminus masking the GEF domain (Feng et al., 2012). The deletion of amino acid 300e305 could induce the conformational change of Rabin8 to an open state to bind its catalytic substrate Rab8. The conformational change could also be induced by ERK1/2 phosphorylation. To test this hypothesis, we have developed an assay based on bioluminescence resonance energy transfer (BRET) technology (Ayoub & Pfleger, 2010; Xie, Soutto, Xu, Zhang, & Johnson, 2011; Xu, Piston, & Johnson, 1999). In this chapter, we will describe the application of this approach to test the autoinhibitory conformation of Rabin8 and studying Rabin8 activation induced by ERK1/2 phosphorylation.

1. Method

1. METHOD 1.1 AUTOINHIBITORY CONFORMATION OF Rabin8 1.1.1 Background BRET shares a similar resonance energy transfer mechanism as fluorescence resonance energy transfer (FRET). In BRET, luciferase serves as the donor, and a fluorophore that can be excited by the luminescence will be the acceptor. Luminescence will be generated in the presence of luciferase substrates, and the acceptor fluorophore will be excited by the luminescence if the donor and the acceptor are in close proximity. We use the NanoLuc and HaloTag system (Promega, Inc.) in the BRET assay to analyze the conformational changes of Rabin8. NanoLuc is a small luciferase engineered for optimal performance as a luminescent reporter and has an emission peak at 460 nm. HaloTag is a protein fusion tag that can form covalent bond with synthetic chemical ligands. The BRET system uses NanoLuc luciferase as the BRET energy donor and HaloTag protein labeled with NanoBRET 618 as the energy acceptor. The NanoLuc and the HaloTag are fused to the N- and C-terminus of Rabin8, respectively. When Rabin8 is in a closed conformation, BRET will occur between NanoLuc and HaloTag-NanoBRET, upon the application of the NanoLuc substrate (Figure 1). We have used syntaxin-4 (STX4) as a positive control as it likely adopts a closed conformation based on the previous structural studies of other syntaxins (Fernandez et al., 1998; Hu, Latham, Gee, James, & Martin, 2007). When STX4 switches from an autoinhibitory conformation to an open conformation, the BRET signal is significantly decreased (Figure 2(A)).

HaloTag NanoLuc 460 1

Rabin8 Rabin8

GEF 144

245

NanoLuc

HaloTa HaloTag

Rabin8 1

GEF 144

245

460

FIGURE 1 Schematic diagram showing the bioluminescence resonance energy transfer (BRET) strategy to study Rabin8 conformational change. NanoLuc (donor) and HaloTag (acceptor) are fused to the N- and C-terminus of Rabin8, respectively. BRET will occur when Rabin8 is in its autoinhibitory conformation and the Nand C-terminus are in close proximity. During the activation of the Rabin8, Rabin8 switches to an “open” conformation, and the energy transfer between NanoLuc and HaloTag is significantly reduced. GEF, the catalytic domain of Rabin8 that mediates guanine nucleotide exchange of Rab8. (See color plate)

61


NanoLuc-STX4 NanoLuc-STX4-HaloTag

450 400 350 300 250 200 150 100

50 0 580 590 600 610 620 630 640 650 660

Wavelength (nm)

(B)

1.2 1.0

Luminescence Intensity (RLU)


(A)

Normalized BRET Ratio

62

1400 1200

NanoLuc-Rabin8 NanoLuc-Rabin8-HaloTag

1000 800 600 400 200 0 580 590 600 610 620 630 640 650 660

Wavelength (nm)

*

0.8 0.6 0.4 0.2 0 Rabin8 Rabin8 (300-305)

FIGURE 2 Examine the autoinhibitory conformation of Rabin8 using bioluminescence resonance energy transfer (BRET). (A) Intramolecular BRET signals are detected for STX4 and Rabin8. STX4 and Rabin8 in pF-NLHT vector are expressed and purified from Escherichia coli. STX4 with a closed conformation is used as a positive control to test the BRET system. STX4 and Rabin8 with NanoLuc only are used as negative controls. Both STX4 and Rabin8 show intramolecular BRET signals with peak emission at 620 nm. (B) Rabin8(300e305D) loses the BRET signal. The luminescence/fluorescence intensities at 460 and 620 nm are recorded, respectively. Signals from mixtures without NanoBRET ligands are used as background. The calculation of BRET ratio is shown in Section 1.1.5 (5). (See color plate)

1.1.2 Materials DNA constructs: The open reading frame (ORF) of human Rabin8 isoform 1, STX4, and Rabin8D(300e305) were constructed into the pF-NLHT vector (Promega, Inc.). BL-21 competent Escherichia coli cells, Luria broth with 100 mg/mL Kanamycin, Luria broth agar plate with 100 mg/mL Kanamycin, 2xYT medium broth: To w900 mL of distilled H2O, add 16 g bacto trytone, 10 g bacto yeast extract, 5 g NaCl. Adjust pH to 7.0, followed by adjusting to 1 L with distilled water and autoclaving,

1. Method

PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 100 mM phenylmethylsulfonyl fluoride (PMSF), Elution buffer: 50 mM Imidazole in 1 PBS, pH 7.4, Talon Metal Affinity Resin, Amicon centrifugal filters (Ultracel, 10 K), TEV-6His protease, NanoLuc substrate: Furimazine (Promega), NanoBRET Ligand (Promega), A 96-well plate readerdA Gemini EM fluorescence microplate reader (Molecular Devices, Inc.) was used for this study.

1.1.3 Expression and purification of NanoLuc-HaloTag fusion proteins We use the pF-NLHT vector (Promega, Inc.) to express the recombinant proteins, with NanoLuc fused to their N-termini, and HaloTag fused to their C-termini. The pF-NLHT has a HaloTag followed by a 6His tag, which will be used to purify the recombinant protein. There is also a TEV protease cleavage site between the ORF and HaloTag. This can be used to obtain proteins with only NanoLuc, which can be used as a negative control to test the BRET system. Briefly, pF-NLHT is digested by SgfI and EcoICR I to remove the Barnase coding region and the ORF of Rabin8, Rabin8D(300e305) or STX4 cDNA are amplified by PCR and inserted into SgfI and EcoICR I sites. The plasmids are transformed into BL-21 E. coli strain and the bacteria are grown on Luria broth agar culture plates containing 100 mg/mL Kanamycin. A single colony is picked up and cultured in 3 mL Luria broth with 100 mg/mL Kanamycin at 37 C overnight. The cultures are then transferred to 500 mL 2xYT medium and incubated at 37 C until the optical density at 600 nm (O.D.600) is 0.6. IPTG is added at a final concentration of 0.5 mM and incubation is continued at 18 C for additional 14e16 h. The bacteria are collected by centrifugation at 3000 g for 10 min at 4 C. The pellets are washed once with 40 mL PBS and then resuspended in 30 mL PBS with 1 mM PMSF. The cells are lysed using a sonicator. Then the cell lysates are incubated with 1% Triton-X100 on a rotator at 4 C for 30 min, followed by centrifugation at 15,000 g for 30 min. The supernatants are collected and incubated with Talon metal affinity resin at 4 C for 3 h on a rotator. The resins are washed four times by prechilled PBS with 10 mM imidazole. The proteins are eluted by 50 mM imidazole and dialyzed with prechilled PBS at 4 C overnight. The proteins are then concentrated using 10-kDa centrifugal filters and the concentration are determined by SDS-PAGE. To obtain the proteins with NanoLuc only, purified proteins are incubated with TEV protease in PBS at 4 C overnight. The mixtures are then incubated with Talon metal affinity resin at 4 C for 3 h. The TEV protease we used has a 6His tag fused to the C-terminus and it will bind to the resin together with the Halo-6His fragments cleaved from the recombinant proteins. The resins are centrifuged at 800 g and the supernatants are collected.

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64


1.1.4 BRET assay setup 1. The NanoLuc substrate Furimazine and HaloTag ligand NanoBRET are diluted to 20 mM and 200 nM in PBS, respectively. 2. 2 pmol purified proteins (NanoLuc-STX4-Halo, NanoLuc-STX4, Nano-Rabin8Halo, NanoLuc-Rabin8) are added to 100 mL diluted ligand or PBS. The mixtures are incubated at room temperature for 5 min in a 96-well plate. The proteins with NanoLuc only are taken as a negative control. 3. 100 mL diluted substrate is added to the mixture and mixed thoroughly. 4. The samples are immediately monitored for luminescence and fluorescence reading in a microplate reader. The luminescence or fluorescence intensities (Relative Luminescence Units, RLU) at 460 nm and from 590e650 nm are recorded. 5. The BRET signals are calculated as RLUxxxnm ¼ RLUxxxnm(substrate þ ligand) RLUxxxnm(substrate) (Examples are shown in Figure 2(A)).

1.1.5 BRET assay to detect the conformational change of Rabin8 1. The NanoLuc substrate Furimazine and HaloTag ligand NanoBRET are diluted as described above. 2. 2 pmol purified proteins (NanoLuc-Rabin8-Halo, NaloLuc-Rabin8D(300e305)Halo) are added to 100 mL diluted ligand or PBS. The mixtures are incubated at room temperature for 5 min in a 96-well plate. 3. 100 mL diluted substrate is added to the mixture and mixed thoroughly. 4. The samples are immediately monitored for luminescence and fluorescence reading in a microplate reader. The luminescence or fluorescence intensities at 460 and 620 nm are recorded. 5. The BRET ratio are calculated as BRET Ratio ¼

RLU620 nm ðsubstrate þ ligandÞ RLU620 nm ðsubstrateÞ RLU460 nm ðsubstrate þ ligandÞ RLU460 nm ðsubstrateÞ

To normalize the BRET Ratio, the ratio of Rabin8 wild type is taken as 1. (An example is shown in Figure 2(B).)

1.2 USING BRET ASSAY TO ANALYZE THE CONFORMATIONAL CHANGE INDUCED BY ERK1/2 1.2.1 Background The Ras-MEK-ERK signal pathway is a principle signaling system in cells for many physiological functions (Chang & Karin, 2001). A number of studies have implicated MEK-ERK in the regulation of vesicle trafficking (Farhan & Rabouille, 2011). For exocytosis, it was recently shown that ERK1/2 phosphorylates the exocyst subunit Exo70 and promotes the exocyst complex assembly (Ren & Guo, 2012). We found that Rabin8 is a directly substrate of ERK1/2 (Wang et al., 2015). Rabin8 phosphorylation by ERK1/2 can activate its GEF activity toward Rab8, increase its binding affinity to Rab8 and inhibit its interaction with Rab11.

1. Method

These effects are similar to the gain of function mutant Rabin8D(300e305), which loses the autoinhibitory conformation. The assay described here was used to study the effect of ERK1/2 phosphorylation on the conformation of Rabin8. We used an in vitro kinase assay to phosphorylate Rabin8 and then use BRET assay to detect the conformational change. The constitutively active ERK2 (ERK2-CA) and kinase-dead ERK2 (ERK2-KD) are purified from E. coli using an ERK2-MEK1 coexpression system, whereby MEK1 phosphorylates and activates ERK2 (Khokhlatchev et al., 1997). ERK2-KD is used as a negative control. The purification of NanoLuc-Rabin8-HaloTag has been described above. The activation of Rabin8 is further examined by GDP exchange assay as described (Feng, Wu, Peranen, & Guo, 2015).

1.2.2 Materials DNA constructs: pET-Hisx6-MEK1-R4F-ERK2, NpT7-5-Hisx6-ERK2 (KD), pF-NLHT-Rabin8, BL-21 competent E. coli cells, Luria broth with 100 mg/mL Kanamycin, Luria broth agar plate with 100 mg/mL Kanamycin, 2xYT medium broth: To w900 mL of distilled H2O, add 16 g bactotrytone, 10 g bacto-yeast extract, 5 g NaCl. Adjust pH to 7.0, followed with adjusting to 1 L with distilled water and autoclaving, PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, Elution buffer: 50 mM imidazole in 1 PBS, pH 7.4, Talon Metal Affinity Resin, Amicon centrifugal filters (Ultracel, 10 K), Phosphatase inhibitor, Kinase buffer: 20 mM Hepes (pH 7.5), 100 mM KCl, 5 mM MgCl2, 1 mM ATP, 1 mM DTT, 1 mM NaF, 1 mM PMSF, and phosphatase inhibitor, Furimazine (Promega), NanoBRET Ligand (Promega), A 96-well plate reader capable of detecting emission at 460 and 620 nm. A Gemini EM fluorescence microplate reader was used for this study.

1.2.3 Purification of ERK2 pET-Hisx6-MEK1-R4F-ERK2, NpT7-5-Hisx6-ERK2 (KD) plasmids are transformed into BL-21 cells and cultured on Luria broth agar plates. A single colony is picked and cultured in 3 mL Luria broth overnight at 37 C. The bacteria are then transferred into 500 mL of 2xYT medium and incubated at 37 C until O.D.600 reaches 0.6. The recombinant proteins are induced by 0.5 mM Isopropyl b-D-1-thiogalactopyranoside (IPTG) at 18 C for 14e16 h. The cells are collected by centrifugation at 3000 g for 10 min at 4 C. The pellets are washed once with 40 mL PBS and then resuspended in 30 mL PBS with 1 mM PMSF and phosphatase inhibitor. The proteins are purified as described above.

65


(A)

(B) A D 2-K 2-C RK ERK E 8+ 8+ bin bin Ra Ra

Rabin8 phospho-Rabin8


66

1.2 0.8 0.6 0.4 0.2

in8

b Ra

**

1.0

0

D

+

in8

b Ra

A

2-C

2-K

K ER

+

K ER

FIGURE 3 Examine the conformational change induced by ERK1/2 phosphorylation using bioluminescence resonance energy transfer (BRET). (A) Rabin8 is phosphorylated by ERK2 in vitro. Purified NanoLuc-Rabin8-HaloTag wildtype proteins from pF-NLHT vector are incubated with constitutively active or kinase dead ERK2 in kinase buffer for 30 min at 30 C. The total and the phosphorylated proteins are detected by Rabin8 antibody or phospho-ERK substrate antibody. (B) The Rabin8 proteins phosphorylated in (A) are used in BRET assay. BRET ratios were normalized and compared as described in Section 1.1.5 (5).

1.2.4 Quantification of Rabin8 conformational change induced by ERK2 phosphorylation 1. 5 mg purified Rabin8 proteins and 1 mg purified ERK2-CA or ERK2-KD are mixed in the kinase buffer with a total volume of 50 mL. 2. The mixture is incubated at 30 C for 30 min and placed on ice immediately. 3. The NanoLuc substrate Furimazine and HaloTag ligand NanoBRET are diluted to 20 mM and 200 nM with PBS, respectively. 4. 1 mL of in vitro phosphorylation product is added to 100 mL of diluted NanoBRET or PBS in a 96-well plate. The mixtures are incubated at room temperature for 5 min. 5. 100 mL diluted NanoLuc substrate is added to the mixture and mixed thoroughly. 6. The samples are immediately monitored for luminescence or fluorescence reading in a microplate reader. The luminescence or fluorescence intensities at 460 and 620 nm are recorded. 7. The calculation of BRET ratio is described in Section 1.1.5 (5). An example is shown in Figure 3.

ACKNOWLEDGMENTS We thank Promega, Inc. for technical helps with the BRET technology. This work is supported by National Institutes of Health R01 grant GM111128 to W.G. The authors declare no conflict of interests.

References

REFERENCES Ayoub, M. A., & Pfleger, K. D. (2010). Recent advances in bioluminescence resonance energy transfer technologies to study GPCR heteromerization. Current Opinion in Pharmacology, 10, 44e52. Barr, F., & Lambright, D. G. (2010). Rab GEFs and GAPs. Current Opinion in Cell Biology, 22, 461e470. Bryant, D. M., Datta, A., Rodriguez-Fraticelli, A. E., Peranen, J., Martin-Belmonte, F., & Mostov, K. E. (2010). A molecular network for de novo generation of the apical surface and lumen. Nature Cell Biology, 12, 1035e1045. Chang, L., & Karin, M. (2001). Mammalian MAP kinase signalling cascades. Nature, 410, 37e40. Cherfils, J., & Zeghouf, M. (2013). Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiological Reviews, 93, 269e309. Farhan, H., & Rabouille, C. (2011). Signalling to and from the secretory pathway. Journal of Cell Science, 124, 171e180. Feng, S., Knodler, A., Ren, J., Zhang, J., Zhang, X., Hong, Y., et al. (2012). A Rab8 guanine nucleotide exchange factor-effector interaction network regulates primary ciliogenesis. Journal of Biological Chemistry, 287, 15602e15609. Feng, S., Wu, B., Peranen, J., & Guo, W. (2015). Kinetic activation of rab8 guanine nucleotide exchange factor rabin8 by rab11. Methods in Molecular Biology, 1298, 99e106. Fernandez, I., Ubach, J., Dulubova, I., Zhang, X., Sudhof, T. C., & Rizo, J. (1998). Threedimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A. Cell, 94, 841e849. Hattula, K., Furuhjelm, J., Arffman, A., & Peranen, J. (2002). A Rab8-specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport. Molecular Biology of the Cell, 13, 3268e3280. Hattula, K., Furuhjelm, J., Tikkanen, J., Tanhuanpaa, K., Laakkonen, P., & Peranen, J. (2006). Characterization of the Rab8-specific membrane traffic route linked to protrusion formation. Journal of Cell Science, 119, 4866e4877. Hu, S. H., Latham, C. F., Gee, C. L., James, D. E., & Martin, J. L. (2007). Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins. Proceedings of the National Academy of Sciences of the United States of America, 104, 8773e8778. Huber, L. A., Pimplikar, S., Parton, R. G., Virta, H., Zerial, M., & Simons, K. (1993). Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane. Journal of Cell Biology, 123, 35e45. Hutagalung, A. H., & Novick, P. J. (2011). Role of Rab GTPases in membrane traffic and cell physiology. Physiological Reviews, 91, 119e149. Khokhlatchev, A., Xu, S., English, J., Wu, P., Schaefer, E., & Cobb, M. H. (1997). Reconstitution of mitogen-activated protein kinase phosphorylation cascades in bacteria. Efficient synthesis of active protein kinases. Journal of Biological Chemistry, 272, 11057e11062. Knodler, A., Feng, S., Zhang, J., Zhang, X., Das, A., Peranen, J., et al. (2010). Coordination of Rab8 and Rab11 in primary ciliogenesis. Proceedings of the National Academy of Sciences of the United States of America, 107, 6346e6351. Peranen, J. (2011). Rab8 GTPase as a regulator of cell shape. Cytoskeleton (Hoboken), 68, 527e539.

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Pfeffer, S. R. (2013). Rab GTPase regulation of membrane identity. Current Opinion in Cell Biology, 25, 414e419. Ren, J., & Guo, W. (2012). ERK1/2 regulate exocytosis through direct phosphorylation of the exocyst component Exo70. Developmental Cell, 22, 967e978. Stenmark, H. (2009). Rab GTPases as coordinators of vesicle traffic. Nature Reviews Molecular Cell Biology, 10, 513e525. Wang, J., Ren, J., Wu, B., Feng, S., Cai, G., Tuluc, F., et al. (2015). Activation of Rab8 guanine nucleotide exchange factor Rabin8 by ERK1/2 in response to EGF signaling. Proceedings of the National Academy of Sciences of the United States of America, 112, 148e153. Westlake, C. J., Baye, L. M., Nachury, M. V., Wright, K. J., Ervin, K. E., Phu, L., et al. (2011). Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome. Proceedings of the National Academy of Sciences of the United States of America, 108, 2759e2764. Xie, Q., Soutto, M., Xu, X., Zhang, Y., & Johnson, C. H. (2011). Bioluminescence resonance energy transfer (BRET) imaging in plant seedlings and mammalian cells. Methods in Molecular Biology, 680, 3e28. Xu, Y., Piston, D. W., & Johnson, C. H. (1999). A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proceedings of the National Academy of Sciences of the United States of America, 96, 151e156.

CHAPTER

Simple in vitro assay of Arf GAPs and preparation of Arf proteins as substrates

6

Pei-Wen Chen, Xiaoying Jian, Ruibai Luo, Paul A. Randazzo1 Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 1


CHAPTER OUTLINE Introduction .............................................................................................................. 70 1. Methods .............................................................................................................. 71 1.1 Preparation of MyrArfs ........................................................................... 71 1.1.1 Background ....................................................................................... 71 1.1.2 Materials ............................................................................................ 72 1.1.3 Buffers ............................................................................................... 72 1.1.4 Expression of MyrArfs ......................................................................... 72 1.1.5 Purification of myristoylated Arfs ......................................................... 73 1.2 GAP Assay ............................................................................................ 74 1.2.1 Background ....................................................................................... 74 1.2.2 Materials ............................................................................................ 74 1.2.3 Preparing large unilamellar vesicles .................................................... 75 1.2.4 Exchanging GXP for [a32P]GTP........................................................... 75 1.2.5 GAP assay.......................................................................................... 75 1.3 Results and Analysis of GAP Assays ........................................................ 76 1.3.1 Calculation of fractional conversion of Arf$GTP to Arf$GDP and estimation of relative enzymatic activity ........................................ 76 Summary .................................................................................................................. 78 Acknowledgment....................................................................................................... 78 References ............................................................................................................... 78

Abstract Defining the interaction of Arf GAPs with specific Arfs is important for understanding their functions in the endocytic system. Cell-based approaches have been valuable for identifying Arfs and Arf GAPs active in the endocytic compartment; however, the cell-based assays have some limitations in establishing relationships among the Arfs and Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.03.021 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 6 In vitro assay of Arf GAPs

ArfGAPs. Here we describe a simple in vitro assay that will provide a means for comparing Arfs as substrates and serve to complement cell-based studies.

INTRODUCTION The endocytic compartment is a dynamic system of endomembranes through which numerous proteins transit with specific itineraries (D’Souza-Schorey & Chavrier, 2006; Grant & Donaldson, 2009). The function and organization of the compartment is important for many cellular processes. The ADP-ribosylation factors (Arfs) are members of a family of guanine nucleotide-binding proteins that regulate membrane traffic including that in the endocytic compartment (Kahn et al., 2006). Humans have 5 genes encoding Arf proteins. They are divided into 3 classes based on primary structure: Class 1 (Arf1 and 3); class 2 (Arf4 and 5) and; class 3 (Arf6). All three classes of Arfs function in the endocytic compartment (Donaldson & Jackson, 2011; Gillingham & Munro, 2007; Grant & Donaldson, 2009; Hickson et al., 2003; Maldonado-Baez, Williamson, & Donaldson, 2013). The effects of Arfs are mediated by proteins that bind to Arfs (Donaldson & Jackson, 2011; D’Souza-Schorey & Chavrier, 2006; East & Kahn, 2011; Gillingham & Munro, 2007; Kahn, 2009). Important classes of Arf effector proteins include vesicle coat proteins, such as clathrin adaptors AP-1 and AP-3, and lipid metabolizing proteins, such as phosphatidylinositol 4-phosphate 5-kinase. The proteins that regulate the Arfs, Arf-GTPase-activating proteins (GAPs), also have a critical role in determining the function of the Arfs. Thirty-one genes encode Arf GAPs in Human, outnumbering Arfs by more than 6-fold (Kahn et al., 2008, 2006). Many of the Arf GAPs have multiple splice variants. In contrast to the Arfs, in which a particular isoform may have more than one site of action, the Arf GAPs may be site specific. Recent investigations have revealed that effects of particular isoforms of Arf on itineraries of proteins that transit the endocytic pathway may be determined, in part, by the site-specific Arf GAP (Chen, Luo, Jian, & Randazzo, 2014). An understanding of the complex and specific itineraries of proteins that transit the endocytic pathway requires characterization of the Arf-ArfGAP pairs functioning in the compartment. Determining the GAP that is catalytic for a specific Arf is difficult using cell-based assays. There are multiple interactions that could confound the interpretations of experiments using whole cells. For example, ASAP1 binds to FIP3, an Arf6-binding protein. Consequences of the ASAP1FIP3 interaction are that Arf6 may coimmunoprecipitate and colocalize with ASAP1 due to binding to FIP3 and, potentially, Arf6$GTP levels are affected because Arf6$GTP is stabilized by the complex with FIP3. Furthermore, the elevated Arf6$GTP potentially can affect the activity of Arf exchange factors such as ARNO, thereby affecting Arf1$GTP levels (Cohen et al., 2007; DiNitto et al., 2007). Thus, the effect of the GAP on Arf$GTP levels in cells could be in part independent of GAP catalytic function.

1. Methods

Simple in vitro GAP assays are a valuable complement to cell-based assays in work defining the role of Arfs and Arf GAPs in endocytic traffic. One important factor in GAP assays is the quality of the Arfs used as substrates. Fortunately for Arfologists, full length Arfs from the three major classes can be prepared in native form as we describe here. Also important is that experiments are designed to provide information about relative enzymatic power, which we also describe in this chapter.

1. METHODS 1.1 PREPARATION OF MyrArfs 1.1.1 Background Arf proteins are Ras-superfamily members. Like other Ras-superfamily members, Arfs are comprised of a nucleotide-binding fold with two motifs, called switch 1 and switch 2, whose conformation is sensitive to nucleotide in the binding pocket. With GTP bound, switch 1 and switch 2 adopt a conformation that interacts with effector proteins and GAPs. Unique to the Arfs is an N-terminal extension of approximately 16 amino acids from the nucleotide-binding fold. The glycine at position 2 in the N-terminus is covalently modified with myristic acid. The N-terminal extension and the C-terminus of Arfs are the most variable between isoforms, and the myristoylated N-terminus may be considered as a third switch motif. In Arf$GDP, the N-terminus associates with Arf between switch 1 and switch 2. In Arf$GTP, the N-terminus is displaced from Arf and associates with membranes. In another Arf family member, Arl2, the amino terminus is part of the interface with the effectors. We have not found the myristate to be critical for interaction with GAPs (in sharp contrast to ArfGEFs) (Chen, Jian, Luo, & Randazzo, 2012; Jian, Gruschus, Sztul, & Randazzo, 2012; Randazzo, 1997). The myristoylated protein is easier to load with nucleotide but myristoylated protein is more difficult to prepare. However, other alterations of the N-terminus are often not tolerated for the GAP assay (Jian, Cavenagh, Gruschus, Randazzo, & Kahn, 2010; Yoon et al., 2004). Deletion of the N-terminus from Arf can reduce activity with some Arf GAPs by 10,000-fold, and substrate specificity that is obvious when examining full-length proteins is lost. Fusion of GST to the N-terminus results in an Arf that binds poorly to nucleotide and poorly interacts with GAPs (Jian et al., 2010). In short, either deletion or fusion of protein to the N-terminus of Arf may yield a protein that is not useful for GAP assays. The other region of Arf that must be considered when comparing Arf isoforms is the C-terminus, which also varies between Arfs and associates with the membrane surface when Arf is bound to GTP (Liu, Kahn, & Prestegard, 2010). We have found that fusion of short tags or GFP directly to the C-terminus, in general, interferes with GAP activity (Jian et al., 2010). The one exception is a 6His tag, although we have not extensively examined this modification. In cell-based studies, the linker between Arf and GFP has been found to have a critical effect (Dejgaard, Luo, Randazzo, & Presley, submitted for publication). However, we have not

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extensively examined the various Arf-GFP recombinants for in vitro assays, so we cannot comment on them. Taking these considerations together, we recommend using full length Arf, either myristoylated or nonmyristoylated, for examination of Arf GAPs. There is no evidence that a His tag will influence interpretation of experiments examining the GAPs, but preparation of the untagged Arfs is straightforward (Randazzo, Weiss, & Kahn, 1995), so the use of the His tag is not necessary. Here we briefly describe the preparation of recombinant Arf1, Arf5, and Arf6 modified with myristic acid. We have not tried Arf3. We have had limited success with Arf4 in that we have not been able to prepare chemical amounts but have been able to prepare enough for GAP assays. We do not describe preparation of Arf3 or Arf4 in this chapter.

1.1.2 Materials BL21 (DE3) Escherichia coli cotransformed with plasmids for expression of Arf1, Arf5, or Arf6 with yeast N-myristoyltransferase (Randazzo, Terui, et al., 1995; Randazzo, Weiss, et al., 1995). Luria broth with 100 mg/mL ampicillin and 25 mg/mL kanamycin. Luria broth/agarose plates with 100 mg/mL ampicillin and 25 mg/mL kanamycin. Isopropyl b-D-1-thiogalactopyranoside. Complete protease inhibitor cocktail tablets (Roche, Cat#11697498001). HiLoad 16/10 phenyl Sepharose HP column (GE Healthcare Life Sciences). Hiload 26/60 Superdex 75 column (GE Healthcare Life Sciences). SDS-PAGE apparatus. Coomassie blue dye. Cell disruptor (Microfluidics M-110P). Refrigerated ultracentrifuge with Ti45 fixed-angle rotor (Beckman). 5-mL HiTrap Q HP column (GE Healthcare Life Sciences). AKTA FPLC (GE Healthcare Life Sciences). Amicon centrifugal filters (Ultracel 10K).

1.1.3 Buffers T20N100M1D1: 20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 1 mM DTT. T20N3000M1D1: 20 mM Tris, pH 8.0, 3000 mM NaCl, 1 mM MgCl2, 1 mM DTT. T20N100M1D1G10: 20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 1 mM DTT, 10% Glycerol. T20N25M1D1G10: 20 mM Tris, pH 8.0, 25 mM NaCl, 1 mM MgCl2, 1 mM DTT, 10% Glycerol.

1.1.4 Expression of MyrArfs We use the same protocol to express representative Arfs from each class, Arf1, Arf5, and Arf6, modified with myristate, described elsewhere in detail (Chen et al., 2012). Briefly, BL21 (DE3) bacteria are grown on Luria-broth agar culture plates containing 100 mg/mL ampicillin and 25 mg/mL kanamycin. A single colony is used for a

1. Methods

100 mL culture in Luria Broth with 100 mg/mL ampicillin and 25 mg/mL kanamycin. The culture is incubated at 37 C until the optical density (OD) at 600 nm is 0.6. The bacteria are collected by centrifugation. They can be used immediately or stored overnight at 4 C. The collected bacteria are used to start a 4 L culture in Luria Broth with 100 mg/mL ampicillin and 25 mg/mL kanamycin at 37 C. When the OD is 0.6, myristic acid is added to achieve a final concentration of 10 mM. For myrArf6, IPTG is added to achieve a final concentration of 1 mM. Incubation is continued for an additional 3 h at 37 C. For myrArf1 and myrArf5, IPTG is added to a final concentration of 0.1 mM and incubation is continued at room temperature overnight. Bacteria are collected by centrifugation and can be used immediately or stored at 80 C.

1.1.5 Purification of myristoylated Arfs The specific purification protocol depends on the isoform, as we briefly describe here. More complete protocols have been previously described (Chen et al., 2012). Human myrArf1 (myristoylated Arf1) and myrArf5 protein are purified by a method modified from Randazzo (1997). The pellet from 2 to 4 L of bacteria cell culture that coexpresses Arf and N-myristoyltransferase is lysed using a cell disruptor (M-110P cell disruptor, Microfluidics) in T20N100M1D1 buffer and complete protease inhibitor cocktail tablets (Roche). Cell lysates are clarified by centrifugation at 100,000 g for 60 min at 4 C. The lysate is then passed through two consecutive 5 mL HiTrapQ HP columns. The material that did not adhere to the column (flow through fraction) is confirmed to contain protein using the BioRad protein assay. The flow through fraction is then adjusted to 3000 mM NaCl by adding solid NaCl and applied to a phenyl-Sepharose HP column with a bed volume of 20e22 mL. The column is developed in a 100 mL gradient from 3000 to 20 mM NaCl. MyrArf1 typically elutes in the center of the gradient. Fractions containing myrArf1 are further purified by size exclusion using a Hiload 26/60 Superdex G75 column in T20N100M1D1G10. The fractions containing MyrArf are combined and concentrated by centrifugal filtration with an Amicon Ultracel 10K to around 1.0 mL (final protein concentration should be 1e2 mg/mL). The purified protein is aliquotted and then snap-frozen in a dry ice/ethanol bath. The protein is stored at 80 C. MyrArf6 is purified as described previously (Chen et al., 2012; Jian et al., 2012). MyrArf6 is extracted from BL21(DE3) bacteria cotransformed with expression plasmids for Arf6 and yeast N-myristoyltransferase. First, bacteria are lysed in T20N100M1D1 plus protease inhibitors, and then the pellet is collected and washed in T20N100M1D1. MyrArf6 is extracted from the pellet into T20N25M1D1G10 containing 1% Triton X-100. The protein is precipitated from the detergent extract using ammonium sulfate, dissolved in T20N25M1D1G10 plus 1% Triton X-100, and dialyzed against T20N25M1D1G10 plus 0.1% Triton X-100. After dialysis, the sample is passed through a 5 mL HiTrapQ column preequilibrated with the same buffer as used for dialysis. Like myrArf1 and myrArf5, myrArf6 does not bind to the Q column and is typically recovered in the flow-through fraction. The

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myrArf6 prepared in the manner is typically in the GTP-bound form. The GTP can be exchanged for [a32P]GTP, which is used as a tracer to follow the conversion of GTP to GDP in the GAP assay.

1.2 GAP ASSAY 1.2.1 Background The assay described here follows the hydrolysis of GTP bound to Arf using a radioactive tracer, P32, in the a position of GTP. We and others have also used an assay in which the conversion of Arf$GTP to Arf$GDP is followed by a change in tryptophan fluorescence in Arf. However, this indirect assay can be confounded by a number of factors, sometimes leading to dramatically different estimates of enzymatic parameters than those obtained by directly following GTP hydrolysis (Luo, Ha, Hayashi, & Randazzo, 2009; Luo & Randazzo, 2008). The assay described here involves first exchanging unlabeled nucleotide bound to Arf with [a32P]GTP. Arf$[a32P]GTP is added to the reaction containing variable concentrations of Arf GAP. A plot ln(Arf$GTP0/Arf$GTP)/t versus [ArfGAP], where Arf$GTP0 is Arf$GTP present in the absence of GAP and Arf$GTP is the amount after incubation with Arf GAP, will have a linear portion at low GAP concentrations with a slope equal to the enzymatic power. Alternatively, the amount of GAP required to reach 50% hydrolysis of the GTP on Arf is inversely proportional to enzymatic power and can be used for comparisons. The assay and analysis is described in more detail below. The reaction occurs on a hydrophobic surface. The surface can be provided by large unilamellar vesicles (LUVs). We describe the preparation of LUVs below.

1.2.2 Materials Lipid hydration buffer: 25 mM HEPES, pH 7.4, 100 mM NaCl, 1 mM DTT (can be stored for 1e2 weeks at 4 C) 5 Exchange buffer: 125 mM HEPES, pH 7.4, 5 mM EDTA, pH 8.0, 2.5 mM MgCl2, 500 mM NaCl, 5 mM DTT, 5 mM ATP (store at 20 C) 5 GAP reaction buffer: 125 mM HEPES, pH 7.4, 500 mM NaCl, 10 mM MgCl2, 5 mM DTT, 5 mM GTP (store at 20 C) Wash buffer: 25 mM Tris$HCl, pH 8.0, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT (can be stored for 1e2 weeks at 4 C) Lipids and Lipid extruder (Avanti Polar Lipids): Phosphatidylcholine (PC, chicken egg), Phosphatidylethanolamine (PE, bovine liver), Phosphatidylserine (PS, porcine brain), Phosphatidylinositol (PI, bovine liver), Phosphatidylinositol 4,5-bisphosphate (PIP2, porcine brain), Phosphatidylinositiol 3,4,5-trisphosphate (PIP3), Cholesterol. Nitrocellulose filters: Protran BA85, 0.45 mm pore size, 25 mm from Whatman PEI-cellulose plastic backed thin layer chromatography plates.

1. Methods

Phosphorimager: StormImager, GE Healthcare Lifesciences.

1.2.3 Preparing large unilamellar vesicles Mix lipids in chloroform/methanol in a 12 75 mm siliconized glass tube at the desired molar ratio in a quantity sufficient for making a 5- to 10-fold concentrate of vesicles to be added to an assay. Typically, the mixture of 40% PC, 25% PE, 15% PS, 10% PI, and 10% cholesterol is used. The amount of PI is reduced and replaced with PIP2 and PIP3 dependent on the Arf GAP. For example, for ASAP1 or AGAP1, 9% PI and 1% PIP2 was used, whereas for ARAP2, 8% PI, 1% PIP2, and 1% PIP3 was used. Chloroform is evaporated under a gentle stream of nitrogen for 30e60 min at room temperature in a fume hood. Residual chloroform is removed with a lyophilizer with a pressure of Set Scale. The conversion factor is specific for every microscope and objective setup and can be obtained from the manufacturer or measured with the aid of a calibration slide. Selection of the “global calibration” option will automatically apply this scale conversion to every image opened in that particular session of Fiji. 3. Set the foreground color to white and the background color to black through the Edit menu: Edit > Options > Colors. 4. Run the Tubeness plugin from the Plugin menu: Plugins > Analyze > Tubeness. Set the desired sigma factor and select the “use calibration information” option in the dialog box. We found for our studies that the default sigma value was most appropriate. However, this value should be empirically determined for every experiment. 5. Threshold the new “tubed” image through the Image menu: Image > Adjust > Threshold. Select the “Yen” thresholding algorithm, and the “dark background” and “stack histogram” options. The upper and lower limits of thresholding can be set with the sliders in the dialog box or can be typed in to the ten-thousandths place. Note that these values should be applied to every image and need to be empirically determined for each unique experiment. Another thresholding algorithm can be used if necessary, but we found of the ones provided in Fiji, that the Yen algorithm worked best. 6. Convert the thresholded image to an 8-bit segmented binary image through the Process menu: Process > Binary > Make Binary. or Process > Binary > Make Mask. It is strongly recommended that the image be saved at this point. 7. On the segmented image, filter the image objects for linear or tubular elements using Analyze Particles through the Analyze menu: Analyze > Analyze Particles. Enter the appropriate circularity cutoff value and select the “add to manager” option. Circularity is a measure of an object’s linearity, with 0 representing a straight line and 1 representing a perfect circle. This value should be empirically determined and used for every image. For our purposes, we found a cutoff of 0.4 worked well. The image objects that meet the circularity requirements can be viewed in the ROI manager window. 8. Create a new 8-bit image with a black background that matches the pixel dimensions of the original micrograph through the File menu: File > New > Image. 9. Transfer the filtered tubular elements in the ROI manager to the new image. Select the new blank image. Go to the ROI manager window and click on the “show all” option to make the objects appear on the blank image in outline form. In the ROI manager window click on the “more” button, and select the “fill” command to fill in the object outlines with white foreground color.

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10. Save the new filtered 8-bit binary image of the tubules and tubular endosomes and proceed with the Sholl analysis outlined below. 11. Note that if the membrane tubules in the micrograph make a closed loop, the loop will be filled in at step 9. One must decide if the resulting filled in space is problematic or not. If so, it can be fixed on a per image basis. a. Open the binary image obtained at the end of step 6. b. Carefully examine the image or cells of interest for closed loops. It may help to compare the binary image to the problematic filtered tubule image to quickly find the filled spaces. c. Set foreground color to black (see step 3) and use the pencil tool with a 1e3 pixel width setting to draw a line through the loop. This will break the loop and prevent the filled spaces from appearing at the end. d. Save the image and proceed with the rest of the image processing/analysis. 2.2.1.1.2 Sholl analysis 1. Open the binary image obtained from step 10 in the previous section. Ensure that the image scale is set correctly. Using the straight-line tool, draw a line on the binary image from the center of the cell to just past the outermost object in that cell. 2. Run the Sholl analysis plugin through the Analyze menu: Analyze > Sholl Analysis. In the dialog box, set the starting radius and desired radius step size. The ending radius should already be set, as it is based on the length of the line drawn in step 1. The other options can be set based upon experimental needs. All options should be empirically determined. For our purposes, we utilized a linear Sholl method with a start radius of 0 and a step size of 0.5 mm without curve fitting or normalizations. 3. Of the several new dialog boxes that open, one is a table with the radius step size and the number of intersections measured at each radius. Transfer these data to spreadsheets and save for subsequent analysis. To make comparisons between treatment groups, it may be helpful to compare areas under the curve. 4. If there are multiple cells within the image that are too close together (such that the line drawn in step 1 overlaps another cell), each cell needs to be singled out. We have found the following to be the easiest way to achieve this: a. Outline each cell in the image using either the free hand or polygon selection tool, and save the cell outlines to the ROI manager. b. Select one cell from the ROI manager list to cause the cell outline to appear on the image. Delete the rest of the image through the Edit menu: Edit > Clear Outside. c. Run the Sholl analysis on that cell and transfer the data to a spreadsheet. d. Without closing the image, revert the image back to its original state through the File menu: File > Revert. e. Repeat steps 4be4d until all cells in the image have been measured. Please note that when interpreting the resulting Sholl data, the nature of the membrane tubules being analyzed has to be taken into account. Endosome membrane tubules are often short and originate from not only the cell center, but also from the cell periphery. Therefore, one cannot interpret the Sholl analysis results

2. In vivo endosome membrane tubule formation

in the same manner as an analysis performed on Golgi membrane tubules, as an increase in object intersections in the cell periphery does not correlate with continuous, longer endosome membrane tubules. Instead, a Sholl analysis performed on endosome membrane tubules would indicate a change in the quantity of membrane tubules at a given distance from the cell center as demonstrated in Figure 4.

2.2.1.2 A Fiji macro script for semiautomated endososme membrane tubule segmentation The following macro script was written to semiautomate the membrane tubule segmentation process, rendering images ready for subsequent Sholl analysis. All images to be processed must be placed into one folder. Parameters used for the scripted plugins were empirically determined and will need to be customized for each experiment. The following script can be copied into a plain text file and placed into the macros folder of Fiji. To run the script, install it from the Plugins > Macros menu. Note that prior to running the script, the xey scale has to be set. // // // // //

"Tubeness Processor" This macro batch processes all images in a folder with the tubeness plugin, resulting in a binary image that can be used for the Sholl Analysis.

d = getDirectory("Choose a Directory "); list = getFileList(d); setBatchMode(true); for (i=0; i500 for 3T3 cells), such a quantitative analysis does not require many cells to reach statistical significance. The results obtained here with four cells are indeed mostly identical to our previous study using 24 cells (Derivery et al., 2012). The nododazole treatment is necessary to scatter the endosomes and ensure reliable 3D endosome segmentation. Although we do not have any evidence that it is the case, one cannot rule out the possibility that the physiology of endosome domains is affected by this nocodazole treatment. To control for this, this analysis may be performed in the absence of nocodazole on peripheral endosomes, since Microtubule (MT) depolymerization is mostly required to improve the segmentation of endosomes clustered around the nucleus. Current limitations in the acquisition speed in 3D limit the use of this quantitative analysis to fixed samples. But microdomains are very dynamic at the surface of endosomes, as exemplified in Figure 1, where a 3D rotation is observed. This is likely an important aspect of the physiology of microdomains that must be taken into account. Future hardware developments will likely allow the combination of the techniques developed in this part and the previous one, in order to analyze the dynamics of the compartmentalization of endosomal membranes in 3D.

CONCLUSION In this chapter, we presented methods to study two aspects of WASH physiology: the fission of transport intermediates, reflected in tubulation when defective, and the compartmentalization of endosome membranes into sorting platforms. These two phenomena being intrinsically related, a major question for the future is to understand how these two aspects are coordinated to drive endosomal sorting. A thorough understanding of WASH-mediated endocytic sorting will require the direct observation of WASH-mediated endosomal fission. This constitutes a major challenge, due to the fast dynamics of endosomes in 3D, which precludes the use of Total Internal Reflection Fluorescence (TIRF) microscopy, the only current method combining speed and high signal to noise ratio. However, the Betzig lab has recently pioneered lattice light sheet microscopy (Chen et al., 2014), a technique that combines 3D isotropic resolution and greatly enhanced temporal resolution compared to conventional spinning

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disk confocal microscopy. This novel imaging technique is thus poised to provide key insights into WASH-mediated endosomal fission in the near future. The techniques presented in this chapter can be applied beyond WASH to examine the organization and dynamics of other endosome domains, such as the ones defined by Rab proteins for instance. The next fascinating step will be to understand how distinct domains of different composition coexist at the surface of endosomes, and how this is regulated in space and time. This could be addressed by extending the methods developed here to three channels, in order to follow two distinct domains at the surface of the same endosome. Combined with novel imaging techniques and recent advances in gene-editing using clustered regularly interspaced short palindromic repeats (CRISPR) technology to label endogenous markers of endosomal microdomains, we expect that the approaches developed here will increase our understanding of trafficking in quantitative and molecular terms.

ACKNOWLEDGMENTS We thank Violaine David for critical reading of the manuscript. We acknowledge support from the Human Frontier Science Program (to E.D.) and from the Agence Nationale pour la Recherche (ANR-11-BSV2-014 to A.G).

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Roux, A., Cuvelier, D., Nassoy, P., Prost, J., Bassereau, P., & Goud, B. (2005). Role of curvature and phase transition in lipid sorting and fission of membrane tubules. EMBO Journal, 24(8), 1537e1545. Simonsen, A., Lippe, R., Christoforidis, S., Gaullier, J. M., Brech, A., Callaghan, J., et al. (1998). EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature, 394(6692), 494e498. Simons, K., & Ikonen, E. (1997). Functional rafts in cell membranes. Nature, 387(6633), 569e572. Soldati, T., & Schliwa, M. (2006). Powering membrane traffic in endocytosis and recycling. Nature Reviews Molecular Cell Biology, 7(12), 897e908. Sonnichsen, B., De Renzis, S., Nielsen, E., Rietdorf, J., & Zerial, M. (2000). Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11. Journal of Cell Biology, 149(4), 901e914. Stenmark, H., Parton, R. G., Steele-Mortimer, O., Lutcke, A., Gruenberg, J., & Zerial, M. (1994). Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO Journal, 13(6), 1287e1296. Stenmark, H., Vitale, G., Ullrich, O., & Zerial, M. (1995). Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell, 83(3), 423e432. Vonderheit, A., & Helenius, A. (2005). Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biology, 3(7), e233. Zech, T., Calaminus, S. D., Caswell, P., Spence, H. J., Carnell, M., Insall, R. H., et al. (2011). The Arp2/3 activator WASH regulates a5b1-integrin-mediated invasive migration. Journal of Cell Science, 124(Pt 22), 3753e3759. Zerial, M., & McBride, H. (2001). Rab proteins as membrane organizers. Nature Reviews Molecular Cell Biology, 2(2), 107e117.

CHAPTER

Rab GTPases and kinesin motors in endosomal trafficking

15

Ce´dric Delevoye*, x,1, Bruno Goud*, {, 1 *Institut Curie, PSL Research University, Paris, France CNRS UMR 144, Structure and Membrane Compartments, Paris, France { CNRS UMR 144, Molecular Mechanisms of Intracellular Transport, Paris, France x

1

Corresponding authors: E-mail: [email protected]; [email protected]

CHAPTER OUTLINE Introduction ............................................................................................................ 236 1. Screen for Rab GTPaseseKinesin Interactions ..................................................... 236 1.1 Methods ............................................................................................. 236 1.2 Results............................................................................................... 237 2. Spatiotemporal Distribution of Kinesins and Rab GTPases..................................... 237 2.1 Material ............................................................................................. 237 2.2 Methods ............................................................................................. 238 2.3 Results............................................................................................... 238 3. Role of the Microtubule Cytoskeleton in the Dynamics of Kinesin and Rab GTPases...................................................................................................... 239 3.1 Material ............................................................................................. 239 3.2 Methods ............................................................................................. 239 3.3 Results............................................................................................... 239 4. Functional Assays .............................................................................................. 239 4.1 Kinesin Depletion................................................................................ 240 4.1.1 Material............................................................................................ 240 4.1.2 Methods........................................................................................... 240 4.1.3 Results............................................................................................. 241 4.2 Ultrastructural Localization of Internalized Tf ........................................ 241 4.2.1 Material............................................................................................ 241 4.2.2 Methods........................................................................................... 241 4.2.3 Results............................................................................................. 242 4.3 Analysis of the Slow Tf Recycling Route ................................................ 242 4.3.1 Material............................................................................................ 242 4.3.2 Methods........................................................................................... 243 4.3.3 Results............................................................................................. 244 Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.05.004 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 15 Rab GTPases and kinesin motors in endosomal trafficking

4.4 Biochemical Analysis of the Maturation of Early Endosomes ................... 245 4.4.1 Material............................................................................................ 245 4.4.2 Methods........................................................................................... 245 4.4.3 Results............................................................................................. 245 Conclusions............................................................................................................ 245 References ............................................................................................................. 246

Abstract The endocytic pathway is composed of distinct types of endosomes that vary in shape, function, and molecular composition. In addition, endosomes are highly dynamic structures that continuously receive, sort, and deliver molecules to other organelles. Among organizing machineries that contribute to endosomal functions, Rab GTPases and kinesin motors play critical roles. Rab proteins define the identity of endosomal subdomains by recruiting set of effectors among which kinesins shape and transport membranous carriers along the microtubule network. In this review, we provide detailed protocols from live cell imaging to electron microscopy and biochemical approaches to address how Rab and kinesin proteins cooperate molecularly and functionally within the endocytic pathway.

INTRODUCTION The endocytic pathway consists in a highly complex network of organelles that constantly exchange cargo molecules (Klumperman & Raposo, 2014). Molecular motors like kinesins or membrane organizers like Rab GTPases play key endosomal functions by their ability to couple the sorting of molecules to their transport via membranous intermediates. Investigating the role of kinesins or Rab proteins within the endocytic pathway requires several complementary approaches to decipher where candidate proteins localize, how they function and with which molecular players they cooperate. Here we describe a nonexhaustive list of methods for establishing the functional links between a kinesin (KIF13A) and a Rab GTPase (Rab11) in the endocytic pathway.

1. SCREEN FOR Rab GTPaseseKINESIN INTERACTIONS The aim is to identify a Rab or a set of Rab proteins with which a given kinesin interacts. An attractive approach is the yeast two-hybrid system that represents a fast and easy way to test the interactions between a protein of interest (a kinesin as a bait) and many candidate proteins (Rab GTPases as preys).

1.1 METHODS The yeast strain Y187 (MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, gal4D, gal8D, met-, URA3::GAL1UAS-GAL1TATAlacZ MEL1) is transformed with the pLexA-Rab constructs (wild-type, dominant active (Q/L mutation), and dominant

2. Spatiotemporal distribution of kinesins and Rab GTPases

inactive (S or T/N mutation) forms for each Rab). Transformants are selected on synthetic medium lacking tryptophan. Yeast stocks in glycerol are distributed on 96-well plates and kept at 80 C. Before experiments, plates are replica-plated onto YPDrich medium in 120 120 mm square Petri dishes, and then onto selective medium. The pGADGH-KIF13A constructs are transformed into the yeast strain L40DGal4 (MATa, ura3-52, his3-200, ade2-101, trp1-901, leu2-3, gal4D, gal80D, URA3:: opLexA-LacZ, LYS2:opLEXA-HIS3) and selected on synthetic medium lacking leucine. After an overnight incubation on rich medium at 30 C, Y187 and L40 strains form diploids. Diploid cells containing the pLEX and pGADGH plasmids are selected on synthetic medium lacking leucine and tryptophan, and then replicaplated onto synthetic medium also lacking histidine and incubated for a further 3e6 days. Hisþ colonies are patched on selective plates and assayed for b-galactosidase activity. Each positive interaction should be retested individually by cotransforming the pLexA-Rab constructs with the pGADGH prey construct into the L40 yeast strain (Janoueix-Lerosey, Jollivet, Camonis, Marche, & Goud, 1995). More details on this method are given in (Lindsay, Miserey-Lenkei, & Goud, 2015).

1.2 RESULTS We have used the yeast two-hybrid assay to test the interactions between several KIF13A constructs (KIF13A-ST lacking the motor domain, KIF13A-T corresponding to the tail domain and KIF13A-S lacking the motor and tail domains) and all human Rab GTPases. Interactions were notably found with the dominant active forms of Rab11a and Rab11b (Rab11a/b Q70L) as well as with Rab25 but not with their respective dominant negative mutants (Rab11a/b S25N and Rab25 T26N) (Delevoye et al., 2014).

2. SPATIOTEMPORAL DISTRIBUTION OF KINESINS AND Rab GTPases The aim is to decipher the intracellular localization and the dynamic behavior of kinesins and Rab GTPases. The visualization of endogenous kinesins by immunofluorescence (IF) may be difficult due to the lack of good specific antibodies and to their distribution between a cytosolic but inactive pool and an active membraneassociated pool. Coexpressing fluorescent protein (FP)-tagged version of kinesins in combination with FP-Rab GTPases can be used to address their localization. In addition, live cell imaging brings useful information to appreciate the dynamics of both kinesins and Rab GTPases.

2.1 MATERIAL For live cell imaging, cells are incubated 30 min before image acquisition in Optimem medium without phenol red supplemented with 10 mM HEPES (Delevoye et al., 2009, 2014).

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2.2 METHODS HeLa cells (5 105) are seeded in 6-well tissue culture plates containing glass coverslips (for IF) or in tissue culture dishes with cover glass bottom (for live cell imaging). Transfection with plasmids encoding kinesin or Rab GTPase constructs is performed the day after using jetPRIME (Polyplus transfection) and by following manufacturer’s procedure except for Rab GTPase encoding plasmids that are used at 1/10th of the proposed quantity. Live cell imaging is performed 24 h after transfection using a spinning-disk microscope equipped with a thermostated chamber set at 37 C.

2.3 RESULTS HeLa cells coexpressing KIF13A fused to YFP at its carboxy-terminal part (KIF13AYFP) and Rab11a fused to mCherry at its amino-terminal part (mCherry-RAB11a) codistributed on highly dynamic tubular structures (Figure 1(AeB)). KIF13A, a plus-ended directed motor (Nakagawa et al., 2000) was enriched at the tip of Rab11a positive tubules (Figure 1(B), arrows). This result shows that kinesin KIF13A and Rab11 are dynamically associated to recycling endosomes. Coexpression of the motor-less version of KIF13A (KIF13A-ST) fused to GFP showed that a fraction of Rab11a still codistributed with the kinesin on endosomes but on punctate

FIGURE 1 Live cell imaging on cells coexpressing KIF13A and Rab11a. (A) Spinning-disk confocal microscopy was used to capture concomitant movements of KIF13A-YFP and mCherry-Rab11A in HeLa cells. KIF13A generates long tubular structures decorated by Rab11a that extend toward the cell periphery. (B) Magnified insets (of boxed area) of consecutive time-lapse single and merged images are presented as a gallery (one image/7 s) and show that KIF13A is enriched at the tip of the growing recycling tubules (arrows). (C) KIF13A deleted from its motor domain (KIF13A-ST) codistributes with Rab11a positive vesicles. (D) KIF13A positive endosomal tubules do not contain Rab5, demonstrating the specific localization of KIF13A to recycling endosomes. Two-colors stacks of 2e4 z-slices (0.2 mm) were acquired using 150 ms exposure for 50 or 100 time points under Metamorph. Scale Bars: 10 mm. (See color plate)

4. Functional assays

structures (Figure 1(C)), indicating that the motor domain of KIF13A is required to generate tubular recycling endosomal structures decorated by Rab11a. As control, other endosomal Rab GTPases such as mCherry-Rab5a known to associate with early sorting endosomes can be coexpressed. KIF13A positive tubules were negative for Rab5a (Figure 1(D)). All together, these results showed KIF13A and Rab11 are specifically and dynamically associated to recycling endosomes.

3. ROLE OF THE MICROTUBULE CYTOSKELETON IN THE DYNAMICS OF KINESIN AND Rab GTPases The aim is to perturb the integrity of the microtubule cytoskeleton to investigate how the dynamics and localization of kinesin and Rab proteins are impacted.

3.1 MATERIAL HeLa cells are grown in complete medium supplemented with 20 mM HEPES containing 10 mM nocodazole. The same concentration of dimethyl sulfoxide (DMSO) is used for controls.

3.2 METHODS Cells are first rinsed in cold PBS and incubated 90 min at 4 C to depolymerize microtubules in complete medium supplemented with 20 mM HEPES. Cells are then washed in cold PBS and incubated in preheated (37 C) medium containing nocodazole for 90 min in order to depolymerize remaining stable microtubules.

3.3 RESULTS Cells treated with nocodazole (Noco) showed a punctate distribution of KIF13AYFP as compared to a more tubular pattern in control (DMSO) condition (Figure 2). KIF13A was still localized to endosomal structures positive for internalized transferrin (Tf) and its receptor (TfR), showing that microtubule cytoskeleton is not required for the endosomal association of KIF13A but necessary for the tubulation of recycling endosomes and their redistribution toward the cell periphery.

4. FUNCTIONAL ASSAYS The aim is to identify the transport pathways in which kinesins are involved. This can be achieved by depleting the expression of a given kinesin by siRNA and by monitoring its impact on various transport markers. For a kinesin associated with endosomal compartments (KIF13A), classical markers are the transferrin receptor (TfR), the epidermal growth factor (EGF) receptor (EGFR), and their ligands

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FIGURE 2 Microtubule depolymerization inhibits the formation of recycling tubules by KIF13A but not its endosomal localization. KIF13A-YFP expressing HeLa cells that internalized TfA647 were incubated for 90 min at 4 C and treated or not (dimethyl sulfoxide (DMSO)) with 10 mM of nocodazole (Noco) to depolymerize microtubules. Cells were processed for immunofluorescence and labeled with an antibody against transferring receptor (TfR). Nocodazole treatment prevented the KIF13A-dependent formation of recycling endosome tubules without impacting on the endosomal localization of KIF13A. Single, merged, and magnified insets of boxed areas were shown. Scale Bars: 10 mm. (See color plate)

(Tf, EGF). Transport of cargoes can be followed by combining electron microscopy (EM) with several biochemical methods.

4.1 KINESIN DEPLETION 4.1.1 Material Oligofectamine solution: 10 mL Oligofectamine þ 20 mL Optimem (per well of 6-well tissue culture plates). siRNA solution: 2 mL siRNA (20 mM) þ 168 mL Optimem (per well of 6-well tissue culture plates).

4.1.2 Methods HeLa cells (2 105) are seeded on day 1 in 6-well tissue culture plates. At least two independent kinesin-specific siRNAs are used. On day 2, cells are washed three times in Optimem (1 mL) and starved for 30 min in 800 mL Optimem. Meanwhile, siRNAs and oligofectamine (Invitrogen) are prepared as followed. SiRNAs (2 mL at 20 mM) are diluted in 168 mL of Optimem and incubated for 20 min at room temperature (RT). In the meantime, 10 mL of oligofectamine is added drop by drop on top of 20 mL of Optimem without touching the plastic tube (20 min at RT). The oligofectamine solution is deposited drop by drop on top of the siRNA solution and incubated for 20 min at RT. The transfection mix (200 mL) is deposited on cells and the plates are carefully shaken. The plates are then incubated for 4 h at 37 C and 1 mL of complete medium (supplemented with fetal bovine serum (FBS) (2x concentrated)) is added overnight. On day 3, transfection medium is replaced by complete medium and experiments are performed 24 or 48 h later.


4.1.3 Results Following this protocol, we obtained a nearly complete extinction of endogenous KIF13A in different cell lines (Delevoye et al., 2009, 2014).

4.2 ULTRASTRUCTURAL LOCALIZATION OF INTERNALIZED TF 4.2.1 Material Culture medium: DMEM supplemented with 25 mg/mL of Tf-HRP (Horse Radish Peroxidase). Fixation buffer: 0.2 M Phosphate buffer (PB), 2% Paraformaldehyde, 0.25% Glutaraldehyde (Glut), pH 7.4. Postfixation buffer: 0.1 M PB, 2.5% Glut. OsO4 buffer: 1% OsO4, 1.5% Ferrocyanure, 0.1 M Cacodylate, pH 7.4. Tris/HCl buffer: 50 mM Tris HCl pH 7.6. DAB buffer: 1.5 mg/mL DAB, 50 mM Tris HCl, pH 7.6. DAB precipitation buffer: 1.5 mg/mL DAB, 0.03% H2O2, 50 mM Tris HCl, pH 7.6. EPON resin: 66 g EPON, 100 g DDSA (solution A); 100 g EPON, 84 g NMA (solution B); both at 20 C.

4.2.2 Methods HeLa cells (2 105) are seeded in 6-well plates containing glass coverslips and grown in complete medium. Cell supernatants are removed and cells are washed once in warmed FBS (2 mL), rinsed twice in 2 mL of DMEM and starved for 45 min in DMEM at 37 C to remove endogenous Tf contained in FBS. Individual coverslips are transferred in 24-well tissue culture plates and incubated for 30 min with 150 mL of DMEM supplemented with Tf-HRP (25 mg/mL). Cells are washed twice with 500 mL of DMEM at RT, and then incubated for 2 h (RT) in fixation buffer (dilution 1:1 in DMEM), washed (3 5 min) in 500 mL of Tris/HCl buffer, incubated 10 min (RT) in 500 mL of DAB buffer and another 10 min in DAB precipitation buffer (RT). Dark brown DAB precipitates can be observed by using binocular microscope. Then cells are washed twice with 500 mL of Tris/HCl buffer (RT). Washing steps need to be performed quickly and well per well in order to avoid the drying of cells. Cells are then fixed again in postfixation buffer at 4 C. This step lasts at least 2 h and at maximum 2 days at 4 C. For incubation times longer than 2 h, plates are protected from light and sealed to avoid evaporation of fixatives. Cells are incubated for 45 min at 4 C in OsO4 buffer and protected from light. Cells are washed in distilled water (3 5 min, RT) and dehydrated at RT in 500 mL of solution with increased concentrations of ethanol (EtOH) (diluted in water) 5 min in 30% EtOH solution; 5 min in 50%; 5 min in 70%; 5 min in 80%; 2 10 min in 95%, and 3 6 min in 100% EtOH solution. Use a new or recent 100% EtOH bottle to avoid hydration of this solution. Start to thaw EPON resin (solutions A and B) at the beginning of the dehydration step, mix 3 mL of solution A with 7 mL of solution B when starting the incubation in the 95% EtOH solution and add catalyzer

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(DMP130 or DMAE; 170 mL/10 mL of resin) when starting the 100% incubation. EPON solution is mixed under rotation to get homogenous EPON resin, a yellow gold color is appearing when adding the catalyzer. To get rid of air bubbles present in the resin, centrifuge the tube 1 min at 2,000 rpm (RT). Using plastic pipette, deposit a drop of resin on top of a glass slide, then remove the coverslip from the EtOH solution using tweezers and quickly add a drop of resin on top of coverslip (on the side where cells have adhered) and deposit the coverslip on the slide covered with the drop of resin. Cells should be on the upper face of the coverslip. Repeat this step for each coverslip and let the slides overnight under a hood to evaporate the EtOH. The remaining EPON resin is frozen and thawed the day after. Gelatin capsule is filled with EPON resin, reversed on top of the coverslip and let again under the hood for a day. The morning after, slides covered by coverslips and capsules are incubated in an oven at 60 C for at least 2 days to polymerize and harden the resin. Note that all of these steps detailed in this section have to be done under a hood due to the toxicity of the chemical products used. Samples are then processed for ultrathin sectioning, contrasting (4% uranyl acetate in water), and electron micrographs acquisitions.

4.2.3 Results In control cells, brown DAB precipitates were observed in tubulo-vesicular structures corresponding to recycling endosomes (Figure 3(A), arrowheads), showing that Tf-HRP internalized for 30 min was predominantly sorted out of the vacuolar domain of sorting endosomes (arrow) and loaded into recycling endosome tubules. In contrast, following KIF13A depletion, very little positive tubules were observed (Figure 3(B), arrowheads) and Tf-HRP accumulated into vacuolar structures containing a few or several intraluminal vesicles reminiscent of sorting or multivesicular endosomes (arrows). This shows that, upon KIF13A depletion, internalized Tf can reach sorting endosomes but is inefficiently sorted into recycling tubules and thus trapped in maturing endosomes. This suggests that KIF13A controls the sorting of Tf into recycling endosomal tubules.

4.3 ANALYSIS OF THE SLOW TF RECYCLING ROUTE Internalized Tf follows two recycling routes: the main one (70e80% of total internalized Tf), called the fast recycling route, occurs at the level of sorting endosomes; the other one (20e30% of the total internalized Tf) is named the slow recycling route and uses the recycling endosomes as an endosomal intermediate.

4.3.1 Material Washing medium: 10 mL DMEM, 1% BSA, 20 mM HEPES (4 C). Endocytosis medium: 10 mL DMEM, 1% BSA, 20 mM HEPES (37 C). Binding medium: 2 mL DMEM, 1% BSA, 20 mM HEPES, 5 mg/mL Tf-A488 (4 C). Stripping buffer: 50 mM Glycine, 100 mM NaCl, pH 3.


FIGURE 3 Ultrastructural localization of Tf-HRP following KIF13A depletion. Conventional electron microscopy on control (A) and KIF13A-depleted HeLa cells (B) that internalized Tf-HRP were processed for DAB/H2O2 cytochemistry. (A) In control condition, electron-dense Tf-HRP localizes to tubulo-vesicular structures (arrowheads), characteristics of recycling endosomes. Vacuolar sorting endosomes (SE, arrow) were rarely positive for TfHRP. (B) KIF13A-depleted cells concentrate electron-dense Tf-HRP in vacuolar organelles (arrows) with morphological features of sorting endosomes (SE) or multivesicular endosomes (MVE). Few electron-dense tubular structures (arrowheads) were observed upon KIF13A depletion. Lyso: Lysosome; Golgi: Golgi apparatus; Mito: Mitochondria. Scale Bars: 500 nm.

4.3.2 Methods HeLa cells are grown to confluence on Petri dishes (10 cm diameter). One dish is used for one experiment corresponding to nine tubes for FACS (“Fluorescence Activated Cell Sorter”) analysis. Cells are first washed in 5 mL of DMEM and starved by incubating them for 30 min in DMEM (37 C). Cells are washed once in PBS and harvested by incubating them in 1 mL of Versene (Gibco) or EDTA solution for 5 min at 37 Cddo not use trypsin-based solution to avoid removal of functional TfR from the cell surface. Cells are suspended in 9 mL of DMEM (4 C) and transfer to a 15 mL conical tube. Cells (10 mL) are centrifuged (2 min at 1,200 rpm), and cell pellets resuspended in 2 mL of cold binding medium and incubated for 1 h on ice to allow binding of fluorescent Tf to TfR at the cell surface. Cells are gently mixed each 10 min by pipetting to allow an efficient and homogenous cell surface Tf binding. Meanwhile, 9 FACS tubes are prepared,

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labeled, and kept on ice (Tube 1 ¼ fluorescence background; Tube 2 ¼ maximum internalized Tf, time point 0 min; Tube 3e9 ¼ time points 2, 4, 6, 8, 10, 15, and 30 min of Tf recycling); cold PBS (2 mL) is added to Tubes 3e9. Cells are then centrifuged (1 min, 1,200 rpm, 4 C), and resuspended in cold washing medium (10 mL) from which 1 mL is transferred to Tube 1. Remaining cells (9 mL) are centrifuged (1 min, 1,200 rpm, 4 C), and resuspended in 2 mL of prewarmed endocytosis medium and incubated at 37 C in a water bath for 6 min. 10 mL of cold PBS is added to cells that are transferred on ice to stop Tf internalization. These cells as well as Tube 1 are centrifuged and kept on ice. Cell pellets are resuspended in 1 mL (15 mL tube) or 500 mL (Tube 1) of cold stripping buffer and incubated on ice for 4 min to remove cell surface associated Tf. Cells in Tube 1 corresponds to the fluorescence background. Cold PBS (7 mL) is added to the 15 mL tube and 2 mL to Tube 1. The maximum amount of Tf is measured by Tf 1 mL from the 15 mL tube to Tube 2. All the tubes are then centrifuged. Recycling of fluorescent Tf is performed by discarding supernatants and adding 300 mL of cold PBS in Tubes 1 and 2 (kept on ice) and 3.5 mL of prewarmed endocytosis medium (37 C) to the 15 mL tube incubated at 37 C in a water bath. Cells (500 mL) suspended in endocytosis medium are collected at the indicated time points (2e 30 min) and transferred on ice in the corresponding 2 mL cold PBS-containing FACS tubes (Tubes 3e9). Cells need to be well mixed before pipetting, since cells deposit at the bottom of the tube by sedimentation. Tubes 3e9 are centrifuged, resuspended in 300 mL of cold PBS and kept on ice before analysis of at least 1 105 cells per point by a flow cytometer. Data are expressed as a percentage of surface-bound fluorescence relative to the total internal fluorescence after acid wash (Tube 2). Background fluorescence (Tube 1) is measured from acidwashed cells just after the binding step and the value is subtracted from all time points.

4.3.3 Results KIF13A depletion impacted only modestly Tf recycling (15% decrease as compared to control) (Delevoye et al., 2014), suggesting that KIF13A functions along the slow recycling route rather than the fast one. Tf recycling in KIF13A-depleted cells segregated from control cells after 6 min of chase. This time window corresponds to the step where Tf is sorted from the vacuolar to tubular domains of early endosomes, namely from sorting to recycling endosomes (Willingham, Hanover, Dickson, & Pastan, 1984). Such a result indicates that KIF13A is primarily acting at an early step of the Tf slow recycling pathway, at the time where Tf segregates from sorting endosomes to be loaded into recycling endosomes. Remarkably, it fits perfectly well with EM observations (Figure 3, see above). For instance, comparable experiments performed on KIF16B-depleted cells have shown that the recycling of Tf was impaired up to 3 min of chase (Hoepfner et al., 2005), suggesting that KIF16B functions at the sorting endosomal level rather than at recycling endosomes. This shows that such analysis is suitable to decipher which endosomal functions are controlled by proteins of the kinesin or Rab family.

Conclusions

4.4 BIOCHEMICAL ANALYSIS OF THE MATURATION OF EARLY ENDOSOMES The aim is to investigate sorting from early to late endosomes by measuring the degradation of EGFR induced by EGF internalization.

4.4.1 Material EGF medium: DMEM with 50 ng/mL of unlabeled EGF. Lysis buffer: 50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, 10 mM EDTA, protease inhibitor cocktail, pH 7.2 (4 C).

4.4.2 Methods HeLa cells (2 105) are seeded on day 1 in a 6-well tissue culture plates. Five time points are required (0, 30, 60, 120, and 180 min), one plate corresponding to one time point. On day 2, cells are transfected with siRNAs for 2 days. On day 4, cells are starved at 37 C for 4 h in DMEM in order to degrade remaining endogenous EGF and pulsed with EGF medium for 30 min (37 C). Exogenous EGF is chased in DMEM for the indicated time points (30e180 min) and cells are lysed in 200 mL of lysis buffer/well. Cells are scrapped; lysates are collected and incubated on ice for 10 min, centrifuged twice at 10,000 rpm for 5 min (4 C). Protein extracts are quantified by the Bradford assay and equal amount of supernatants (for each time point and each siRNA condition) is prepared for western blot loading (5e10% of the whole cell lysate) and analysis. Membranes are probed with antibodies specific for EGFR to evaluate its degradation over chase.

4.4.3 Results Western blot analysis showed that KIF13A or KIF16B depletion does not have the same effect on the degradation of EGFR (Delevoye et al., 2014; Hoepfner et al., 2005). Whereas KIF13A depletion does not affect the expression level of EGFR overtime (Delevoye et al., 2014), KIF16B depletion slightly accelerates its degradation after 2 h of chase (Hoepfner et al., 2005), showing that KIF13A and KIF16B function at different levels along the endocytic pathways.

CONCLUSIONS The diversity and plasticity of endosomal compartments conjugated to the multiple parallel or overlapping endocytic routes make the analysis of the functions of kinesin, Rab GTPase, and other proteins, a very challenging task. A plethora of methods is available but only their combination is instrumental for unraveling which role(s) a given protein plays along the endocytic pathway. In particular, recent work has highlighted the power of correlating light to electron microscopy on live or fixed samples (Brown, Van Weering, Sharp, Mantell, & Verkade, 2012; Heiligenstein et al., 2014).

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This method is adapted to the study of the endocytic pathway and should be included in our methodological arsenal in future studies.

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CHAPTER

Measuring receptor recycling in polarized MDCK cells

16 Luciana Gallo, Gerard Apodaca1

Departments of Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA 1


CHAPTER OUTLINE Introduction ............................................................................................................ 248 1. Objectives and Rationale .................................................................................... 250 1.1 Labeling Surface Receptors.................................................................. 251 1.2 Internalization of Receptor ................................................................... 252 1.3 Measurement of Receptor Recycling ..................................................... 252 2. Materials and Instruments .................................................................................. 253 2.1 Reagents to Iron Load Canine apo-Tf..................................................... 253 2.2 Reagents to Iodinate Canine holo-Tf...................................................... 254 2.3 Reagents to Culture MDCK Cells........................................................... 256 2.4 Reagents to Measure 125I-Tf Recycling ................................................. 256 2.5 Reagents to Perform Tricholoracetic Acid Precipitation........................... 256 2.6 Equipment ......................................................................................... 257 3. Methods ............................................................................................................ 257 3.1 Loading apo-Tf with Iron ...................................................................... 257 3.2 Iodinating holo-Tf................................................................................ 258 3.3 Culturing MDCK Cells on 12-mm Transwells.......................................... 259 3.4 Experimental Strategy to Measure Receptor Recycling............................ 260 3.4.1 Background and objective ................................................................ 260 3.4.2 Flow of experiment ........................................................................... 260 Summary ................................................................................................................ 266 References ............................................................................................................. 267

Abstract Recycling of proteins such as channels, pumps, and receptors is critical for epithelial cell function. In this chapter we present a method to measure receptor recycling in polarized MadineDarby canine kidney cells using an iodinated ligand. We describe a technique to iodinate transferrin (Tf), we discuss how 125I-Tf can be used to label a cohort of


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CHAPTER 16 Measuring receptor recycling in polarized MDCK cells

endocytosed Tf receptor, and then we provide methods to measure the rate of recycling of the 125I-Tf-receptor complex. We also show how this approach, which is easily adaptable to other proteins, can be used to simultaneously measure the normally small amount of 125 I-Tf transcytosis and degradation.

INTRODUCTION Epithelial tissues are comprised of continuous sheets of adherent epithelial cells that cover the body surfaces and cavities, form glands, and line the inner and outer surfaces of sac- and tube-shaped organs (Apodaca & Gallo, 2013; Apodaca, Gallo, & Bryant, 2012). Epithelial cells characteristically have an apicalebasolateral asymmetry or polarity that is marked by compositionally, functionally, and morphologically distinct membrane domains: an apical one that is contiguous with the external milieu or faces the body cavities, a lateral domain that promotes cellecell interactions, and a basolateral domain that abuts the underlying tissues (Figure 1). By separating the external environment from the internal one, epithelial cells form barriers to water, ions, and pathogens, carry out immune surveillance, perform sensory transduction, and play critical roles in vectorial water, ion, and peptide transport (Apodaca & Gallo, 2013). The execution of these epithelial functions depends on endocytosis. For example, water channels recycle to the cell surface in response to vasopressin (Wilson, Miranda, & Knepper, 2013), and absorption of Naþ ions in the kidney depends, in part, on apical recycling of the epithelial sodium channel (Butterworth et al., 2012). Recycling is also critical at the basolateral surface of the epithelial cell where it can perform important signaling and housekeeping functions. For example, the uptake of iron, which is a key component of catalase, cytochromes, and hemoglobin, occurs at this surface. Because the human body lacks a dedicated mechanism to rid itself of iron (other than cell sloughing), the chief mechanism of control is to regulate its uptake (Winter, Bazydlo, & Harris, 2014). The latter is accomplished by the transferrin receptor (TfR)-1, which is a ubiquitous type II membrane protein, comprised of two identical 85 kDa subunits linked by a disulfide bond, that recycles at the basolateral surface of most, but not all polarized epithelial cells (e.g., in proximal tubule epithelial cells the TfR is found apical) (Fuller & Simons, 1986; Perez Bay, Schreiner, Benedicto, & Rodriguez-Boulan, 2014). In the two well-studied MadineDarby canine kidney (MDCK) cell lines, high-resistance type I and lowerresistance type II, TfR has a basolateral:apical distribution of 800:1 and 300:1, respectively (Fuller & Simons, 1986). Overexpression of the TfR in MDCK cells can result in a considerable amount of missorting, indicating a sorting machinery that may be easily saturated (Odorizzi, Pearse, Domingo, Trowbridge, & Hopkins, 1996; Sheff, Daro, Hull, & Mellman, 1999; Sheff, Kroschewski, & Mellman, 2002). At the basolateral surface, and in the neutral pH of the extracellular fluid (pH 7.4), the homo-dimeric TfR binds with nanometer affinity to two molecules of holo-Tf, an w80 kDa serum protein, primarily secreted by the liver, that is bound

Introduction

FIGURE 1 Endocytic pathways in polarized MDCK cells. General pathways for endocytosed cargo: Proteins, lipids, and fluid are internalized from the apical (step 1a) or basolateral (step 1b) surfaces, and delivered to apical early endosomes (AEEs) or basolateral early endosomes (BEEs). Cargoes can be recycled to the cell surface (steps 2a/b), be delivered to late endosomes (steps 3a/b), from which they will be delivered to lysosomes (not shown), or they can be delivered to the common recycling endosome (CRE; steps 4a/b). There may be a direct pathway from the AEE to the apical recycling endosome (ARE, step 4c). CRE cargoes are shuttled to the basolateral cell surface (step 5), or delivered to the ARE (step 6), from where they gain access to the apical cell surface (not shown). Pathways for transferrin receptor (TfR) recycling in polarized MDCK cells (marked with red arrows): The TfR, bound to holo-Tf, is internalized at the basolateral surface of the cell and then delivered to BEEs (step 1b). In response to a decrease in pH, which results from the activity of the Hþ-ATPase, Fe3þ is released from Tf. The Fe3þ is reduced by the activity of STEAP3, and the resulting Fe2þ is transported into the cytosol by the action of DMT1. While some TfR with bound apo-Tf rapidly recycles from the BEE (step 2b), a significant fraction is delivered to the CRE, where further dissociation and recovery of iron occurs. The TfR, with bound apo-Tf, is then recycled to the basolateral cell surface (step 5). At the basolateral cell surface, apo-Tf dissociates from the TfR, which is now primed to bind holo-Tf anew and resume additional rounds of recycling. (See color plate)

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to two Fe(III) atoms with high affinity (Ka w1023 L/mol) (Frazer & Anderson, 2014). The iron atoms are coordinated by way of two Tyr, a His and an Asp residue, and along with a bidentate carbonate anion (Cheng, Zak, Aisen, Harrison, & Walz, 2004), and iron binding is accompanied by a conformational change in the Tf molecule. Tf binding to its receptor can be species specific. For example, bovine Tf binds poorly to the human Tf receptor (Young & Garner, 1990). The amount of TfR in the cell is regulated by the iron response/regulatory element (IRE) binding protein, which in the presence of iron binds to a hairpin-like structure (the IRE) in the 30 untranslated region of the TfR mRNA, preventing its degradation (Frazer & Anderson, 2014). The basolateral TfReholo-Tf complex is endocytosed at clathrin-coated pits along with cargoes that are targeted for degradation and those that will be delivered to the opposite pole of the cell in a process termed transcytosis (Apodaca et al., 2012). Cargo initially enters basolateral early endosomes (BEEs), where the drop in pH stimulates the release of the bound Fe(III) (Fe3þ) atoms from holo-Tf. The iron atoms, reduced to Fe2þ by STEAP3, are then transferred from the lumen of the endosome to the cytosol by way of the divalent metal transporter DMT1 (Frazer & Anderson, 2014) (Figure 1). Upon loss of its bound iron, the resulting apo-Tf remains attached to the TfR until the receptor ligand complex is recycled to the basolateral cell surface. This recycling can occur directly from BEEs or from the downstream common recycling endosome (CRE), which derives its name from being a site of intermixing of cargoes, both apical and basolateral and from the biosynthetic and endocytic pathways (Apodaca et al., 2012) (Figure 1). Upon arrival at the basolateral surface, and in response to exposure to pH 7.4, apo-Tf rapidly dissociates from the TfR. In this chapter, we describe methods to measure recycling in polarized MDCK type II cells, using the TfR as a paradigm to understand how such measurements are made. For this assay, MDCK cells are grown on porous filter supports to form wellpolarized monolayers with tight junctions. 125I-Tf is internalized from the basolateral pole of the cell and the amount of ligand recycling is measured using a sensitive, reproducible, and relatively easy to perform biochemical assay (Gallo et al., 2014; Leung, Deng, DasGupta, Whiteheart, & Apodaca, 1998; Leung et al., 1999; Maples, Ruiz, & Apodaca, 1997; Rojas, Ruiz, Leung, Jou, & Apodaca, 2001).

1. OBJECTIVES AND RATIONALE The goal of many investigations is to understand the fate of internalized receptors, the membrane trafficking machinery that controls receptor recycling, or the physiological regulatory mechanisms that control these events. To measure recycling, cell biologists employ assays that allow them to measure the return of internalized surface receptors (and or their cargoes) to the cell surface. Integral to these assays is (1) a technique to label the receptor at the cell surface; (2) a step that allows the receptor to be internalized,

1. Objectives and rationale

and for remaining cell-surface receptors to be removed; and (3) a method to measure the return of receptors to the cell surface. While we focus on receptor recycling, the techniques described can be applied to other trafficking steps and other proteins as well.

1.1 LABELING SURFACE RECEPTORS An ideal method to track receptor recycling is by following a native, tagged ligand, or if available one can use a labeled antagonist or agonist. In the case of the TfR, one can use Tf that has been tagged with biotin (e.g., NHS-SS-biotin), fluorophores (such as FITC/TRITC), horseradish peroxidase (HRP), or 125I. If no ligand is available, then one can label the receptor directly by techniques that include cell-surface biotinylation (Gottardi, Dunbar, & Caplan, 1995; Matter, Stieger, Klumperman, Ginsel, & Hauri, 1990; Sargiacomo, Lisanti, Graeve, Le Bivic, & RodriguezBoulan, 1989). An indirect method to label a receptor is by use of primary antibodies (which are tagged using biotin, fluorophores, HRP, or 125I). However, antibodies are better substituted with Fab fragments, which contain only one binding site and are therefore less likely to stimulate receptor cross-linking (Matter et al., 1990). Alternatively, one can engineer a binding site into a receptor (e.g., an SNAP tag, CLIP tag, coiled-coil tag, Halo tag, or a-bungarotoxin tag), which can be detected using the appropriate antibodies or fluorescently labeled ligand derivatives (Gautier et al., 2008; Hannan, Wilkins, Thomas, & Smart, 2013; Wilkins, Li, & Smart, 2008; Yano, Kawano, Omae, & Matsuzaki, 2012; Yano & Matsuzaki, 2009). An important caveat associated with the use of some of these methods is that they can induce cross-linking or unanticipated changes in receptor structure, either of which may affect the fate of internalized receptor. Ditto for methods used to label ligands, which are often sensitive to the degree of labeling. Thus, the investigator must confirm that any new (or existing) labeling method does not provide erroneous interpretations. Moreover, the investigation must also take into consideration that some native ligands, and nonnative ligands (such as antibodies), dissociate from the receptor, often in response to the change in pH as the receptor and its bound cargo enter the early endosome. In such cases, one is measuring the fate of the ligand and not necessarily the receptor itself. In this chapter we provide methods to label the TfR by binding to its native ligand, which has been iodinated using a technique (the iodine monochloride (ICl) method) that does not expose the ligand to oxidation and shows excellent recycling kinetics, with little transcytosis or degradation (Gallo et al., 2014; Leung et al., 1998, 1999; Maples et al., 1997; Rojas et al., 2001). Thus, it is an ideal method to follow the recycling of the TfR, and can be easily adapted to follow the post-endocytic fate of other receptor and ligand pairs that are recycled, transcytosed, or degraded (Gallo et al., 2014; Leung et al., 1998, 1999; Maples et al., 1997; Rojas et al., 2001). When designing a receptor-recycling assay, one must also take into consideration whether one will measure a single round of receptor recycling or whether one will follow the recycling of a larger cohort of receptors. In the case of the former, one typically performs the initial receptor-labeling step at 4 C, which prevents

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endocytosis. However, this technique requires that there be sufficient receptors at the surface to allow subsequent detection. In the case of MDCK cells, only a small fraction of the TfR is at the basolateral surface at any one time, and as a result following a single round of recycling is difficult. Instead, we typically label a large cohort of TfR by continuously supplying 125I-Tf to the basolateral cell surface for 45 min at 37 C, and thus Tf/TfR gains access to BEEs and CREs. One can also perform the labeling steps with ligands, antibodies, or biotin at reduced temperatures such as 18 C, or in the presence of nocodazole, which in either case will maintain the receptors in BEEs (Apodaca, Katz, & Mostov, 1994; Song, Bomsel, Casanova, Vaerman, & Mostov, 1994).

1.2 INTERNALIZATION OF RECEPTOR Upon labeling the receptor, one must then allow the receptor to be internalized. If one is performing a single round of recycling, this typically involves warming up the cells to 37 C. The length of warm-up can vary, but typically one chooses a time of sufficient length to allow the majority of receptor to be internalized from the surface, but short enough that significant receptor recycling is not observed. If the goal is to measure the fate of a larger cohort of receptor, then the internalization step can be performed at 37 C, or any other desired temperature. In such cases, the duration of warm up is not as critical, but is typically continued until one achieves a steady state. The latter is determined by showing that no additional receptor accumulates intracellularly after the given incubation time. Such measurements in MDCK cells show that Tf reaches a steady state after 45 min at 37 C. Because one measures recycling by quantifying the return of the endocytosed pool of receptor to the cell surface, it is critical that all traces of surface receptor be removed following the internalization step. This can be accomplished by a number of different techniques. In the case of an antibody or ligand, one can use incubation in an acidic solution or exposure to proteinases (which surprisingly do not cause the epithelial cells to dissociate from their membrane supports), or in the case of NHS-SS-biotin use of a reducing agent such as glutathione or MESNA (Elferink & Strick, 2005; Gottardi et al., 1995; Lisanti, LeBivic, Sargiacomo, & Rodriguez-Boulan, 1989; Sargiacomo et al., 1989; Shah, Baterina, Taupin, & Farquhar, 2013). In the specific case of Tf, some investigators incubate cells in buffer at w pH 5.0 and then buffer at pH 7.4, and/or include the iron chelator desferrioxamine (also known as desferoxamine B) in the bathing medium (Dunn, McGraw, & Maxfield, 1989; Ghosh, Gelman, & Maxfield, 1994; Roberts & Bomford, 1988). However, we and others have found that after several washes at 4 C, incubating for 2.5 min at 37 C is sufficient to remove cell-surface disposed receptors by promoting their internalization.

1.3 MEASUREMENT OF RECEPTOR RECYCLING The final step in the process is to measure the amount of receptor that recycles once cell-surface receptor has been removed in the preceding step. Ideally, the method

2. Materials and instruments

should be easily quantifiable and the results should be highly reproducible. Some methods measure the loss of receptor signal. For example, once receptors are labeled with NHS-SS-biotin, the cells are typically incubated at 37 C for several minutes to allow receptor recycling, and then cooled down at 4 C. Control cells are left untreated, while experimental ones are treated with a reducing agent, which removes biotin moiety from the receptor. The amount of recycling is estimated by quantifying the loss of biotinylated receptor in the experimental versus control cell populations (Elferink & Strick, 2005; Shah et al., 2013). An additional example is the loss of fluorescence signal that occurs when fluorescently-tagged Tf ligand is recycled to the cell surface and dissociates. Other methods directly measure the amount of ligand that is recycled. In our case, recycled 125I-Tf ligand that has dissociated from the TfR is recovered in the medium bathing the basolateral surface of the cells (Gallo et al., 2014; Leung et al., 1998, 1999; Maples et al., 1997; Rojas et al., 2001). Importantly, this method is highly reproducible and the amount of recycled 125I-Tf is readily quantified by direct counting of the samples in a gamma-counter. A shortfall of the biotinylation approach described in the preceding paragraph is that it only measures cell-surface receptors that have recycled at the instant that the cells are cooled down. As a result, it is likely to underestimate the amount of recycling. However, if the biotin approach is coupled with cell-surface immunoprecipitation or proteolysis (performed by adding antibodies or proteinases to the culture medium at 18 C or even 37 C), it can provide a more integrated estimate of the amount of recycling. Other techniques that employ indirect labeling of the receptor by binding of labeled antibodies or Fab fragments can also underestimate the amount of recycling. This is because these reagents may not dissociate completely from the receptor. Again, cell-surface immunoprecipitation or proteolysis may obviate such concerns, or use of low pH treatments. In the case of the TfR, this is less of a concern as 125I-apo-Tf rapidly dissociates from its receptor as soon as the recycling receptor encounters the pH 7.4 medium. While there is no one method that will work for all receptors, iodinated Tf ligand is an ideal way to measure recycling of the TfR.

2. MATERIALS AND INSTRUMENTS Unless otherwise specified, all reagents are from SigmaeAldrich (St. Louis, MO).

2.1 REAGENTS TO IRON LOAD CANINE apo-Tf •

Canine apo-Tf powder (T6011dunfortunately, Sigma has recently discontinued this product and will no longer carry this product when all current stock is sold). An alternative is to use MDCK cells expressing the human TfR and then use human apo-Tf. However, as noted above, overexpression of the TfR can result in receptor missorting. • 250 mM Tris base titrated to pH 8.0 using HCl.

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• 10 mM sodium bicarbonate (freshly prepared in H2O). • Fe/NTA solution: 100 mM nitrilotriacetic acid and 12 mM FeCl3$6H2O dissolved in H2O and titrated to pH 7.5 with 10 N NaOH. Store aliquots at 20 C • Phosphate-buffered saline (10X stock ¼ 1.37 M NaCl, 27 mM KCl, 80.6 mM Na2HPO4, 14.7 mM KH2PO4 prepared in H2O). Store at room temperature. When diluted to 1X, the resulting solution will have a pH of 7.4. • PD10 (Sephadex G-25) disposable desalting column (GE Healthcare Life Sciences, product number 17-0851-01). • 1.5 mL Eppendorf (microcentrifuge) tubes. • Centrifugation concentrator with 30,000-molecular weight cutoff. Although we traditionally used Centricon products from Amicon, they are now discontinued. Thermo Scientific now offers a similar product (#88502).

2.2 REAGENTS TO IODINATE CANINE holo-Tf • • • • • •

• • • •

Zeba 2-mL spin desalting column with 7000-molecular weight cutoff (Thermo Scientific #89889). 1X PBS. PBS/bovine serum albumin (BSA): 1% (w/v) BSA dissolved in 1X PBS. Prepare fresh. 1 M Tris base titrated to pH 8.0 using HCl. Stored at room temperature. 2 M NaCl prepared fresh in H2O. ICl: N.B. This reagent must be prepared as described previously (Oztan, Rondanino, & Apodaca, 2008), and in our experience cannot be substituted with ICl powder purchased from chemical supply houses such as SigmaeAldrich. The preparation of ICl should be carried out in a fume hood. Because ICl is a corrosive chemical, gloves and a lab coat should be worn during preparation. Dissolve 0.15 g solid NaI in 8 mL 6 N HCl. Then, forcibly inject 2 mL NaIO3 (49.5 mg/mL in H2O) into the NaI solution using a 5-mL syringe fitted with a 20-gauge needle. Bring the solution up to 40 mL with water and place in a clean separatory funnel. Add 10 mL CCl4 to the mixture and shake vigorously. CCl4 is a carcinogen: avoid breathing vapors or skin contact. Discard the lower pink organic phase and repeat the extraction with CCl4 until the organic phase is colorless. Transfer the extracted aqueous layer to a clean Erlenmeyer flask and aerate with moist air for 8e10 h to remove any trace of CCl4 from the aqueous phase (see Figure 2 in Oztan et al. (2008)). Prepare 0.5-mL aliquots and store away from light at 20 C. The solution is stable for several years at 20 C. 125 I radionuclide (10 mCi, specific activity 17.4 Ci/mg; Perkin Elmer, Boston, MA, product number NEZ033010MC). NaI/PBS/BSA: Dissolve 5 mg/mL of NaI in PBS/BSA. Can be aliquoted and stored at e20 C. 1.5-mL self-standing, screw-cap microcentrifuge tube (USA Scientific, Ocala, FL, product number 1415e9704). Iron-loaded Tf.


FIGURE 2 Culture of polarized MDCK cells. (A) MDCK cells cultured on 10-cm dish. (B) MDCK cells cultured on 12-mm Transwell. (C) Formation of a polarized epithelial sheet is confirmed by labeling the cell nuclei with ToPro-3 (green) and an antibody that recognizes the tight junction-associated protein ZO-1. The upper panel is a projection of a confocal Z-series. The bottom panel shows an XeZ section taken from the same stack. Bar ¼ 10 mm. (See color plate)

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2.3 REAGENTS TO CULTURE MDCK CELLS • •

• • • • • • •

MDCK type II cells (we use the EMBO strain, obtained from Dr Kai Simons, MPI-CBG, Germany). Complete minimum essential medium (MEM): The MEM with Earle’s salts and Ò L-glutamine (MEM; Corning, Cellgro , Mediatech, Manassas, VA, product number 10-010-CV) supplemented with 10% fetal bovine serum (FBS; Thermo Scientific; HyClone, Logan, UT, product number SH30071.03) and 1% antibioticeantimycotic (100 U/mL penicillin G sodium, 100 mg/mL streptomycin sulfate, and 250 ng/mL amphotericin B as FungizoneÒ; Thermo Scientific, Hyclone, product number SV30079.01). Sterile PBS (Corning, Cellgro, product ID 21-040-CV). Trypsin/ethylenediaminetetraacetic acid (EDTA): 0.25% (w/v) trypsin with 1 mM EDTA (Corning, Cellgro, product number 25-053-CI). Freezing medium: 10% (v/v) dimethyl sulfoxide (DMSO) and 90% (v/v) FBS. 70% (v/v) ethanol. 10-cm tissue culture dishes (100 10 mm, polystyrene, non-pyrogenic dishes, Corning, Corning, NY, product number 353003). Freezing vials (1.8 mL cryotube vials, Thermo Scientific, product number 125542). TranswellÒ permeable supports, 12-mm diameter, 0.4-mm pore size (Corning, Corning, NY, product number 3401).

2.4 REAGENTS TO MEASURE •

• • • • • •

125

I-Tf RECYCLING

MEM/BSA: Mix 20 mL of 1 M HEPES-NaOH, pH 7.6 and 10 mL antibiotice antimycotic in 900 mL of water. To this solution, add 10.7 g of MEM powder (containing Hanks’ salts, L-glutamine, and nonessential amino acids, but lacking NaHCO3, Sigma product number M1018), 0.35 g NaHCO3 and 24 g BSA. Adjust to pH 7.4 (using either HCl or NaOH), bring up to 1 L with H2O, and store at 20 C in clean, autoclaved bottles. Thaw prior to use. It is important to swirl the bottles to disperse the components after thawing the medium. Thawed MEM/BSA is stable at 4 C for about 1 week. Tissue culture plates: 12-well, flat-bottom FalconÒ (Corning). 125 I-Tf stored at 4 C in a leaded pig. It is stable for about 1 month. Collection tubes: 1.2-mL polypropylene microdilution tubes with conical bottom (USA Scientific, Ocala, FL). PBSþ: In 100 mL ice-cold 10X PBS, add 0.1 g anhydrous CaCl2 and 0.1 g MgCl2$6H2O. Bring up to 1 L with ice-cold water and stir to dissolve. Store at 4 C. Kimwipes. Fisherbrand cork (size 00, Fisher Scientific, catalog number 07782B).

2.5 REAGENTS TO PERFORM TRICHOLORACETIC ACID PRECIPITATION •

TCA: 100% (w/v) in water. TCA is very caustic; handle with gloves in a ventilated enclosure. Store at 4 C.

3. Methods

• •

75-mm polypropylene test tubes and 12-mm cap, rib plug type (Fisher Scientific, Pittsburgh, PA, catalog number 14-377-843). Fisherbrand cork.

2.6 EQUIPMENT • • • • • • • • • • •

• •

pH meter, such as SevenMulti pH Meter (Mettler, Toledo). Centrifuges, including microfuge and low-speed centrifuge. Lead foil and/or lead-impregnated Plexiglass shield (Fisher Scientific). Lead apron (Fisher Scientific). Goggles/spectacles (Fisher Scientific). Latex gloves. Geiger counter with scintillation probe (Ludlum Measurements Inc., Sweetwater, TX). Gamma-counter, such as Cobra II auto-gamma-counting system, Packarde Perkin Elmer, Boston, MA. 20 and 70 C freezers. Tissue culture incubator. Circulating water bath: We use a 30.5 50.8 21.6 cm (width length height) bath with temperature regulator/circulator (VWR, West Chester, PA). A 22.5 26 cm aluminum plate is placed on top of test tube racks, and the water in the bath is adjusted so that the aluminum plate is covered by about 5 mm H2O. Timer. Pipettors (10, 200, and 1000 mL capacity) with appropriate tips.

3. METHODS 3.1 LOADING apo-Tf WITH IRON Our experience is that commercial preparations of holo-Tf do not work as well as preparations that we have loaded. Regardless of species, we load apo-Tf with Fe3þ using the following technique. Add 1 mL of 10 mM NaHCO3 to 1 mL of 250 mM TriseHCl, pH 8.0 buffer. In 400 mL of this buffer, dissolve apo-Tf to give a final concentration of w5e6 mg/mL. To this solution, add 8 mL of Fe/NTA solution and incubate for 30 min at 37 C. This solution will turn a light salmon/pink color during this incubation. The loaded holo-Tf must next be separated from uncoupled Fe3þ, a feat that is accomplished by loading the reaction on top of a 2-mL PD10 desalting column (Sephadex G-25 or equivalent) that has been equilibrated with 10 column volumes of 1X PBS. Using 1X PBS as an eluent, collect 0.5 mL fractions in 1.5-mL Eppendorf tubes. Those tubes containing salmon/pink color (and have a high OD280 and OD470) are collected. The percentage of iron loading can be estimated by measuring the solutions’ OD470 and then calculating the concentration of Tf using its extinction coefficient of 87,200 L/mol$cm. This value can then be

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compared to the known starting concentration (the OD280 of Tf does not appreciably change during iron loading). Because it is important that the concentration of holoTf be high during the iodination step, we typically pool the fractions containing holo-Tf and concentrate the sample to 5e6 mg/mL using a centrifugation concentrator with a 30,000 molecular weight cutoff. The holo-Tf can be stored in small aliquots at 70 C for years.

3.2 IODINATING holo-Tf While there are several different techniques to iodinate ligands, we routinely use the ICl method, as the 125I is oxidized prior to addition of the ligand (Helmkamp, Goodland, Bale, Spar, & Mutschler, 1960; McFarlane, 1958). We find that this technique works well for most proteins, including low-density lipoprotein particles, which are highly sensitive to oxidation. Remove the top and bottom caps of Zeba 2-mL desalting column, and then secure the column in a vise clamp. While these columns are nominally spin columns, we typically allow them to flow by gravity. The column is equilibrated with four column volumes of PBS/BSA. At the end of the last wash, allow the buffer to almost completely enter the column, and then cap off the column to prevent it from drying out. Place 50e100 mg holo-Tf in a tube labeled “ligand” and store in an ice container in a fume hood designated for iodination use. The total volume of ligand should not exceed 10e20 mL for an efficient iodination reaction. Add 100 mL TriseHCl (pH 8.0) to a tube labeled “reaction” and store this in the ice container as well. Prepare a 1:50 dilution of ICl reagent in 2 M NaCl and then add 20 mL of this mixture to the “reaction” tube containing the Tris buffer. The contents should be mixed by trituration and then kept on ice. At this point the investigator should wear a protective lab coat and lead apron, eyewear (e.g., goggles), and a double pair of disposable gloves, which should be changed often. A Geiger counter with scintillation probe is useful for monitoring the dose of radiation and potential contamination. Lead foil or lead-impregnated Plexiglass can be used to further shield the investigator from the 125I. Add 4 mL sodium 125I (w1.4 mCi) to the “reaction” tube, mix by trituration, and incubate the reaction for 1 min on ice. The 125I is oxidized during this reaction. At the end of the 1-min incubation, transfer the holo-Tf ligand to the “reaction” tube. Mix gently by trituration and incubate for 10 min on ice. At the end of the 10-min incubation, stop the reaction by adding 100 mL NaI/PBS/BSA to the “reaction” tube. Carefully transfer the iodination mixture from the “reaction” tube and place on the surface of the washed desalting column. Once the iodination mixture has entered the column, wash the column by adding 300 mL PBS/BSA to the column bed. Collect any eluent in a tube labeled “waste.” This initial eluent usually contains little iodinated ligand (this can be confirmed with the Geiger counter) and can be discarded. Replace the “waste” tube with the screw-cap tube (with cap removed) and collect the 125I-Tf by eluting the column with an additional 600 mL PBS/BSA. Gently mix the tube containing 125I-Tf to disperse the ligand equally in the eluent

3. Methods

and then count a 1-mL aliquot in a gamma-counter. A typical reaction contains 300,000 to 500,000 cpm 125I-Tf/mL. Store the iodinated ligand, in a lead pig, at 4 C. The ligand is typically stable for up to 4 weeks, but rapidly loses function afterward.

3.3 CULTURING MDCK CELLS ON 12-MM TRANSWELLS In our studies, we use MDCK type II cells, which are maintained in complete MEM in a 37 C tissue culture incubator gassed with 5% CO2/95% air. Cell culture is performed as follows (Oztan et al., 2008). We routinely grow the cells in 10-cm diameter tissue culture-treated dishes, but flasks are an acceptable alternative (Figure 2(A)). To passage the cells, the medium is aspirated from a confluent 10-cm dish of cells, and the cells are washed with 10 mL warm (37 C) sterile PBS (which lacks Ca2þ or Mg2þ). The PBS is aspirated, 1 mL of trypsin/EDTA is added, and the cells are incubated at 37 C in the tissue culture incubator until the cells detach from the plate. It is important to not over-trypsinize the cells, or they will die. When the cells detach from the plate, 5 mL complete MEM is added to the dish, and the cells are resuspended by trituration using a 5-mL pipet until there are no visible cell clumps remaining. The cell suspension is centrifuged at 100 g for 5 min a room temperature to remove the trypsin-containing medium, and then resuspended in 10 mL of complete medium. For routine passaging, 1 mL of cell suspension is added to a 10-cm dish containing 9 mL complete MEM (1:10 dilution). The cells take about 4e5 days to reach confluence. To freeze cells, a confluent dish of MDCK cells is trypsinized, the cells are resuspended in 5 mL complete MEM, and the cells are then recovered by centrifugation at 100 g for 5 min at room temperature. Following aspiration of the medium, the cells are carefully resuspended by triturition in 1.5 mL warm (37 C) freezing medium. Aliquots (0.5 mL) of the cell suspension are placed in freezing vials, capped, and then placed in a room temperature Styrofoam container. The Styrofoam container is placed in a 70 C freezer. The cells are stable at 70 C for some time but can be transferred after 2e3 days to liquid nitrogen for extended storage. Cells are thawed by partially immersing the cell vial containing the frozen cells in a 37 C water bath. The cell vial is removed from the water bath, dried, rinsed with ethanol, and the cap removed in a cell culture hood. The thawed cell suspension is transferred to a 10-cm dish, and 10 mL complete MEM is slowly added to the cells. To minimize osmotic shock, the first 2 mL of MEM should be added at a rate of about 1 mL/min. The next day, the medium is aspirated and replaced with 10 mL complete MEM. The cells typically achieve confluence by the 2nd day of thawing. We generally thaw new cells every 1e2 weeks. The following is a protocol for plating the MDCK cells on 12-mm Transwells (Figure 2(B)). A confluent dish of MDCK cells is trypsinized, and the cells are resuspended in 5 mL complete MEM. The cells are then recovered by centrifugation at 100 g for 5 min at room temperature. The trypsin-containing medium is aspirated, and the cell pellet is resuspended in 12 mL complete MEM. Next, place 0.5 mL cell

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suspension (containing w1 106 cells) into the apical chamber of each 12-mm Transwell. Add 1 mL complete MEM to the well that faces the basolateral surface of the Transwell unit. The cells typically form a tight, polarized monolayer by 3 day post-plating and can be used at this time (Figure 2(C)). After the 3rd day, the cells are fed daily by aspirating the basolateral, then apical medium and adding 0.5 mL complete MEM to the apical chamber and then 1.5 mL medium to the basolateral chamber. The order of medium aspiration and addition is important to prevent hydrostatic pressure-induced cell lifting.

3.4 EXPERIMENTAL STRATEGY TO MEASURE RECEPTOR RECYCLING 3.4.1 Background and objective The goal of the following protocol is to show how to measure basolateral recycling of the TfR in polarized MDCK cells. As described above, this requires a technique to label the receptor, a protocol to allow for internalization of the receptor, and a method to follow recycling of the receptor back to the cell surface. Furthermore, this method can be used to simultaneously measure if the receptoreligand pair is undergoing transcytosis or degradation. An increase in transcytotic delivery can indicate a treatment or condition that alters polarized sorting of the TfR. We also note that under most conditions, little ligand-bound TfR is degraded (w1e3%). However, we have found that some treatments can cause missorting of some receptors to the degradation pathway (e.g., see Maples et al. (1997)). The product of receptor degradation is small peptides that are released from the pole of the cell from which they were internalized. Thus, it is prudent to precipitate the nominally recycled (and transcytosed) ligand with trichloroacetic acid (TCA), which allows one to differentiate between intact, recycled 125I-ligand versus that which was degraded. Following centrifugation, intact 125I-ligand is found in the pellet, while degraded 125I-ligand-derived peptides are found in the supernatant.

3.4.2 Flow of experiment 3.4.2.1 Labeling cohort of TfR with 125I-Tf, and ensuring their internalization Prior to performing the recycling assay one needs to internalize a cohort of 125I-Tf into the cells. The Transwell-grown MDCK cells are first washed with warm (37 C) MEM/BSA two times to remove the cell culture medium. Washing is accomplished by transferring the Transwells to a 12-well dish, aspirating the medium (basolateral first, then apical), and then adding 1 mL MEM/BSA to the apical and then basolateral chambers of the Transwell. After the second wash, 1 mL of MEM/BSA is added to each chamber and the cells are incubated at 37 C for 90 min to allow for recycling of previously internalized Tf. Just prior to the end of this incubation, prepare a humid labeling chamber by placing a piece of moistened filter paper in a storage container with a removable lid. A sheet of Parafilm is cut and placed on top of the moistened filter paper. Wear appropriate safety gear (disposable gloves and lab coat). For each filter, dilute 3e4 mL 125I-Tf in a total of 30 mL MEM/BSA

3. Methods

and place the drop of diluted 125I-Tf on the sheet of Parafilm, avoiding air bubbles. Close the chamber and place it in the water bath to keep it at 18e37 C. Lead foil can be placed over the labeling chamber to shield the investigator during this incubation. After the cells are incubated for 90-min in MEM/BSA for depleting Tf, the medium is aspirated, and 300 mL MEM/BSA are added to the apical chamber of the Transwell. A Kimwipe is used to carefully remove any MEM/BSA adhering to the rim of plastic surrounding the porous filter on the basolateral side of the Transwell. The Transwell filter is placed on the 30-mL drop of 125I-Tf in the humid labeling chamber and pulse labeled (e.g., for 120 min at 18 C or 45 min at 37 C). Lead foil can be placed over the labeling chamber to shield the investigator during this incubation. Unbound ligand is removed by washing. Return the Transwell filters to a 12-well plate and add 1 mL ice-cold MEM/BSA to the basolateral surface of each Transwell filter. Remove the basolateral medium by aspiration. Wash the basolateral surface an additional two times, and then perform three more washes, three times 10 min each, using ice-cold MEM/BSA. Discard the MEM/BSA wash liquid generated in these and subsequent steps in suitable radioactive waste disposal containers.

3.4.2.2 Removal of cell-surface bound 125I-Tf Aspirate any remaining apical and basolateral media and then add 1 mL of prewarmed, 37 C MEM/BSA to the apical and then basolateral chamber of each Transwell. Place the 12-well plate containing Transwells in a 37 C water bath (Figure 3(A)). Incubate the cells for 2.5 min to allow for nonspecifically-bound ligand to dissociate from the filter and for cell-surface ligand to be internalized and “chased” into the cells. During the chase, carefully discard the sheet of Parafilm containing the 125I-Tf. If required, the filters can be left at 4 C in MEM/BSA until one is ready for the next step.

3.4.2.3 Perform recycling assay This assay is similar to that we have previously described to measure transcytosis (Oztan et al., 2008). Label a total of 11 1.2-mL collection tubes for each Transwell filter: five are used for apical medium collected at 7.5, 15, 30, 60, and 120 min; five are used for basolateral medium collected at 7.5, 15, 30, 60, and 120 min; and one is for the filter, which is cut out at the end of the experiment. Place 500 mL of prewarmed, 37 C MEM/BSA into the wells of a 12-well plate. You will need five wells of MEM/BSA for each individual Transwell filter (one for each of five time-points). We typically perform each experimental treatment group in triplicate or quadruplicate. If performing multiple treatment groups, it is easier to use a separate 12-well plate for each time-point. Place the 12-well plates containing MEM/BSA atop the aluminum plate in the water bath to keep them warm before sample collection starts. When ready, aspirate the medium from both sides of the cells in step 3.4.2.2 and add 500 mL warm, 37 C MEM/BSA to the apical chamber. Place the filters in the 12-well plates containing MEM/BSA. Start a timer as the filters are placed in the wells of MEM/BSA. After 7.5 min, begin collecting samples. Lift an individual

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FIGURE 3 Assay to measure recycling of 125I-Tf. (A) Circulating water bath containing elevated aluminum plate and 12-well dish containing filter-grown MDCK cells labeled with 125I-Tf. (B) Sampling the apical medium of MDCK cells pulse-labeled with 125I-Tf. (C) Apical medium, containing 125I-Tf, is transferred into collection tube. (See color plate)

Transwell from the 12-well plate, quickly recover the apical medium using a 1-mL pipettor fitted with the appropriate tip (taking care not to puncture the Transwell filter) (Figure 3(B)), and transfer the contents to the labeled 1.2-mL collection tubes prepared above (these are the 7.5-min apical samples) (Figure 3(C)). Eject the tip and use a new tip to gently transfer 500 mL MEM/BSA to the apical chamber of the Transwell. Place the same Transwell in the next well containing 500 mL MEM. Repeat the process for all Transwells in the experiment. After all of the Transwells are transferred to new wells, collect the MEM/BSA from the basal wells of the previous 7.5-min time-point and place in the 1.2-mL collection tubes (these

3. Methods

are the 7.5-min basolateral samples). Repeat the collection process at 15, 30, 60, and 120 min. At the end of 120-min chase period, you will have five apical samples (7.5, 15, 30, 60, and 120 min) and five basolateral samples (7.5, 15, 30, 60, and 120 min) for each Transwell filter. Finally, wash the filter and associated cells with ice-cold PBSþ two times to remove MEM/BSA, cut the filter from its holder using a scalpel, and place the filter into a 1.2-mL counting tube (this is the cell-associated Tf fraction for each sample). At this point, you can begin counting the samples in the gammacounter, or if you are going to determine the amount of degradation, then continue to the next step.

3.4.2.4 TCA precipitation Place the collection tubes containing medium on ice. Add 50 mL TCA to each tube. Vortex each tube for 15 s to thoroughly mix the sample. As noted above, TCA will precipitate intact 125I-Tf ligand but not degraded fragments of 125I-Tf released from the cell into the culture medium. Incubate samples on ice for 30e45 min and then centrifuge the samples in a microfuge at 14,000 g for 10 min at 4 C. The collection tubes are placed in a rack, and the supernatant is carefully removed using a 3-mL syringe fitted with a 22-gauge needle. The pellets are retained as these contain the undegraded 125I-Tf. For quantifying the total amount of degraded 125I-Tf, pool all of the basolateral and apical supernatants for each Transwell in w75 mm polypropylene test tubes. If the aim is to measure the kinetics of degradation, then place the supernatant of each apical and basolateral time-points into separate collection tubes. Cap all collection tubes (containing pellets, supernatants, or filter) with a cork stopper. Place the samples in a gamma-counter and count the 125I associated with the filter (cell-associated Tf), apical TCA pellets (transcytosed Tf), basolateral TCA pellets (recycled Tf), and the TCA supernatants of the apical and basolateral samples (degraded Tf).

3.4.2.5 Data analysis 1. For each Transwell, sum the total cpm associated with the filter, apical and basolateral TCA pellets, and the apical and basolateral TCA supernatants. This is the total ligand that was internalized during the pulseechase labeling protocol. In Figure 4, we show data for a single experiment performed in triplicate. The total amount of internalized 125I-Tf was on average 3575 cpm. 2. For each Transwell, calculate the percentage of total ligand associated with the filter, the apical and basolateral TCA pellets for each time-point, and the pooled apical and basolateral TCA supernatants (Figure 4). N.B. If examining the kinetics of degradation, calculate the individual percentages associated with the TCA supernatant for each time-point. 3. Calculate a running sum of percentages for the apical TCA pellets (transcytosis) and then for the basolateral TCA pellets (recycling) (Figure 4). If examining the kinetics of degradation, also calculate the running sum of the apical and basolateral TCA supernatants.

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FIGURE 4 Data from three filters showing cpm of 125I-Tf released into the apical medium following TCA precipitation (apical pellet ¼ ligand that was transcytosed), 125I-Tf released into the basolateral medium following TCA precipitation (basolateral pellet ¼ ligand that was recycled), 125I-Tf remaining in the cell after the 120-min chase, and 125I-Tf found in the pooled supernatant following TCA precipitation (TCA supernatant ¼ ligand that was degraded). Average data (mean SD) are shown in the rightmost columns.

4. Calculate the averages and standard deviation for each experimental treatment and plot the data using a graphing program such as Excel or Prism. 5. After performing several separate experiments one can calculate the average mean value and the standard error of the mean, again using Excel or Prism. It is worth noting that Tf recycling fits a single exponential function, thus it is possible to provide descriptive statistics of the rate, time constant, and half-time for completeness (see, e.g., Figure 5).

3.4.2.6 Considerations During the iodination protocol Na125I is oxidized to 125I2 (molecular iodine) in preparation for the iodination reaction. This step is time sensitive and should be accurately timed. In a typical reaction, more than 99% of the recovered iodinated products are TCA precipitable, indicating that there is little free iodine in the final eluate. This test can be performed by adding 1 mL of the column eluate to 0.5 mL MEM/BSA, adding TCA to 10% (v/v), incubating for 30 min on ice, centrifuging for 15 min at 4 C, and counting the resulting supernatant and pellet. If the reaction is less than 100,000e200,000 cpm/mL, it is usually considered poor and is discarded. There are several considerations when performing MDCK cell culture. Trypsinization usually takes 10e30 min depending on how long the cells have been cultured on the dish. An easy way to test whether the trypsinization reaction is complete is to rock the dish slowly back and forth. When the cells are trypsinized, they readily come off the dish and enter the moving fluid phase. Do not over-trypsinize the cells

3. Methods

FIGURE 5 Endocytic fate of 125I-Tf in polarized MDCK cells. (A) The average data, obtained from the amount of 125I-Tf that was recycled or transcytosed in Figure 4, are plotted versus time. The line shows the best fit of the data to a single exponential curve. (B) MDCK cells were incubated in serum-free medium for 4 h, and then pulse-labeled with unlabeled Tf (25 mg/mL, using the same protocol described in this chapter). At t ¼ 0, 15, or 120 min, the cells were fixed and then immunolabeled with a polyclonal anti-Tf antibody and a rat monoclonal antibody against ZO-1. Upper panel: Single XeY confocal section at the level of the tight junction showing Tf in CRE. Bottom panel: XeZ section through entire cell layer. (See color plate)

as this can lead to cell death. When passaging cells the amount of cell dilution affects the time it takes for the cells to become confluent. At a dilution of 1:2, the cells will become confluent overnight. When diluted 1:5, the cells take about 3 days to reach confluence. At 1:20, the cells take about 7e8 days to reach confluence. After trypsinization and centrifugation of the cells, it is important to make sure the cell pellet is completely resuspended to ensure proper monolayer formation. Improper resuspension, or plating too many cells, results in cells that adhere to the apical surface of the monolayer or stratification. The formation of a tight monolayer can be monitored by measuring the transepithelial resistance across the monolayer using an EVOM meter (Warner Instruments, Hamden, CT). A Transwell lacking cells but otherwise incubated in complete MEM medium is used to correct for series resistance. Although there is some variation between clones, type II MDCK cells typically achieve a transepithelial resistance of 110e150 U cm2 once they are polarized and have formed a tight monolayer.

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During the pulse-labeling step, the recycling assays, and the TCA precipitation step (if employed) it is important to note the following. Avoid air bubbles during the pulse-labeling step. This can be accomplished by first touching the edge of the Transwell near the edge of the drop, and then slowly releasing the Transwell onto the globule of ligand. During the recycling assay, it is important to be rhythmic when collecting the samples. A useful strategy is to stagger the collection of each sample by about 10e15 s. Because degraded, iodinated peptides are membrane permeable and diffuse from the cell into the MEM/BSA, we find that the majority of cell-associated ligand remaining in the filter at the end of the experiment is TCA precipitable. As such, we do not routinely perform TCA precipitation of the filter-associated cells. Use of radioactivity, and 125I in particular, can initially cause some anxiety to the investigator. However, if appropriate precautions are taken, which are described above, then it is very rare to observe any uptake of 125I into one’s thyroid (the major site of uptake in the body) after performing these procedures. And, any uptake we have experienced has been limited to very small doses that are only a few counts above background. Still, every attempt should be made to avoid unnecessary exposure to this radioactive nuclide.

3.4.2.7 Results In this experiment we observe the following: 1. The majority of basolaterally internalized 125I-Tf (w84%) is rapidly recycled to the basolateral cell surface with a t1/2 ¼ 10 min (Figure 5). 2. A small fraction of basolaterally internalized 125I-Tf (w5%) is released from the apical pole of the cell (transcytosed), with a t1/2 ¼ 9 min (Figure 5). 3. The amount of ligand degraded is also small (w4%), and the balance remaining in the cell after a 120-min incubation is w7% (Figure 4).

SUMMARY Finally, while there are many techniques that can be used to label a receptor and follow its recycling, we present methods that allow the investigator to follow receptor recycling using an iodinated ligand. While there can be shortfalls with this approach (Butt, 1984), the methods we describe have several benefits over other methods: (1) Perhaps the chief benefit is that the investigator in one experiment can quantify the amount of ligand uptake, and the degree to which it is degraded, transcytosed, and recycled. (2) Using the ICl protocol it is possible to obtain ligands that retain their native structure and biological activity. (3) The iodinated ligand can be used at low physiological concentrations. (4) The iodinated ligand is easily detectable, as gamma-counting is highly efficient. (5) The assay described is highly reproducible and can be easily repeated. (6) The iodinated ligand has a reasonable

References

lifetime of w30 days at 4 C, and the relatively short half-life of 125I (w60 days) means that there is no build-up of radioactive material in the long term.

REFERENCES Apodaca, G., & Gallo, L. I. (2013). Epithelial polarity. In I. R. Nabi (Ed.), Colloquim series on building blocks of the cell: Cell structure and function (pp. 1e4). Morgan & Claypool Life Sciences. Apodaca, G., Gallo, L. I., & Bryant, D. M. (2012). Role of membrane traffic in the generation of epithelial cell asymmetry. Nature Cell Biology, 14(12), 1235e1243. http://dx.doi.org/ 10.1038/ncb2635. Apodaca, G., Katz, L. A., & Mostov, K. E. (1994). Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes. Journal of Cell Biology, 125, 67e86. Butt, W. R. (1984). Problems of iodination. In W. R. Butt (Ed.), Practical immunoassay: The state of the art (Vol. 14, pp. 19e33). New York, N.Y: Marcel Dekker, Inc. Butterworth, M. B., Edinger, R. S., Silvis, M. R., Gallo, L. I., Liang, X., Apodaca, G., et al. (2012). Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC). American Journal of Physiology e Renal Physiology, 302(5), F581eF590. http://dx.doi.org/10.1152/ajprenal.00304.2011. Cheng, Y., Zak, O., Aisen, P., Harrison, S. C., & Walz, T. (2004). Structure of the human transferrin receptor-transferrin complex. Cell, 116(4), 565e576. Dunn, K. W., McGraw, T. E., & Maxfield, F. R. (1989). Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome. Journal of Cell Biology, 109, 3303e3314. Elferink, L. A., & Strick, D. J. (2005). Functional properties of rab15 effector protein in endocytic recycling. Methods in Enzymology, 403, 732e743. http://dx.doi.org/10.1016/S00766879(05)03064-8. Frazer, D. M., & Anderson, G. J. (2014). The regulation of iron transport. Biofactors, 40(2), 206e214. http://dx.doi.org/10.1002/biof.1148. Fuller, S. D., & Simons, K. (1986). Transferrin receptor polarity and recycling accuracy in “tight” and “leaky” strains of MadineDarby canine kidney cells. Journal of Cell Biology, 103, 1767e1779. Gallo, L. I., Liao, Y., Ruiz, W. G., Clayton, D. R., Li, M., Liu, Y. J., et al. (2014). TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells. Molecular Biology of the Cell. http://dx.doi.org/10.1091/mbc.E13-10-0604. Gautier, A., Juillerat, A., Heinis, C., Correa, I. R., Jr., Kindermann, M., Beaufils, F., et al. (2008). An engineered protein tag for multiprotein labeling in living cells. Chemistry & Biology, 15(2), 128e136. http://dx.doi.org/10.1016/j.chembiol.2008.01.007. Ghosh, R. N., Gelman, D. L., & Maxfield, F. R. (1994). Quantification of low density lipoprotein and transferrin endocytic sorting HEp2 cells using confocal microscopy. Journal of Cell Science, 107(Pt 8), 2177e2189. Gottardi, C. J., Dunbar, L. A., & Caplan, M. J. (1995). Biotinylation and assessment of membrane polarity: caveats and methodological concerns. American Journal of Physiology, 268(2 Pt 2), F285eF295. Hannan, S., Wilkins, M. E., Thomas, P., & Smart, T. G. (2013). Tracking cell surface mobility of GPCRs using alpha-bungarotoxin-linked fluorophores. Methods in Enzymology, 521, 109e129. http://dx.doi.org/10.1016/B978-0-12-391862-8.00006-5.

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Helmkamp, R. W., Goodland, R. L., Bale, W. F., Spar, I. L., & Mutschler, L. E. (1960). High specific activity iodination of gamma-globulin with iodine-131 monochloride. Cancer Research, 20, 1495e1500. Leung, S.-M., Deng, D., DasGupta, B., Whiteheart, S. W., & Apodaca, G. (1998). SNAP-23 requirement for endocytic traffic in streptolysin-o permeabilized MadineDarby canine kidney cells. Journal of Biological Chemistry, 273, 17732e17741. Leung, S.-M., Rojas, R., Maples, C., Flynn, C., Ruiz, W. G., Jou, T.-S., et al. (1999). Modulation of endocytic traffic in polarized MDCK cells by the small GTPase RhoA. Molecular Biology of the Cell, 10, 4369e4384. Lisanti, M. P., LeBivic, A., Sargiacomo, M., & Rodriguez-Boulan, E. (1989). Steady-state distribution and biogenesis of endogenous MadineDarby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery. Journal of Cell Biology, 109, 2117e2127. Maples, C. J., Ruiz, W. G., & Apodaca, G. (1997). Both microtubules and actin filaments are required for efficient postendocytic traffic of the polymeric immunoglobulin receptor in polarized MadineDarby canine kidney cells. Journal of Biological Chemistry, 272, 6741e6751. Matter, K., Stieger, B., Klumperman, J., Ginsel, L., & Hauri, H. P. (1990). Endocytosis, recycling, and lysosomal delivery of brush border hydrolases in cultured human intestinal epithelial cells (Caco-2). Journal of Biological Chemistry, 265(6), 3503e3512. McFarlane, A. (1958). Efficient trace-labelling of proteins with iodine. Nature, 182(4627), 53. Odorizzi, G., Pearse, A., Domingo, D., Trowbridge, I. S., & Hopkins, C. R. (1996). Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism. Journal of Cell Biology, 135(1), 139e152. Oztan, A., Rondanino, C., & Apodaca, G. (2008). Transcytosis of polymeric immunoglobulin a in polarized MadineDarby canine kidney cells. Methods in Molecular Biology, 440, 157e170. http://dx.doi.org/10.1007/978-1-59745-178-9_12. Perez Bay, A. E., Schreiner, R., Benedicto, I., & Rodriguez-Boulan, E. J. (2014). Galectin4-mediated transcytosis of transferrin receptor. Journal of Cell Science, 127(Pt 20), 4457e4469. http://dx.doi.org/10.1242/jcs.153437. Roberts, S., & Bomford, A. (1988). Chelation of transferrin iron by desferrioxamine in K562 cells. The partition of iron between ferrioxamine and ferritin. Biochemical Journal, 254(3), 869e875. Rojas, R., Ruiz, W. G., Leung, S. M., Jou, T. S., & Apodaca, G. (2001). Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized MDCK cells. Molecular Biology of the Cell, 12, 2257e2274. Sargiacomo, M., Lisanti, M., Graeve, L., Le Bivic, A., & Rodriguez-Boulan, E. (1989). Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells. Journal of Membrane Biology, 107(3), 277e286. Shah, M., Baterina, O. Y., Jr., Taupin, V., & Farquhar, M. G. (2013). ARH directs megalin to the endocytic recycling compartment to regulate its proteolysis and gene expression. Journal of Cell Biology, 202(1), 113e127. http://dx.doi.org/10.1083/jcb.201211110. Sheff, D. R., Daro, E. A., Hull, M., & Mellman, I. (1999). The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions. Journal of Cell Biology, 145, 123e139. Sheff, D. R., Kroschewski, R., & Mellman, I. (2002). Actin dependence of polarized receptor recycling in MadineDarby canine kidney cell endosomes. Molecular Biology of the Cell, 13(1), 262e275. http://dx.doi.org/10.1091/mbc.01-07-0320.

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Structural and functional analysis of endosomal compartments in epithelial cells

17

Andres E. Perez Bay, Ryan Schreiner, Enrique Rodriguez-Boulan1 Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY, USA 1


CHAPTER OUTLINE Introduction ............................................................................................................ 272 1. Objectives and Rationale .................................................................................... 275 2. Experimental Protocols....................................................................................... 275 2.1 Microscopy-Based Protocols for Endosomal Labeling in Polarized Epithelial Cells ................................................................................... 275 2.1.1 Apical sorting endosomes ................................................................. 276 2.1.2 Basolateral sorting endosomes.......................................................... 278 2.1.3 Common recycling endosomes ......................................................... 279 2.1.4 Apical recycling endosome ............................................................... 279 2.1.5 Late endosomes and lysosomes ........................................................ 280 2.2 Microscopy-Based Trafficking Assays in Polarized Epithelial Cells ........... 281 2.2.1 Trafficking of internalized cargo proteins to specific endosomal compartments .................................................................................. 281 2.2.2 Microscopy-based apical transcytosis assay ...................................... 282 2.3 Biochemical-Based Assay .................................................................... 283 2.3.1 Sulfotag biotin-Tf recycling and transcytotic assay ............................. 283 References .............................................................................................................285

Abstract Epithelial cells display segregated early endosomal compartments, termed apical sorting endosomes and basolateral sorting endosomes, that converge into a common late endosomal-lysosomal degradative compartment and common recycling endosomes (CREs). Unlike recycling endosomes of nonpolarized cells, CREs have the ability to sort apical and basolateral plasma membrane proteins into distinct apical and basolateral recycling Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.06.019 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 17 Endosomal trafficking assays

routes, utilizing mechanisms similar to those employed by the trans Golgi network in the biosynthetic pathway. The apical recycling route includes an additional compartment, the apical recycling endosomes, consisting of multiple vesicles bundled around the basal body. Recent evidence indicates that, in addition to their role in internalizing ligands and recycling their receptors back to the cell surface, endosomal compartments act as intermediate stations in the biosynthetic routes to the plasma membrane. Here we review methods employed by our laboratory to study the endosomal compartments of epithelial cells and their multiple trafficking roles.

INTRODUCTION Endosomal compartments in epithelial cells participate in many polarized signaling and nutrient delivery functions; to carry out these functions they are endowed with the ability to traffic and sort plasma membrane (PM) receptors, for example, transferrin (TfR), low-density lipoprotein (LDLR), epidermal growth factor receptor (EGFR) and megalin, in the endocytic, recycling, and biosynthetic pathways (Perret et al., 2005). Early studies in nonepithelial cells have shown that these receptors are internalized from the PM into peripheral sorting endosomes (SE), where the acidic environment dissociates the ligands (except for transferrin (Tf), which loses the iron while remaining bound to TfR) (Maxfield & McGraw, 2004). Within minutes, SE cease to receive incoming vesicles from the cell surface and mature into late endosomes (LE), a process characterized by microtubule motor-facilitated translocation toward the cell center and exchange of the small guanosine triphosphatase (GTPase) Rab5 by Rab7 (Poteryaev et al., 2010; Rink, Ghigo, Kalaidzidis, & Zerial, 2005). LE then fuse with lysosomes thus exposing the luminal material to hydrolases for degradation (Puertollano et al., 2001). Most membrane receptors in SE (exception EGFR) are incorporated into narrow tubules, in part through the action of sorting nexins (Traer et al., 2007), (1) quick return to the cell surface (fast-recycling loop) or (2) microtubule- and dynein-mediated traffic to perinuclear Rab11-positive recycling endosomes (RERab11) (Shah, Baterina, Taupin, & Farquhar, 2013), and Rab11/myosinVb-dependent return to the cell surface (slow recycling loop) (Lapierre et al., 2001; Ren et al., 1998; Ullrich, Reinsch, Urbe, Zerial, & Parton, 1996) (Figure 1(A)). Because polarized epithelia have distinct apical and basolateral PM domains, separated by tight junctions, their endosomal organization and function are more complex. The basolateral PM displays nutrient receptors, such as TfR and LDLR, which pick up the respective ligands from the blood and are then internalized into basolateral SE (BSE). The apical PM faces the lumen of the organs and participates in organ-specific endocytic processes. For example, epithelial cells in the proximal tubule of the kidney display the fast recycling receptors megalin and cubilin at the apical PM, which act as a Velcro mechanism to recover proteins in the glomerular ultrafiltrate and are internalized into apical SE (ASE), localized immediately under the apical surface. ASE and BSE mature into common LE targeting the fluid phase and luminal ligands for lysosomal degradation (Bomsel, Prydz, Parton, Gruenberg,

Introduction

FIGURE 1 Model. This cartoon displays the various endosomal compartments and endosomal trafficking pathways in nonepithelial (A) and polarized epithelial (B) cells. The basolateral pathways and mechanisms of polarized epithelial cells are shown in red and the apical in blue. (See color plate)

& Simons, 1989). Apical receptors in ASE and basal receptors in BSE are recycled via complex mechanisms. They are either returned by a fast recycling loop to the respective cell surface or transported via tubular carriers to TfR-rich perinuclear common recycling endosomes (CRETfR) where they are sorted via specific mechanisms and recycled via distinct carrier vesicles back to the correct PM domain (slow recycling loop) (Hughson & Hopkins, 1990). Basolateral receptors are sorted via basolateral signals similar to endocytic motifs, bound by clathrin adaptors such as AP-1B (Folsch, Ohno, Bonifacino, & Mellman, 1999; Gan, McGraw, & RodriguezBoulan, 2002; Gravotta et al., 2007), and incorporated into clathrin-coated vesicles for delivery to the basolateral PM (Deborde et al., 2008). The recycling pathway of apical receptors has not been fully characterized yet. Based mainly on the route of transcytotic receptors such as polymeric IgA receptor (pIgR) (Apodaca, Katz, & Mostov, 1994; Brown et al., 2000; Wang, Kumar, Navarre, Casanova, & Goldenring, 2000), neonatal Fc receptor (FcRn) (Tzaban et al., 2009), and TfR in epithelial cells lacking AP-1B (Perez Bay et al., 2013), they are believed to be sorted at CRETfR via specific apical signals (e.g., N-glycans, for pIgR and TfR) (Luton et al., 2009; Perez Bay et al., 2014) into vesicles for microtubule-dependent transport to a subapical endosomal compartment enriched in Rab11, the apical recycling endosome (ARErab11) (Apodaca et al., 1994; Brown et al., 2000; Golachowska, Hoekstra, & van, 2010; Perez Bay et al., 2013; Perret et al., 2005), and then delivered to the apical PM via a Rab11, myosin Vb-dependent mechanism (Thuenauer, Rodriguez-Boulan, & Romer, 2014; Wang et al., 2000) (Figure 1(B)).

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Based on early experiments, sorting of apical and basolateral PM proteins in the biosynthetic route is believed to occur at the trans Golgi network (TGN) (Matlin et al., 1983; Misek, Bard, & Rodriguez-Boulan, 1984; Rodriguez Boulan & Sabatini, 1978; Rodriguez-Boulan, Paskiet, Salas, & Bard, 1984). Sorting of apical PM proteins at the TGN is mediated by apical sorting signals such as glycosylphosphatidyl inositol anchors (Brown, Crise, & Rose, 1989; Lisanti et al., 1989), transmembrane domains (Kundu et al., 1996; Scheiffele, Roth, & Simons, 1997; Skibbens, Roth, & Matlin, 1989), cytoplasmic determinants (Takeda, Yamazaki, & Farquhar, 2003; Thuenauer et al., 2014), and N- or O-glycans (Delacour et al., 2006; Perez Bay et al., 2014; Stechly et al., 2009). These signals are recognized by membrane microdomains termed lipid rafts, galectins, or microtubule motors. Sorting of basolateral proteins at the TGN is mediated by short peptidic sequences often resembling endocytic motifs (Yxxf; NPYX), which are recognized by the clathrin adaptors AP-1A or AP-1B (Carvajal-Gonzalez et al., 2012; Folsch, Pypaert, Maday, Pelletier, & Mellman, 2003; Gravotta et al., 2012) for incorporation into clathrin-coated vesicles (Deborde et al., 2008). However, over the past decade it has become evident that, in addition to their specific roles in recycling processes, endosomes are also intermediate compartments in the biosynthetic routes of PM proteins. For example, the basolateral PM protein VSV G traverses CRETfR en route to the basolateral domain (Folsch et al., 2003; Gravotta et al., 2007), whereas several apical proteins traverse ARErab11 (i.e., endolin and rhodopsin) (Cresawn et al., 2007; Thuenauer et al., 2014) or ASE (i.e., influenza hemagglutinin (HA)) (Cresawn et al., 2007). The functional significance of transendosomal transport pathways remains unclear. Interestingly, the basolateral adhesion molecule E-cadherin also traverses ARErab11 in its biosynthetic pathway (Lock & Stow, 2005), suggesting the emerging concept that ARErab11 might have apical-basolateral sorting roles such as the TGN and CRE. The exact site of function of AP-1A and AP-1B in basolateral sorting is controversial. Although biochemical sorting assays indicate that AP-1B mediates basolateral sorting at CRETfR and the highly homologous adaptor AP-1A performs basolateral sorting at the TGN, a recent report, on the basis of microscopy experiments using overexpressed tagged adaptor subunits, has suggested that both adaptors localize and function at the TGN and CRETfR (Folsch et al., 2003; Gravotta et al., 2012; Guo et al., 2013; Rodriguez-Boulan, Perez-Bay, Schreiner, & Gravotta, 2013). Full elucidation of the basolateral sorting mechanisms of epithelial cells will require to identify the compartments where the endogenous adaptors operate and to determine how these adaptors carry out their complementary sorting functions. Interestingly, AP-1B is an epithelial-specific adaptor and AP-1A is ubiquitous (Ohno et al., 1999), there are some epithelia that lack AP-1B and hence express a subset of cognate basolateral proteins at the apical PM. These include the retinal pigment epithelium (RPE) and the kidney proximal tubule (Diaz et al., 2009; Schreiner et al., 2010). The mechanisms responsible for the apical localization of basolateral proteins in these epithelia have recently been identified. Using the basolateral recycling receptor TfR as a model, it was shown that, in MDCK cells lacking AP-1B and in RPE and kidney proximal tubule cell lines, TfR is internalized into CRETfR where

2. Experimental protocols

it is sorted apically by an N-glycan signal and galectin-4 into vesicles that are then transported via Golgi-nucleated microtubules and the kinesin KIF16B to ARErab11, from where it is delivered to the apical PM by a Rab11-dependent mechanism (Perez Bay et al., 2013, 2014).

1. OBJECTIVES AND RATIONALE Based on the discussion above, it is clear that a full understanding of the recycling, transcytotic, and biosynthetic pathways of epithelial cells requires the appropriate methodology to study the transport of cargo proteins along these routes. Here we discuss assays used by our laboratory to study endosomal compartments in epithelial cells and the polarized recycling and transcytotic routes of apical and basolateral receptors. Microscopic and biochemical recycling and transcytotic assays provide complementary information. Microscopy assays inform on endosomal localization and morphology as well as the kinetics with which internalized cargo proteins traverse specific endosomes. Biochemical assays also provide kinetic data on the endocytic and transcytotic pathways, but in a much higher quantitative manner, since this approach allows one to analyze millions of cells at the same time. Here we provide protocols for microscopy and biochemical assays that allow one to (1) specifically label the multiple endosomal compartments of epithelial cells, (2) determine the trafficking kinetics of internalized cargo proteins to these compartments, and (3) determine the trafficking kinetics of cargo proteins to the apical and basolateral PM in the recycling and transcytotic pathways.

2. EXPERIMENTAL PROTOCOLS 2.1 MICROSCOPY-BASED PROTOCOLS FOR ENDOSOMAL LABELING IN POLARIZED EPITHELIAL CELLS The endosomal compartments of epithelial cells can be labeled with different approaches: 1. Internalization of fluorescently labeled antibodies that recognize a luminal epitope of a receptor or internalization of fluorescently labeled specific ligands (e.g., Tf for TfR). In order to label the desired compartment, fluorescent antibodies/ligands are applied to the apical and/or basolateral PM domain for specific times. 2. Immunofluorescence of endosomal markers (e.g., proteins highly enriched in specific endosomes) or expression of these markers tagged with fluorescent proteins (FP) (Table 1). Internalized antibodies/ligands are usually more specific than endosomal markers, for example, 5 min basolateral internalization of Tf labels BSE and

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Table 1 Ligands and Endosomal Markers to Label Endosomal Compartments in Polarized Epithelial Cells

Endosomal Compartment

Fluorescent Ligand

ASE

Flu-WGA (apical uptake. 50 ) Flu-Tf (basolateral uptake. 50 ) Flu-Tf (basolateral uptake. 10þ50 ) e e 10 kDa Flu-Dx (Uptake. 60þ600 )

BSE CRETfR ARERab11 LE Lys

Endosomal Marker (Immunofluorescence) EEA1, Rab4, Rab5

Endosomal Marker (FPFusion Protein Expression)

TfR

EEA1-FP, Rab4FP, Rab5-FP TfR-FP

Flu-Tf (Basolateral uptake. 150 ) Rab11 Rab7 Lamp1

Rab11-FP Rab7-FP Lamp1-FP

5 min apical internalization of wheat germ agglutinin (WGA) labels ASE, whereas immunofluorescence of the early endosomal antigen EEA1 labels both BSE and ASE. On the other hand, expression of endosomal markers fused to FP are specially useful for life imaging experiments, as opposed to fluorescent ligands that usually require to fix the cells at a specific time to prevent them from labeling other compartments. Therefore, fluorescent ligands should be used to achieve higher specificity and FP-tagged endosomal markers for high-temporal resolution kinetic experiments. Section 2.1 describes protocols for fluorescent ligand-based endosomal labeling and provides a list of endo/lysosomal markers for immunofluorescence and FPtagged expression (Table 1).

2.1.1 Apical sorting endosomes 2.1.1.1 Background and objective ASE in polarized MDCK cells appear as small vesicular compartments located immediately beneath the apical PM (Figure 2(A), green fluorescent signal). ASE rapidly exchange apical PM proteins and lipids with the apical PM domain (w2 min for internalization and w5 min for recycling). This can be exploited by adding fluorescent WGA (Flu-WGA, SigmaeAldrich, Saint Louis, MO, USA), which binds to highly glycosylated proteins abundant in the apical PM (Cresawn et al., 2007; Perez Bay et al., 2013).

2.1.1.2 Buffers composition Hanks balanced salt solution (HBSS): 1.26 mM CaCl2, 0.49 mM MgCl2, 0.41 mM MgSO4, 5.3 mM KCl, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 137.9 mM NaCl, 0.338 mM Na2HPO4, 5.55 mM dextrose.


FIGURE 2 (A) ASE and ARERab11 labeling. Polarized WT MDCK cells were incubated for 5 min with Alexa488-WGA (green) from the apical surface, washed with N-methyl-D-glucosamine (NMDG) and immunostained for Rab11. (B) BSE and CRETfR labeling. Polarized WT MDCK cells were incubated for 15 min from the basolateral surface with Alexa633-Tf (green) followed by a 5 min chase, with CF594-Tf (red). Orthogonal view (top) and two confocal sections at the supranuclear (middle) and nuclear (bottom) regions are shown. Scale: 10 mm. (See color plate)

Phosphate buffer saline (PBS): 137 mM NaCl; 2.7 mM KCl; 10 mM Na2HPO4; 2 mM KH2PO4.

2.1.1.3 Experimental protocol 1. Plate MDCK cells at 300,000 cells/cm2 on 12-mm polycarbonate Transwell filtersÒ (Corning, Union City, CA, USA), in Dulbecco Modified Eagle Medium (Corning, Union City, CA, USA), supplemented with 5% fetal bovine serum (5% FBS DMEM). Culture MDCK cells for 4 days, to allow polarization. 2. Rinse twice (apically and basolaterally) with HBSS buffer, supplemented with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (37 C). 3. Add apically 25 mg/mL Flu-WGA in prewarmed HBSS supplemented with 20 mM HEPES (37 C, 5 min). This step allows Flu-WGA internalization into ASE. 4. Incubate three times with 100 mM N-acetyl-D-glucosamine (SigmaeAldrich, Saint Louis MO, USA) in HBSS (4 C, 10 min). This step strips Flu-WGA bound to the apical PM. 5. Rinse with HBSS (4 C), fix with 4% paraformaldehyde in PBS (4 C, 10 min), and process for immunofluorescence.

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2.1.2 Basolateral sorting endosomes 2.1.2.1 Background and objective Like ASE and the apical PM, BSE are small vesicular compartments intimately related to the basolateral PM (Figure 2(B), red fluorescent signal) with which it exchanges components at a very high rate. Since TfR is strictly basolateral in most epithelial cells (i.e., MDCK, CaCo, CaLu, and FRT cells), BSE can be efficiently labeled by exposure of MDCK cells grown on Transwell filtersÒ to fluorescent TfR (Flu-Tf, SigmaeAldrich, St Louis, MO, USA) added to the basolateral compartment. This approach is also achievable in AP-1B-deficient RPE cells, since a large fraction of TfR remains basolateral in these cells. It is worth noting that MDCK cells express low levels of TfR, compared to the extensively used TrvBi cells (McGraw, Greenfield, & Maxfield, 1987); therefore, overexpression of TfR might be necessary to perform these experiments. Alternatively, labeling Tf with bright and photostable fluorophores yields enough signal to detect endogenous Tf in MDCK cells (in our laboratory, we have successfully used the fluorophores CF594 (Biotium, Hayward, CA, USA) and SeTau647 (Seta Biomedicals, Urbana, IL, USA), among others (Perez Bay et al., 2013, 2014).

2.1.2.2 Experimental protocol 1. Plate MDCK cells at 300,000 cells/cm2 density on 12-mm polycarbonate Transwell filtersÒ (Corning, Union City, CA, USA), in 5% FBS DMEM (Corning, Union City, CA, USA) and culture for 4 days to allow polarization. 2. Rinse twice (apically and basolaterally) with HBSS buffer supplemented with 20 mM HEPES (37 C). 3. Incubate with HBSS buffer supplemented with 20 mM HEPES (37 C, 60 min). This step removes unlabeled Tf from TfR. 4. Add basolaterally 5e10 mg/mL Flu-Tf in HBSS supplemented with 20 mM HEPES and 1% bovine serum albumin (BSA) (4 C, 60 min). This step allows for basolateral surface binding of Flu-Tf. Avoid concentrations higher than 20 mg/mL of Flu-Tf, since this increases the likelihood of fluid phase endocytosis. 5. Incubate three times with HBSS buffer supplemented with 20 mM HEPES and 1% BSA (4 C, 5 min). 6. Incubate with prewarmed HBSS buffer supplemented with 20 mM HEPES and 1% BSA (37 C, 5 min). This allows Flu-Tf internalization into BSE. 7. Rinse (HBSS, 4 C), fix with 4% paraformaldehyde in PBS (4 C, 10 min) and process for immunofluorescence.

2.1.2.3 Other markers of ASE and BSE Several additional markers, such as early endosome antigen 1 (EEA1) and the small GTPases Rab4 and Rab5, are used to label SE in nonpolarized cells, either by immunofluorescence or by tagging them with FP. However, polarized epithelial cells display segregated sets of ASE and BSE, which are specifically labeled with apical internalization of Flu-WGA and basolateral internalization of Flu-Tf, as described above.


2.1.3 Common recycling endosomes 2.1.3.1 Background and objective Notably, there is currently no specific marker for CRETfR. These endosomes have been originally defined by electron microscopy experiments as a peri/supranuclear compartment where apically internalized concanavalin A and basolaterally internalized Tf mix, 10e15 min after internalization (Hughson & Hopkins, 1990). Although Flu-Tf is the gold standard for CRETfR labeling, continued basolateral incubation of Flu-Tf for 10 or more minutes labels both BSE and CRETfR. Therefore, we developed a two-color Flu-Tf approach to dissect CRETfR and BSE in the same WT MDCK cell (Perez Bay et al., 2013). As shown in Figure 2(B), CRETfR appear as large tubule-vesicular structures concentrated in the supranuclear region in WT MDCK cells (green fluorescent signal).

2.1.3.2 Experimental protocol 1. Plate WT MDCK cells at 300,000 cells/cm2 density on 12-mm polycarbonate Transwell filtersÒ (Corning, Union City, CA, USA), in 5% FBS DMEM (Corning, Union City, CA, USA) and culture for 4 days to allow polarization. 2. Rinse twice (apically and basolaterally) with HBSS buffer supplemented with 20 mM HEPES (37 C). 3. Incubate with HBSS buffer supplemented with 20 mM HEPES (37 C, 60 min). 4. Add basolaterally 5e10 mg/mL Flu-Tf in HBSS supplemented with 20 mM HEPES and 1% BSA (4 C, 60 min). 5. Incubate with prewarmed HBSS buffer supplemented with 20 mM HEPES and 1% BSA (37 C, 15 min). 6. Add basolaterally 5e10 mg/mL Flu-Tf (labeled with a different fluorophore than the one used in step 4) in HBSS supplemented with 20 mM HEPES and 1% BSA (4 C, 60 min). 7. Incubate three times with HBSS buffer supplemented with 20 mM HEPES and 1% BSA (4 C, 5 min). 8. Incubate with prewarmed HBSS buffer supplemented with 20 mM HEPES and 1% BSA (37 C, 5 min). 9. Rinse (HBSS, 4 C), fix with 4% paraformaldehyde in PBS (4 C, 10 min) and process for immunofluorescence.

2.1.4 Apical recycling endosome 2.1.4.1 Background and objective In contrast to ASE, BSE, and CRETfR, ARERab11 are best labeled with immunofluorescence of the endosomal marker Rab11 or by expressing Rab11-FP fusion protein. Immunofluorescence conditions should be optimized according to the cell type and antibody used. As shown in Figure 2(A), ARErab11 appear as a characteristic large spot located just beneath the center of the apical PM in MDCK cells. The appearance of ARErab11 may vary in different epithelial cells.

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2.1.4.2 Experimental protocol The following is one of many possible immunofluorescence protocols. 1. Plate MDCK cells at 300,000 cells/cm2 density on 12-mm polycarbonate Transwell filtersÒ (Corning, Union City, CA, USA), in 5% FBS DMEM (Corning, Union City, CA, USA) and culture for 4 days to allow polarization. 2. Rinse twice (apically and basolaterally) with HBSS buffer supplemented with 20 mM HEPES (37 C). 3. Fix with 4% paraformaldehyde in PBS (room temperature, 10 min). 4. Quench paraformaldehyde with 50 mM ammonium chloride in PBS (room temperature, 15 min). 5. Block with 1% BSA-PBS (room temperature, 40 min). 6. Incubate with rabbit anti-Rab11 (Invitrogen, Carlsbad, CA, USA) in 1% BSAPBS (1 in 100 dilution; room temperature, 60 min). 7. Wash three times with 1% BSA-PBS (room temperature, 5 min). 8. Incubate anti-rabbit fluorescently labeled secondary antibody in 1% BSA-PBS (room temperature, 45 min). 9. Wash three times with 1% BSA-PBS (room temperature, 5 min).

2.1.5 Late endosomes and lysosomes 2.1.5.1 Background and objective LE/Lys can be labeled with immunofluorescence of the endo/lysosomal markers Rab7 (LE) and Lamp1 (Lys) or expression of these proteins tagged with FP; however, these markers are not completely specific since small amounts of Rab7 can be found in Lys and Lamp1 in LE. Lys can be selectively labeled with internalization assays of fluorescent fluid phase solutes, such as fluorescently labeled low-molecular-weight dextrans (10 kDa Flu-Dx; Invitrogen, Carlsbad, CA, USA). There exist other commercially available probes to label Lys based on a pH-sensitive dye, which is almost nonfluorescent at neutral pH and fluoresces in the acidic environment of the Lys. This section describes a classical protocol for Lys labeling with 10 kDa Flu-Dx.

2.1.5.2 Experimental protocol 1. Plate MDCK cells at 300,000 cells/cm2 density on 12-mm polycarbonate Transwell filtersÒ (Corning, Union City, CA, USA), in 5% FBS DMEM (Invitrogen, Carlsbad, CA, USA) and culture for 4 days to allow polarization. 2. Rinse twice (apically and basolaterally) with HBSS buffer supplemented with 20 mM HEPES (37 C). 3. Add apically and basolaterally 0.1 mg/mL 10 kDa Flu-Dx in HBSS supplemented with 20 mM HEPES and 1% BSA (37 C, 60 min). Note that high concentration of the fluorophore is used to maximize fluid phase endocytosis. 4. Rinse three times with HBSS buffer supplemented with 20 mM HEPES and 1% BSA (37 C).


5. Incubate with HBSS buffer supplemented with 20 mM HEPES and 1% BSA (37 C, 60 min). This step allows the accumulation of internalized 10 kDa FluDx into Lys and the clearance from ASE and BSE. 6. Rinse (HBSS, 4 C), fix with 4% paraformaldehyde in PBS (4 C, 10 min), and process for immunofluorescence.

2.2 MICROSCOPY-BASED TRAFFICKING ASSAYS IN POLARIZED EPITHELIAL CELLS This section describes assays to determine the time of arrival of a basolaterally internalized PM protein to a specific endosomal compartment (2.2.1) and the transcytosis of a basolaterally internalized PM protein in polarized MDCK cells with a microscopy-based approach (2.2.2).

2.2.1 Trafficking of internalized cargo proteins to specific endosomal compartments 2.2.1.1 Background and objective This assay requires the combination of an internalization assay for the PM cargo protein and an endo/lysosomal labeling protocol. As an example, this section describes a protocol to study the trafficking of basolaterally internalized TfR to ARERab11 in WT and AP-1B KD MDCK cells. As we had previously reported (Perez Bay et al., 2013), Figure 3 shows that CF594-Tf internalized from the basolateral side for 10 min avoids ARERab11 in WT MDCK cells and reaches this compartment in

FIGURE 3 Trafficking of basolaterally internalized TfR to ARERab11. Polarized WT and AP-1B KD MDCK cells were incubated for 10 min with CF594-Tf (red) from the basolateral surface and immunostained with anti Rab11 antibody (green). Orthogonal view (top) and two confocal sections at the supranuclear (middle) and nuclear (bottom) regions are shown. Scale: 10 mm. (See color plate)

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AP-1B KD MDCK cells. For protocols on TfR trafficking to other compartments such as ASE or Lys, please see the original papers (Perez Bay et al., 2013, 2014).

2.2.1.2 Experimental protocol 1. Plate MDCK cells at 300,000 cells/cm2 density on 12-mm polycarbonate Transwell filtersÒ (Corning, Union City, CA, USA), in 5% FBS DMEM (Corning, Union City, CA, USA) and culture for 4 days to allow polarization. Prepare one filter for each time point to be studied in one experiment. 2. Rinse twice (apically and basolaterally) with HBSS buffer supplemented with 20 mM HEPES (37 C). 3. Incubate with HBSS buffer supplemented with 20 mM HEPES (37 C, 60 min). 4. Add basolaterally 5e10 mg/mL Flu-Tf in HBSS supplemented with 20 mM HEPES and 1% BSA (4 C, 60 min). 5. Add basolaterally 5e10 mg/mL Flu-Tf in prewarmed (37 C) HBSS supvplemented with 20 mM HEPES and 1% BSA (add the same medium without FluTf to the apical chamber). 6. Incubate for different times. Typical incubation times are 0, 5, 10, 15, and 30 min. 7. Rinse (HBSS, 4 C), fix with 4% paraformaldehyde in PBS (4 C, 10 min). 8. Immunostain ARERab11 using Rab11 as endosomal marker and the immunofluorescence protocol described above. 9. In order to estimate the time of arrival of TfR to ARERab11, carry out colocalization analysis of Rab11 with TfR in the different time points studied. For an excellent review on colocalization analysis, please see (Dunn, Kamocka, & McDonald, 2011).

2.2.2 Microscopy-based apical transcytosis assay 2.2.2.1 Background and objective This section describes a protocol to measure apical transcytosis of TfR luminally tagged with green fluorescent protein (GFP) (TfR-GFP), in WT and AP-1B KD MDCK cells. We have used this approach to study apical transcytosis of transiently transfected TfR-GFP mutated in each of TfR glycosylation sites (Perez Bay et al., 2014). Briefly, a fluorescently labeled primary rabbit anti-GFP antibody (MBL International, Des Plaines, IL, USA) is applied to the basolateral side and a fluorescently labeled secondary anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA) to the apical side. Therefore, basolateral TfR-GFP internalization will be proportional to the fluorescent signal of the primary antibody and apical transcytosis will be proportional to the fluorescent signal of the secondary antibody.

2.2.2.2 Experimental protocol 1. Plate MDCK cells at 300,000 cells/cm2 density on 12-mm polycarbonate Transwell filtersÒ (Corning, Union City, CA, USA), in 5% FBS DMEM (Corning, Union City, CA, USA) and culture for 4 days to allow polarization.


2. 3. 4. 5. 6. 7.

8. 9. 10.

11.

Prepare one filter for each time point to be studied in one experiment and two additional filters for negative controls. On day 3, transfect TfR-GFP (2 mg DNA and 2 mL Lipofectamine in 200 mL Optimem (Invitrogen, Carlsbad, CA). Apply the mix overnight. On day 4, rinse twice (apically and basolaterally) with HBSS buffer supplemented with 20 mM HEPES (37 C). Incubate with HBSS buffer supplemented with 20 mM HEPES (37 C, 60 min). Add basolaterally 2.5 mg/mL rabbit 647-aGFP in HBSS supplemented with 20 mM HEPES (4 C, 120 min). Rinse three times with HBSS supplemented with 20 mM HEPES (4 C). Incubate apically with 2.5 mg/mL 568-aRabbit in prewarmed (37 C) HBSS supplemented with 20 mM HEPES for different times (add the same medium without 568-aRabbit to the basolateral chamber). This step promotes the apical transcytosis of rabbit 647-aGFP in AP-1B KD MDCK cells, which is subsequently detected by 568-aRabbit. Rinse three times (apically and basolaterally) with HBSS supplemented with 20 mM HEPES (4 C). Fix with 4% paraformaldehyde in PBS (4 C, 10 min). Apical transcytosis can be estimated in several ways: (1) mean intensity fluorescence (MIF) of 568-aRabbit, (2) 568-aRabbit MIF/647-aGFP MIF, or (3) Manders colocalization coefficient I, which reports the percentage of 647aGFP pixels occupied by 568-aRabbit pixels. For detailed information on quantification and colocalization analysis, please see the original papers (Perez Bay et al., 2013, 2014). Appropriate negative controls for this experiment include: (1) apical application of secondary 568-aRabbit without basolateral application of rabbit 647aGFP primary antibody and (2) basolateral application of rabbit 647-aGFP and apical application of 568-aRabbit antibodies at 4 C.

2.3 BIOCHEMICAL-BASED ASSAY 2.3.1 Sulfotag biotin-Tf recycling and transcytotic assay In this assay, Tf is labeled with both the luminophore Sulfotag and biotin (sb-Tf). To measure TfR recycling and transcytosis in polarized MDCK cells, sb-Tf is applied basolaterally until achieving steady-state occupancy of the TfR in endosomes. After stripping sb-Tf from the basolateral surface, sb-Tf released to the apical and basolateral medium is collected at different time points. The collected mediums are applied to streptavidin-coated 96-well plates (Meso Scale Discovery, Rockville, MD, USA), which allows the capture of sb-Tf to the bottom of the wells and the detection of Sulfotag luminescence using SI2400 Multiplex analyzer (Meso Scale Discovery, Rockville, MD, USA). This assay is similar to the radioactivity-based biochemical assay reported previously (McGraw & Subtil, 2001) except for the fact that Tf is labeled with Sulfotag/biotin instead of 125I. In our hands sb-Tf is detected with higher sensitivity than 125I-Tf.

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2.3.1.1 Experimental protocol 2.3.1.1.1 Tf labeling with Sulfotag and biotin 1. Dissolve NHS-Sulfotag (Meso Scale Discovery, Rockville, MD, USA) in dimethyl sulfoxide (DMSO) and mix with apo-Tf. Use 5X NHS-Sulfotag/apoTf molar ratio. 2. Rotate (room temperature, 60 min). 3. Purify Sulfotag-Tf using PD10 columns (GE Healthcare, Piscataway, NJ, USA). 4. Prepare NHS-biotin (Pierce, Waltham, MA, USA) in DMSO and mix with Sulfotag-Tf. Use 5X NHS-biotin/Sulfotag apo-Tf molar ratio. 5. Rotate (room temperature, 60 min). 6. Purify sb-Tf using PD10 columns (GE Healthcare, Piscataway, NJ, USA). 7. Iron load sb-Tf using standard protocol described previously (McGraw & Subtil, 2001). 2.3.1.1.2 Recycling/transcytosis assay 1. Plate MDCK cells at 300,000 cells/cm2 density on 12-mm polycarbonate Transwell filtersÒ (Corning, Union City, CA, USA), in 5% FBS DMEM (Corning, Union City, CA, USA) and culture for 4 days to allow polarization. Prepare 6 filters for each time point to be studied in one experiment: three replicas for recycling/transcytosis determination and three replicas for background determination. 2. Rinse twice (apically and basolaterally) with HBSS buffer supplemented with 20 mM HEPES (37 C). 3. Incubate with HBSS buffer supplemented with 20 mM HEPES (37 C, 60 min). 4. Add basolaterally 5e10 mg/mL sb-Tf in HBSS supplemented with 20 mM HEPES and 1% BSA (37 C, 60 min). For background determination apply the same solution, with 5e10 mg/mL sb-Tf and 200X unlabeled Tf. 5. Rinse three times with HBSS buffer supplemented with 20 mM HEPES and 1% BSA (4 C). 6. Incubate with pH 5 buffer (200 mM NaCl, 50 mM 2-(N-morpholino) ethanesulfonic acid, pH 5) plus 50 mM deferroxamine (4 C, 30 min). 7. Rinse with HBSS buffer supplemented with 20 mM HEPES (4 C). 8. Incubate with HBSS buffer supplemented with 20 mM HEPES and 200X unlabeled Tf (4 C, 30 min). 9. Rinse three times with HBSS buffer supplemented with 20 mM HEPES and 1% BSA (4 C). 10. Incubate (apically and basolaterally) with 200 mL prewarmed (37 C) HBSS buffer supplemented with 20 mM HEPES, 1% BSA, 50 mM deferroxamine and 200X unlabeled Tf. 11. Collect apical and basolateral medium at the desired time points. 12. Remove filter from the plastic case by cutting through the edges of the case and incubate the filter in 200 mL lysis buffer (40 mM Tris pH 7.6, 150 mM NaCl, 1.5% Triton X-100) with protease inhibitors.

References

13. Collect lysate in a tube (do not transfer the filter to the tube). 14. Centrifuge (4 C, 10 min, 16,000 g) and transfer to new tubes. 15. Apply samples to 96-well streptavidin-coated plates (Meso Scale Discovery, Rockville, MD, USA). 16. Incubate in orbital shaker (room temperature, 60 min). 17. Quantify luminescence in SI2400 Multiplex analyzer (Meso Scale Discovery, Rockville, MD, USA).

REFERENCES Apodaca, G., Katz, L. A., & Mostov, K. E. (1994). Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes. Journal of Cell Biology, 125, 67e86. Bomsel, M., Prydz, K., Parton, R. G., Gruenberg, J., & Simons, K. (1989). Endocytosis in filter-grown Madin-Darby canine kidney cells. Journal of Cell Biology, 109, 3243e3258. Brown, D. A., Crise, B., & Rose, J. K. (1989). Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science, 245, 1499e1501. Brown, P. S., Wang, E., Aroeti, B., Chapin, S. J., Mostov, K. E., & Dunn, K. W. (2000). Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling. Traffic, 1, 124e140. Carvajal-Gonzalez, J. M., Gravotta, D., Mattera, R., Diaz, F., Perez Bay, A., Roman, A. C., et al. (2012). Basolateral sorting of the coxsackie and adenovirus receptor through interaction of a canonical YXXPhi motif with the clathrin adaptors AP-1A and AP-1B. Proceedings of the National Academy of Sciences of the United States of America, 109, 3820e3825. Cresawn, K. O., Potter, B. A., Oztan, A., Guerriero, C. J., Ihrke, G., Goldenring, J. R., et al. (2007). Differential involvement of endocytic compartments in the biosynthetic traffic of apical proteins. EMBO Journal, 26, 3737e3748. Deborde, S., Perret, E., Gravotta, D., Deora, A., Salvarezza, S., Schreiner, R., et al. (2008). Clathrin is a key regulator of basolateral polarity. Nature, 452, 719e723. Delacour, D., Cramm-Behrens, C. I., Drobecq, H., Le Bivic, A., Naim, H. Y., & Jacob, R. (2006). Requirement for galectin-3 in apical protein sorting. Current Biology, 16, 408e414. Diaz, F., Gravotta, D., Deora, A., Schreiner, R., Schoggins, J., Falck-Pedersen, E., et al. (2009). Clathrin adaptor AP1B controls adenovirus infectivity of epithelial cells. Proceedings of the National Academy of Sciences of the United States of America, 106, 11143e11148. Dunn, K. W., Kamocka, M. M., & McDonald, J. H. (2011). A practical guide to evaluating colocalization in biological microscopy. American Journal of Physiology Cell Physiology, 300, C723eC742. Folsch, H., Ohno, H., Bonifacino, J. S., & Mellman, I. (1999). A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell, 99, 189e198. Folsch, H., Pypaert, M., Maday, S., Pelletier, L., & Mellman, I. (2003). The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains. Journal of Cell Biology, 163, 351e362. Gan, Y., McGraw, T. E., & Rodriguez-Boulan, E. (2002). The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane. Nature Cell Biology, 4, 605e609.

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Golachowska, M. R., Hoekstra, D., & van, I. S. C. (2010). Recycling endosomes in apical plasma membrane domain formation and epithelial cell polarity. Trends in Cell Biology, 20, 618e626. Gravotta, D., Carvajal-Gonzalez, J. M., Mattera, R., Deborde, S., Banfelder, J. R., Bonifacino, J. S., et al. (2012). The clathrin adaptor AP-1A mediates basolateral polarity. Developmental Cell, 22, 811e823. Gravotta, D., Deora, A., Perret, E., Oyanadel, C., Soza, A., Schreiner, R., et al. (2007). AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells. Proceedings of the National Academy of Sciences of the United States of America, 104, 1564e1569. Guo, X., Mattera, R., Ren, X., Chen, Y., Retamal, C., Gonzalez, A., et al. (2013). The adaptor protein-1 mu1B subunit expands the repertoire of basolateral sorting signal recognition in epithelial cells. Developmental Cell, 27, 353e366. Hughson, E. J., & Hopkins, C. R. (1990). Endocytic pathways in polarized Caco-2 cells: identification of an endosomal compartment accessible from both apical and basolateral surfaces. Journal of Cell Biology, 110, 337e348. Kundu, A., Avalos, R. T., Sanderson, C. M., & Nayak, D. P. (1996). Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells. Journal of Virology, 70, 6508e6515. Lapierre, L. A., Kumar, R., Hales, C. M., Navarre, J., Bhartur, S. G., Burnette, J. O., et al. (2001). Myosin vb is associated with plasma membrane recycling systems. Molecular Biology of the Cell, 12, 1843e1857. Lisanti, M. P., Caras, I. W., Davitz, M. A., & Rodriguez-Boulan, E. (1989). A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. Journal of Cell Biology, 109, 2145e2156. Lock, J. G., & Stow, J. L. (2005). Rab11 in recycling endosomes regulates the sorting and basolateral transport of E-cadherin. Molecular Biology of the Cell, 16, 1744e1755. Luton, F., Hexham, M. J., Zhang, M., & Mostov, K. E. (2009). Identification of a cytoplasmic signal for apical transcytosis. Traffic, 10, 1128e1142. Matlin, K., Bainton, D. F., Pesonen, M., Louvard, D., Genty, N., & Simons, K. (1983). Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. I. Morphological evidence. Journal of Cell Biology, 97, 627e637. Maxfield, F. R., & McGraw, T. E. (2004). Endocytic recycling. Nature Reviews Molecular Cell Biology, 5, 121e132. McGraw, T. E., Greenfield, L., & Maxfield, F. R. (1987). Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor. Journal of Cell Biology, 105, 207e214. McGraw, T. E., & Subtil, A. (2001). Endocytosis: biochemical analyses. Current Protocols in Cell Biology. May Issue, (Chapter 15), Unit 15.13. Misek, D. E., Bard, E., & Rodriguez-Boulan, E. (1984). Biogenesis of epithelial cell polarity: intracellular sorting and vectorial exocytosis of an apical plasma membrane glycoprotein. Cell, 39, 537e546. Ohno, H., Tomemori, T., Nakatsu, F., Okazaki, Y., Aguilar, R. C., Foelsch, H., et al. (1999). Mu1B, a novel adaptor medium chain expressed in polarized epithelial cells. FEBS Letters, 449, 215e220. Perez Bay, A. E., Schreiner, R., Benedicto, I., & Rodriguez-Boulan, E. J. (2014). Galectin-4mediated transcytosis of transferrin receptor. Journal of Cell Science, 127, 4457e4469.

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CHAPTER

Analyzing the role of AP-1B in polarized sorting from recycling endosomes in epithelial cells

18 Heike Fo¨lsch1

Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 1


CHAPTER OUTLINE Introduction ............................................................................................................ 290 1. Objectives and Rationale .................................................................................... 291 2. Materials and Instruments .................................................................................. 292 2.1 Materials ............................................................................................ 292 2.1.1 Cell lines .......................................................................................... 292 2.1.2 Reagents and materials .................................................................... 292 2.2 Instruments ........................................................................................ 293 3. Methods ............................................................................................................ 293 3.1 Methods to Determine if a Protein Depends on AP-1B for Basolateral Sorting at Steady State ........................................................................ 293 3.1.1 Analysis of protein sorting in LLC-PK1 cells ....................................... 293 3.1.2 Immunoprecipitation of cargo proteins after chemical crosslinking to AP-1B .......................................................................................... 296 3.2 Methods to Determine if a Protein Depends on AP-1B during Endocytic Recycling, Biosynthetic Delivery, or Both .............................................. 299 3.2.1 Radioactive pulse-chase experiments in MDCK cells depleted of AP-1B .......................................................................................... 299 Summary ................................................................................................................ 303 Acknowledgments ................................................................................................... 303 References ............................................................................................................. 303


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CHAPTER 18 AP-1B in epithelial cell sorting

Abstract Epithelial cells polarize their plasma membrane into apical and basolateral domains where the apical membrane faces the luminal side of an organ and the basolateral membrane is in contact with neighboring cells and the basement membrane. To maintain this polarity, newly synthesized and internalized cargos must be sorted to their correct target domain. Over the last ten years, recycling endosomes have emerged as an important sorting station at which proteins destined for the apical membrane are segregated from those destined for the basolateral membrane. Essential for basolateral sorting from recycling endosomes is the tissue-specific adaptor complex AP-1B. This chapter describes experimental protocols to analyze the AP-1B function in epithelial cells including the analysis of protein sorting in LLC-PK1 cells lines, immunoprecipitation of cargo proteins after chemical crosslinking to AP-1B, and radioactive pulse-chase experiments in MDCK cells depleted of the AP-1B subunit m1B.

INTRODUCTION Recycling endosomes in polarized epithelial cells are important sorting stations that ensure that cargo proteins that travel through them during endocytic recycling or biosynthetic delivery are correctly sorted to either the apical or basolateral membrane domain (Ang & Fo¨lsch, 2012). Correct sorting depends on specific sorting signals decoded in the cargo proteins. In the case of basolateral sorting, these signals resemble endocytic determinants and frequently conform to the consensus sequences of tyrosine-based (YxxØ, FxNPxY) or dileucine-based ([D/E]xxxL[L/I]) sorting signals (Fo¨lsch, 2008). In recycling endosomes of epithelial cells, these signals are recognized and sorted by the cytosolic adaptor complex AP-1B, either alone (YxxØ and [D/E]xxxL[L/I] signals) or in cooperation with the autosomal recessive hypercholesterolemia protein (ARH, FxNPxY signals) (Cancino et al., 2007; Fields et al., 2007; Fo¨lsch, Ohno, Bonifacino, & Mellman, 1999; Fo¨lsch, Pypaert, Maday, Pelletier, & Mellman, 2003; Fo¨lsch, Pypaert, Schu, & Mellman, 2001; Gan, McGraw, & Rodriguez-Boulan, 2002; Kang & Fo¨lsch, 2011). AP-1B is a heterotetrameric clathrin adaptor complex whose expression is mostly confined to columnar epithelial cells (Ohno et al., 1999). AP-1B is composed of two large subunits, the approximately 100 kDa b1-adaptin and g-adaptin, the small approximately 20 kDa s-adaptin, and the approximately 50 kDa medium subunit m1B (Fo¨lsch et al., 1999). Whereas b1-adaptin crosslinks the adaptor complex to clathrin and interacts with ARH, the m subunit interacts with YxxØ sorting motifs, and the g-adaptin/s-adaptin hemicomplex interacts with dileucine-based sorting signals (Doray, Lee, Knisely, Bu, & Kornfeld, 2007; Kang & Fo¨lsch, 2011; Mattera, Boehm, Chaudhuri, Prabhu, & Bonifacino, 2011; Ohno et al., 1999). AP-1B shares most of its subunits with the ubiquitously expressed AP-1A complex. The only exception is the incorporation of their respective medium subunits m1B or m1A (Fo¨lsch et al., 1999). Even though AP-1B and AP-1A are highly similar


with their medium subunits showing an about 80% sequence identity on the amino acid level (Ohno et al., 1999), they have largely nonoverlapping functions in polarized epithelial cells (Carvajal-Gonzalez et al., 2012; Fo¨lsch et al., 1999, 2003, 2001). This can mostly be attributed to the fact that AP-1B exclusively localizes in recycling endosomes, whereas AP-1A functions at the trans-Golgi network (TGN) (Fo¨lsch et al., 2003, 2001; Gravotta et al., 2012). Importantly, AP-1B is essential for basolateral sorting from recycling endosomes and its expression in epithelial cells has profound effects on the nature of recycling endosomes themselves. For example, AP-1B expression triggers the accumulation of PI(3,4,5)P3 in recycling endosomes (Fields, King, Shteyn, Kang, & Fo¨lsch, 2010). As a consequence, AP1B cooperates with a wealth of regulatory factors that are not accessible for AP1A. Among those factors are the small GTPase Arf6 that is most likely involved in AP-1B recruitment (Shteyn, Pigati, & Fo¨lsch, 2011), the coadaptor ARH that is needed for basolateral targeting of cargos with FxNPxY sorting motifs (Kang & Fo¨lsch, 2011), and the mammalian exocyst complex involved in tethering AP-1B vesicles to the target membrane (Fo¨lsch et al., 2003; Grindstaff et al., 1998). Thus, for a deeper understanding of the sorting pathways in a given epithelial cell, it is important to access (1) if AP-1B is expressed in the cell line of interest and (2) if AP-1B is involved in the basolateral targeting of the protein that is being studied. Whereas the expression of AP-1B in a given cell line is easily analyzed via Western blotting or reverse transcription polymerase chain reaction, it can be more tricky to determine if a given protein depends on AP-1B for basolateral sorting. This chapter will discuss methods aimed at determining if a protein depends on AP-1B for localization at the basolateral membrane.

1. OBJECTIVES AND RATIONALE There are multiple pathways by which proteins can reach the basolateral membrane. Some may be sorted by AP-1A at the level of the TGN, especially during biosynthetic delivery (Gravotta et al., 2012). Subsequently, these cargos may enter basolateral early endosomes before surface delivery. Indeed, many receptors that cycle back and forth between the endosomal system and the TGN are exclusively detected at the basolateral membrane (Distel, Bauer, Le Borgne, & Hoflack, 1998; Hunziker, Harter, Matter, & Mellman, 1991; Nabi, Le Bivic, Fambrough, & Rodriguez-Boulan, 1991). These cargos are frequently independent of AP-1B expression for basolateral localizationdat least during biogenesis. However, not all cargos are sorted at the TGN during biosynthetic delivery, but move from the TGN into recycling endosomes to be sorted by AP-1B (Ang et al., 2004; Fo¨lsch, Mattila, & Weisz, 2009). In recycling endosomes, they merge with internalized proteins that depend on AP-1B for correct recycling to the basolateral membrane. Thus, a protein may depend on AP-1B during endocytic recycling alone or both endocytic recycling and biosynthetic delivery. It should be noted that in addition to sorting into distinct transport vesicles for membrane delivery, proteins might

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effectively be retained at the basolateral membrane with or without prior sorting at the TGN or recycling endosomes (Mellman & Nelson, 2008; Rodriguez-Boulan & Zurzolo, 1993). This chapter will discuss how to determine if the localization of a given protein at the basolateral membrane is dependent on AP-1B expression at a steady state using the Lilly Laboratories cell porcine kidney (LLC-PK1) model cell line. Next, a crosslinking approach will be introduced that allows one to directly test if a cargo protein is in physical proximity to AP-1B. Finally, a radioactive pulse-chase experiment followed by surface biotinylation will be presented to determine if a protein is sorted by AP-1B only during endocytic recycling or also during biosynthetic delivery. Taken together, these approaches will provide a comprehensive understanding of a protein’s dependence on AP-1B for basolateral sorting.

2. MATERIALS AND INSTRUMENTS 2.1 MATERIALS 2.1.1 Cell lines LLC-PK1::m1B LLC-PK1::m1A LLC-PK1::m1B-HA LLC-PK1::m1A-HA MadineDarby canine kidney (MDCK) MDCK cells depleted of m1B

2.1.2 Reagents and materials Transwell polycarbonate filter units (12 and 24 mm, 0.4 mm pore size, from Corning costar). Six-well plates. Cell counting chamber. Growth media minimum essential medium (MEM) and aMEM supplemented with fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin, and appropriate selection drugs (geneticin or puromycin). SMEM ¼ MEM without magnesium and calcium. MEM without methionine and cysteine (MEM-met-cys), methionine and cysteine, dialyzed FBS. Defective adenoviruses or plasmid DNA to express the cargo protein of interest. DTSSP ¼ 3,30 -Dithiobis(sulfosuccinimidylpropionate) or comparable crosslinker. 35 S-Express label containing 35S-methionine and 35S-cysteine at about 14 mCi/ mL from New England Nuclear and charcoal. Paraformaldehyde (PFA), NaCl, KCl, KH2PO4, Na2HPO4, Ca2þ, Mg2þ, glycerol, saponin, sucrose, orthovanadate, glycine, TriseHCl, Triton X-100, bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), protease inhibitor cocktail (“complete”

3. Methods

from Roche), sodium dodecyl sulfate (SDS), SDS sample buffer, nitrocellulose membranes. Protein-G beads and appropriate primary and secondary antibodies. Neutravidin beads and biotin reagent.

2.2 INSTRUMENTS Zeiss 510 confocal microscope or equivalent microscopes. Eppendorf microcentrifuges 5415R and 5415D or equivalent microcentrifuges. Beckman tabletop ultracentrifuge and TLA55 rotor or equivalent ultracentrifuge and rotor. SDS gel system and blotting chamber. Western blot developing system. Phosphorimager system.

3. METHODS 3.1 METHODS TO DETERMINE IF A PROTEIN DEPENDS ON AP-1B FOR BASOLATERAL SORTING AT STEADY STATE 3.1.1 Analysis of protein sorting in LLC-PK1 cells 3.1.1.1 Background and objective While most columnar epithelial cells express AP-1B, there are exceptions. A prominent exception is the LLC-PK1 cell line that polarizes in culture, but does not express AP-1B (Ohno et al., 1999). LLC-PK1 cells are porcine kidney cells most likely derived from the proximal kidney tubules that naturally lack AP-1B expression (Schreiner et al., 2010). We, and others, have successfully used this cell line to explore the function of AP-1B in polarized sorting from recycling endosomes (Fields et al., 2007; Fo¨lsch et al., 1999, 2003, 2001; Gan et al., 2002; Gephart et al., 2011; Reales, Sharma, Low, Fo¨lsch, & Weimbs, 2011). To this end, we engineered LLC-PK1 cell lines stably expressing exogenous m1B (LLC-PK1::m1B cells) or extra copies of m1A (LLC-PK1::m1A cells) as a control (Fo¨lsch et al., 1999). Seeded on filter supports to promote polarization, a protein that is sorted basolateral in LLC-PK1::m1B cells, but is nonpolarized or apical in LLC-PK1::m1A cells is dubbed AP-1B-dependent for basolateral sorting. Moreover, internally tagging m1B or m1A creates functional, tagged versions of AP-1B and AP-1A that can be used for either colocalization analysis (not discussed here) or coimmunoprecipitation studies with or without chemical crosslinking (as discussed in Section 3.1.2) (Fo¨lsch et al., 2003, 2001).

3.1.1.2 Flow of experiment 1. Seed 4 105 LLC-PK1::m1B and LLC-PK1::m1A cells on 12 mm polycarbonate filters. Incubate for 3 days at 37 C in the presence of 5% CO2 in aMEM

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containing 10% (w/v) FBS, 2 mM L-glutamine, 0.1 mg/mL of penicillin/ streptomycin and 1 mg/mL of geneticin. Change the medium in the basolateral chamber every day. 2. On day 3 after seeding, infect the cells with defective adenoviruses expressing your protein of interest. Wash the cells in serum-free medium and add 100 mL of serum-free medium with defective adenoviruses at a concentration of 15 pfu/ cells to the apical chamber of the Transwell. Next, gently rock the cells for 2 h in a CO2 incubator at 37 C, wash the virus away, and add fresh growth medium. Further incubate the cells overnight. For a more detailed description of this procedure, see Pigati, Kang, and Fo¨lsch (2013). 3. One day after infection (and four days after seeding), perform a surface staining for your protein of interest, followed by fixation in 3% PFA and incubation with secondary antibodies. For a more detailed description of this procedure, see Pigati et al. (2013) or Cook, Ang, Kang, and Fo¨lsch (2011). 4. Analyze your specimens by confocal microscopy.

3.1.1.3 Considerations Beware that unlike LLC-PK1::m1B cells, parental and LLC-PK1::m1A cells do not form homogenous monolayers (Fo¨lsch et al., 1999). When performing the confocal analysis, it is thus necessary to analyze only regions of the filter in which the cells are grown in a single monolayer. It is also recommended to create stable cell lines using low-expressing vectors to avoid overexpression artifacts. For example, we used pCB6 that led to a twofold to fivefold overexpression of m1B (Fo¨lsch et al., 1999; Sugimoto et al., 2002). Under these conditions, the cells behaved well. However, even with these moderate overexpression levels, we already detected unassembled m1B subunits in gel filtration analyses (Fo¨lsch et al., 1999; Sugimoto et al., 2002). Sometimes, a protein contains more than one basolateral sorting signal. If this is suspected or known, it is recommended to analyze each of the individual sorting signals separately for their dependence on AP-1B for basolateral sorting to gain a comprehensive picture of its sorting itineraries. For example, the low-density lipoprotein receptor (LDLR) contains two sorting signals, a proximal FxNPxY signal and a distal noncanonical YxxØ signal (Koivisto, Hubbard, & Mellman, 2001; Matter & Mellman, 1994). These signals can be analyzed individually by either mutating the critical tyrosine of the FxNPxY signal (LDLR[Y18A] mutant) leaving only the distal sorting signal intact, or by truncating the cytoplasmic tail after the proximal FxNPxY signal to remove the distal sorting determinant (LDLR-CT27 mutant) (Kang & Fo¨lsch, 2011; Matter & Mellman, 1994).

3.1.1.4 Expected results If a protein is dependent on AP-1B for basolateral localization at steady state, it will be localized to the basolateral surface in LLC-PK1::m1B cells, but will be nonpolarized or localized to the apical surface in LLC-PK1::m1A cells (illustrated in Figure 1(A)). A protein that does not depend on AP-1B for basolateral delivery

3. Methods

(A)

(B)

FIGURE 1 LLC-PK1 cell assay to determine if basolateral sorting depends on AP-1B. Depicts expected sorting phenotypes in LLC-PK1::m1A and LLC-PK1::m1B cell lines for (A) cargos that depend on AP-1B for basolateral sorting, and (B) cargos that are sorted to the basolateral membrane independent of AP-1B. Bold lines indicate surface staining of cargos.

will be correctly localized in LLC-PK1::m1A and LLC-PK1::m1B cells (illustrated in Figure 1(B)). Membrane receptors that we determined to be dependent on AP-1B for basolateral sorting using this assay include LDLR and the LDLR-CT27 truncation mutant, transferrin receptor, asialoglycoprotein receptor subunit H1 (AGPRH1) and vesicular stomatitis virus G protein (Fields et al., 2007; Fo¨lsch et al., 1999, 2001; Sugimoto et al., 2002). In contrast, we found that the LDLR(Y18A) mutant, FcII-B2 receptor, and the sodium potassium ATPase reach the basolateral membrane independent of AP-1B expression (Fields et al., 2007; Fo¨lsch et al., 1999).

3.1.1.5 Alternative approaches The LLC-PK1 cell system presents a gain-of-function system. To analyze AP-1B loss of function, m1B may be depleted in MDCK cells (Anderson et al., 2005; Gravotta et al., 2007). The pitfalls for this system are that (1) m1B depletion may not be complete, and (2) because of the close similarity between m1B and m1A, some siRNAs directed against m1B may also deplete m1A to some extent and vice versa (Almomani et al., 2012). Instead of expressing your protein of interest using defective adenoviruses, you may also transiently transfect your cells or even generate stable cell lines expressing your protein of interest in the LLC-PK1 cell lines (Gephart et al., 2011; Reales et al., 2011; Sugimoto et al., 2002).

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3.1.2 Immunoprecipitation of cargo proteins after chemical crosslinking to AP-1B 3.1.2.1 Background and objective In most cases, a protein that was found to depend on AP-1B for steady-state localization at the basolateral membrane is directly sorted by AP-1B and thus will reside in close physical proximity to AP-1B on membranes. However, it is also possible that a protein depends on AP-1B for basolateral localization because the function of a sorting factor is influenced by AP-1B expression (Reales et al., 2011). Therefore, we recommend chemical crosslinking of cargos to AP-1B followed by immunoprecipitation of the crosslink adduct. Because specific antibodies against m1B are not readily available, it is recommended to use cells lines that express HA-tagged m1B (LLC-PK1::m1B-HA) and m1A (LLC-PK1::m1A-HA) as control.

3.1.2.2 Flow of experiment 1. Seed about 2.5 106 LLC-PK1::m1B-HA and LLC-PK1::m1A-HA cells into individual wells of a six-well plate and grow overnight at 37 C in the presence of 5% CO2 in aMEM containing 10% (w/v) FBS, 2 mM L-glutamine, 0.1 mg/mL of penicillin/streptomycin and 1 mg/mL of geneticin. Your cells should be confluent the next day. 2. One day after seeding, infect the cells with defective adenoviruses encoding your protein of interest in serum-free medium (1 mL containing 15 pfu/cell). Remove virus after 1 h, add growth medium, and further incubate at 37 C in a CO2 incubator overnight. For a more detailed description of the virus infection, see Pigati et al. (2013). 3. One day after infecting the cells (and two days after seeding), place the six-well plate onto a metal plate on ice. Wash cells once with 1 mL ice-cold phosphate buffered saline (PBS) containing calcium and magnesium (PBSþþ: 2.67 mM KCl, 1.47 mM KH2PO4, 137.93 mM NaCl, 8.06 mM Na2HPO4, 0.901 mM Ca2þ, and 0.493 mM Mg2þ). 4. Add 1 ml glycerol buffer (10% [w/v] glycerol, 0.1% [w/v] saponin, 1 mM orthovanadate in PBSþþ) to the cells and incubate for 20 min on ice. Wash the cells once with 1 ml of this buffer. 5. Add 1.5 mL crosslinking buffer (0.25 M sucrose, 1 mM orthovanadate in PBSþþ) with either 15 mL of 10 mM DTSSP in DMSO for a final concentration of 100 mM or with 15 mL DMSO without crosslinker as a negative control. Incubate for 2 h on ice. Wash once with 1 mL ice-cold PBSþþ. 6. Add 1 mL of a quenching buffer (100 mM glycine, pH 8.0 in PBSþþ) and incubate on ice for 20 min. Wash once with 1 mL ice-cold PBSþþ. 7. Add 1.25 mL of a lysis buffer (2.5% [w/v] Triton X-100, 10 mM TriseHCl, pH 7.4, 1x protease inhibitor cocktail in PBSþþ), scrape cells with a cell scraper and pass four times through a 22 G1 ½ gauge needle and 1 mL syringe. Transfer into 1.5 mL centrifugation tubes and incubate on ice for 30 min, followed by a clarifying spin at 15,700 g at 4 C for 15 min.

3. Methods

8. Add 500 mL of each supernatant to protein-G beads coupled with specific antibodies against your cargo protein and to beads coupled to nonspecific control antibodies in 500 mL PBSþþ. Rotate end over end for 90 min at 4 C. 9. Wash immunoprecipitates two times with 1 mL lysis buffer containing 1% [w/v] Triton X-100, and once with 1 mL lysis buffer without Triton X-100. Make sure to remove all supernatant after the last wash. Add 25 mL SDS sample buffer to the beads, shake for 20 min, and boil for 5 min at 95 C. 10. Run an SDS gel, blot onto a nitrocellulose membrane, and analyze by Western blotting. To this end, incubate the membrane subsequently with antibodies against HA, g-adaptin, clathrin heavy chain, and the cargo protein that you precipitated.

3.1.2.3 Considerations ˚ as a crosslinker in studies We successfully used DTSSP with a spacer arm of 12 A showing that LDLR is in close proximity to AP-1B (Fo¨lsch et al., 2001). However, other cargos may crosslink better to AP-1B if reagents with longer or shorter spacer arms are selected. Importantly, DTSSP is cleaved in the presence of reducing agents such as b-mercaptoethanol in the SDS sample buffer. Thus, crosslink adducts are separated before the samples are run on SDS gels. If the sorting signal of the analyzed cargo is known, an additional control should be performed with a mutated cargo that carries a nonfunctional sorting motif. This would enhance confidence in the specificity of the crosslinks. When introducing tags into m1B (or m1A), great care should be taken to ensure fully functional AP-1 complexes. We successfully used internal HA tags in m1A and m1B that we showed in numerous publications not to interfere with function (Fo¨lsch et al., 2003, 2001). Caution should be exercised when tagging m1 at its C-terminus, because some tags contain sorting signals themselves that may be recognized by other adaptor complexes and thus have the potential to interfere with the function of the tagged m1. For example, the c-myc-tag (EQKLISEEDL) contains a bona fide dileucine motif, whereas the HA tag (YPYDVPDYA) contains three tyrosine residues.

3.1.2.4 Expected results If a cargo is actively sorted by AP-1B, then AP-1B should be immunoprecipitated with the crosslink adducts of the cargo protein. Furthermore, because AP-1B is a clathrin adaptor, clathrin heavy chains should be readily detectable on Western blots. If the interaction with AP-1B is specific, then AP-1A should not be associated with the cargo in LLC-PK1::m1A cells and cargos with mutated sorting signals should no longer crosslink to AP-1B (illustrated in Figure 2). In the past, we performed these crosslinking assays with LDLR. We found that after crosslinking and precipitation of LDLR with specific anti-LDLR antibodies, AP-1B could be found in the immunoprecipitates only in cell lines that expressed AP-1B, but not in the LLC-PK1::m1A control cell line (Fo¨lsch et al., 2001). In addition to AP-1B, we also found clathrin associated with LDLR in the crosslinks (Fo¨lsch et al., 2001). Importantly, AP-1B was not found in the LDLR crosslink

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FIGURE 2 Chemical crosslinking to show interaction with AP-1B. Schematic representation of expected immunoprecipitations of crosslink adducts in LLC-PK1::m1A (lanes 1 and 2) and LLC-PK1:: m1B cells (lanes 3e6) of wild-type cargo (lanes 1e4) or a cargo with mutated sorting signal (lanes 5 and 6) as detected by Western blotting with antibodies against the cargo protein, clathrin heavy chain, g-adaptin, and the HA-tag.

adducts that were generated with an LDLR mutant in which both proximal and distal sorting determinants were inactivated (Fo¨lsch et al., 2001).

3.1.2.5 Alternative approaches There are a few alternative approaches that merit a brief discussion. If the interactions between your cargo and AP-1B are strong enough, AP-1B may be coimmunoprecipitated with the cargo without prior crosslinking (Fo¨lsch et al., 2001). It is also possible to immunoprecipitate AP-1B via the HA-tag on m1B and then probe for coprecipitated cargo molecules (Almomani et al., 2012). As a variation of this theme, AP-1B-HA vesicles may be immunoisolated with anti-HA antibodies. This may be done with or without prior vesicle purification on sucrose/optiprep gradients. Immunoisolated AP-1B vesicles can then be probed for various cargo proteins (Fo¨lsch et al., 2003). To test if a known sorting motif directly interacts with m1B, a yeast two-hybrid assay may be a good option to obtain some information (Carvajal-Gonzalez et al., 2012; Fields et al., 2007; Gravotta et al., 2012; Guo et al., 2013). However, it should be noted that an observed yeast two-hybrid interaction needs to be verified in cellbased assays. Instead of using defective adenoviruses to express the cargo of interest, transient or stable expression in the LLC-PK1 cell lines may be an option. The benefits of adenoviral expression are high infection rates and flexibility (Pigati et al., 2013).

3. Methods

3.2 METHODS TO DETERMINE IF A PROTEIN DEPENDS ON AP-1B DURING ENDOCYTIC RECYCLING, BIOSYNTHETIC DELIVERY, OR BOTH 3.2.1 Radioactive pulse-chase experiments in MDCK cells depleted of AP-1B 3.2.1.1 Background and objective To fully understand at which stages a protein is dependent on AP-1B for basolateral sorting, it is beneficial to distinguish if sorting by AP-1B occurs only during endocytic recycling or also during biosynthetic delivery. To this end, a radioactive pulse-chase experiment combined with selective biotinylation of either the apical or basolateral surface is still the best way to answer this question. However, this assay cannot be performed with LLC-PK1 cell lines, because they readily open their tight junctions at 4 C and thus are not amicable for selective surface biotinylation, which requires intact tight junctions. Therefore, we recommend using MDCK cells that naturally express AP-1B and MDCK cells depleted of the AP-1B subunit m1B for this assay (Anderson et al., 2005). A protein that is sorted directly to the basolateral membrane in MDCK cells but directly delivered to the apical membrane in MDCK cells depleted of m1B will be identified as a protein that depends on AP-1B and thus moves from the TGN into recycling endosomes during biosynthetic delivery.

3.2.1.2 Flow of experiment 1. Seed 8 105 MDCK cells depleted of m1B or control MDCK cells on 24-mm polycarbonate filters. For a detailed protocol on how to generate MDCK cells depleted of m1B, see Pigati et al. (2013). You will need two filters per time point of the chase to separately biotinylate the basolateral and apical surfaces. Incubate for 4 days at 37 C in the presence of 5% CO2 in MEM containing 7% (w/v) FBS, 2 mM L-glutamine, 0.1 mg/mL of penicillin/streptomycin and 0.4 mg/mL of puromycin. Change the medium in the basolateral chamber every day. 2. On day 4 after seeding, infect the cells with defective adenoviruses to express your protein of interest. Wash the cells with SMEM and add 250 mL SMEM containing the defective adenovirus at a concentration of 15 pfu/cell per apical chamber of the filter units. Incubate on a rocker for 2 h at 37 C in a CO2 incubator, remove virus, add fresh growth medium, and incubate overnight. For a more detailed description of the virus infection, see Pigati et al. (2013). 3. On the day of the cell infection, prepare neutravidin beads. Use 50 mL beads per sample and wash three times with 1 mL of a buffer containing 1% [w/v] BSA and 1% [w/v] Triton X-100 in PBSþþ. After the last wash, carefully remove all supernatant and add 1 mL unlabeled cell lysate and incubate overnight by rotating end over end at 4 C. This will reduce the amount of nonspecific binding of radioactive proteins to the neutravidin beads. 4. One day after the infection (and 5 days after seeding), proceed with cell labeling. Wash the cells once with warm PBSþþ. Subsequently, add

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5.

6. 7.

8.

9.

10. 11.

12.

13.

MEM-met-cys containing 10% [w/v] dialyzed FBS and 2 mM L-glutamine to the cells, and incubate for 30 min at 37 C in a CO2 incubator. Place the filter units on the lid of the six-well plate containing 140 mL of the label reaction (35S-Express label at a final concentration of 2 mCi/mL in MEM-met-cys containing 10% [w/v] dialyzed FBS and 2 mM L-glutamine). Label for 15 min at 37 C in a container with charcoal to absorb radioactivity that may become airborne. After the labeling reaction, transfer samples to a new six-well plate. Wash the basolateral chamber two times and the apical chamber once with warm PBSþþ. Add growth medium plus an excess (5X) of methionine and cysteine to the cells. Chase the cells for 0, 30, 60, and 90 min. After each chase, place the cells in ice-cold PBSþþ in a new six-well plate that is placed on a metal plate on ice, and wash three times for 5 min in ice-cold PBSþþ. To the cell surfaces that are not going to be biotinylated, add ice-cold PBSþþ (1.5 mL to the apical chamber and 2.5 mL to the basolateral chamber). To the surfaces that are going to be biotinylated, add 1 mL of a solution containing 1.5 mg/mL of biotin in PBSþþ. Incubate for 20 min on ice. Aspirate biotin solution and add freshly prepared 1.5 mg of biotin in PBSþþ, followed by incubation for 20 min on ice. Wash once in 100 mM glycine in PBSþþ, followed by two 5 min incubation in 100 mM glycine in PBSþþ, and two 5 min incubation in 20 mM glycine in PBSþþ on ice to quench unreacted biotin. Excise the filters and transfer into a new six-well plate. Add 1.25 mL lysis buffer (1% [w/v] Triton X-100, 0.1% [w/v] SDS, 20 mM glycine, 1x protein inhibitor cocktail in PBS). Scrape cells with a cell scraper and pass four times through a 22 G1 ½ gauge needle and 1 mL syringe. Transfer into 1.5 mL centrifugation tubes and incubate on ice for 30 min, followed by a clarifying spin at 15,700 g at 4 C for 15 min. Take the supernatant and spin at 100,000 g at 4 C for 30 min (TLA55 rotor [Beckman Coulter], tabletop ultracentrifuge). Transfer 900 mL of the supernatant to Protein-G beads coupled to an antibody that recognizes your protein of interest. Incubate overnight by rotating end over end at 4 C. The next day, wash the Protein-G beads three times with 1 mL lysis buffer. Remove all supernatant after the last wash and add 100 mL SDS buffer (20 mM TriseHCl, 100 mM NaCl, 20 mM glycine, 2% [w/v] SDS in H2O). Vortex samples and spin briefly, boil for 2 min, and chill for 30 s on ice. Vortex samples for 30 s, followed by a 2 min spin at 15,700 g at 4 C. Take out 20 mL for your total input. Subsequently, add 1 mL lysis buffer and vortex for 30 s followed by a 2 min spin at 15,700 g at 4 C. Transfer 900 mL of the radioactive immunoprecipitate in lysis buffer to the prepared neutravidin beads (make sure to completely remove the unlabeled cell lysate before adding your samples). Incubate by rotating end over end for 1 h at 4 C. Wash neutravidin beads with your bound sample three times with 1 mL lysis buffer, followed by one wash with 1 mL PBS containing 0.2% [w/v] SDS and

3. Methods

one wash with 1 mL PBS. Follow with two washes in 1 mL high salt buffer (350 mM NaCl, 0.1% [w/v] Triton X-100 in PBS), and one wash with 1 mL PBS. 14. After the last wash, add 50 mL SDS sample buffer and boil for 5 min. Run half of the sample on an SDS gel, dry the gel. Running half the sample leaves you with enough probe in case you have to rerun your gel. 15. Analyze samples with a phosphorimager after incubating your dried gels on phosphorimager plates. Data may be presented as autoradiogram, as percentage of protein reaching each surface relative to the total amount of surface protein at each time point or both.

3.2.1.3 Considerations One of the most important aspects for the success of this experiment is to reduce background activity. This is achieved by (1) quenching reactive biotin at the end of the biotinylation reaction. If this is not done carefully, reactive biotin will remain in the samples during cell lysis resulting in biotinylated cargos that did not reside at the plasma membrane. (2) Prebinding unlabeled proteins to the neutravidin beads before use greatly reduces the background that can be caused by nonspecific binding of proteins to the beads. (3) The high-speed spin in the ultracentrifuge after lysis of the cells will remove any labeled protein that is still associated with ribosomes. Because those translational complexes are rather large, they would come down with and contaminate the immunoprecipitates.

3.2.1.4 Expected results A newly synthesized and hence radiolabeled protein will be biotinylated at the plasma membrane upon arrival. Thus, this method measures the amount of the total labeled protein (internal and surface protein) that reaches the plasma membrane during the chase. A protein that is directly sorted to either apical or basolateral domain will be biotinylated only at the respective domain. Proteins that depend on AP-1B during biosynthetic delivery will be directly delivered to the basolateral membrane in MDCK cells but should be directly missorted to the apical membrane in MDCK cells depleted of m1B (illustrated in Figure 3(A) and (B)). If however a protein is first sorted to the basolateral membrane, but reaches the apical membrane after internalization, radioactive material will first be biotinylated at the basolateral membrane and at later time points the basolateral signal will vanish while a signal will appear in the lanes that contain material biotinylated at the apical membrane. This latter scenario will be observed in MDCK cells depleted of m1B if a cargo is sorted at the TGN independent of AP-1B, but relies on AP-1B during endocytic recycling (illustrated in Figure 3(C)). We successfully used this protocol to show that the neuron-glia cell adhesion molecule (Ng-CAM) reaches the apical membrane via basolateral-to-apical transcytosis (Anderson et al., 2005). Furthermore, we determined that LDLR-CT27 is directly missorted to the apical membrane if the v-SNARE cellubrevin is dysfunctional (Fields et al., 2007). This indicated that LDLR-CT27 moves from the TGN into recycling endosomes to be sorted along the AP-1B pathway during biosynthetic delivery.

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(A)

MDCK cells 0 A

30 B A

60 B A

90 B A

min chase B surface

IP total

(B)

MDCK cells depleted of µ1B, diredt apical delivery 0

30 B A

A

60 B A

90 B A

min chase B surface

IP total

(C)

MDCK cells depleted of µ1B, indiredt apical delivery 0 A

30 B A

60 B A

90 B A

min chase B surface

IP total

FIGURE 3 Radioactive pulse-chase and biotinylation to measure surface arrival. Schematic drawing of the expected results showing autoradiograms of surface arrival (IP lanes) at the basolateral or apical domains relative to the total amount of labeled cargo protein (total lanes) after 0, 30, 60, and 90 min of chase. (A) Represents MDCK cell control, (B) represents expected results in MDCK cells depleted of m1B if a cargo depends on AP-1B during biosynthetic delivery, and (C) depicts expected results in MDCK cells depleted of m1B if a cargo depends on AP-1B only during endocytic recycling. IP, immunoprecipitation; A, apical surface; B, basolateral surface.

3.2.1.5 Alternative approaches There are some alternative approaches that may substitute for some aspects of the radioactive pulse-chase and surface biotinylation assay. For example, if the function of recycling endosomes is selectively ablated by a horseradish peroxidase reaction on ice, newly synthesized proteins that travel through recycling endosomes on their way to the cell surface will be trapped and their arrival at the plasma membrane will be delayed (Almomani et al., 2012; Ang et al., 2004; Cresawn et al., 2007). We found that overexpression of dominant negative Rab13T22N or constitutive active Rab13Q67L after microinjection of their cDNA into the nuclei of polarized MDCK cells severely inhibits the surface delivery of cargo proteins that move from the TGN into recycling endosomes (Nokes, Fields, Collins, & Fo¨lsch, 2008). Thus, this method may be used to determine if a protein travels through recycling endosomes during biosynthetic delivery; however, it requires a microinjection set up and mastering of the technology (Cook et al., 2011). As mentioned for the other procedures described in this chapter, stable or transient expression of the protein of interest may be substituted for the virus infection

References

if it is guaranteed that the protein is expressed in at least 70e80% of the cells (Hunziker et al., 1991; Matter, Hunziker, & Mellman, 1992; Matter, Whitney, Yamamoto, & Mellman, 1993).

SUMMARY The methods in this chapter provide a comprehensive guideline to study AP-1Bdependent sorting from recycling endosomes. In addition to revealing if AP-1B expression is needed for steady-state localization at the basolateral membrane, the described methods help in answering if a cargo depends on AP-1B for basolateral sorting only during endocytic recycling or also during biosynthetic delivery of the protein. Thus, combining the described methods will lead to a comprehensive understanding of the biosynthetic and endocytic pathways of any protein residing at the basolateral membrane.

ACKNOWLEDGMENTS Research in the Fo¨lsch laboratory has been supported by a grant from the National Institutes of Health (GM070736).

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Cook, R. N., Ang, S. F., Kang, R. S., & Fo¨lsch, H. (2011). Analyzing the function of small GTPases by microinjection of plasmids into polarized epithelial cells. Journal of Visualized Experiments, (51). pii: 2645. Cresawn, K. O., Potter, B. A., Oztan, A., Guerriero, C. J., Ihrke, G., Goldenring, J. R., et al. (2007). Differential involvement of endocytic compartments in the biosynthetic traffic of apical proteins. The EMBO Journal, 26, 3737e3748. Distel, B., Bauer, U., Le Borgne, R., & Hoflack, B. (1998). Basolateral sorting of the cationdependent mannose 6-phosphate receptor in Madin-Darby canine kidney cells. Identification of a basolateral determinant unrelated to clathrin-coated pit localization signals. The Journal of Biological Chemistry, 273, 186e193. Doray, B., Lee, I., Knisely, J., Bu, G., & Kornfeld, S. (2007). The gamma/sigma1 and alpha/sigma2 hemicomplexes of clathrin adaptors AP-1 and AP-2 harbor the dileucine recognition site. Molecular Biology of the Cell, 18, 1887e1896. Fields, I. C., King, S. M., Shteyn, E., Kang, R. S., & Fo¨lsch, H. (2010). Phosphatidylinositol 3,4,5-trisphosphate localization in recycling endosomes is necessary for AP-1B-dependent sorting in polarized epithelial cells. Molecular Biology of the Cell, 21, 95e105. Fields, I. C., Shteyn, E., Pypaert, M., Proux-Gillardeaux, V., Kang, R. S., Galli, T., et al. (2007). v-SNARE cellubrevin is required for basolateral sorting of AP-1B-dependent cargo in polarized epithelial cells. The Journal of Cell Biology, 177, 477e488. Fo¨lsch, H. (2008). Regulation of membrane trafficking in polarized epithelial cells. Current Opinion in Cell Biology, 20, 208e213. Fo¨lsch, H., Mattila, P. E., & Weisz, O. A. (2009). Taking the scenic route: biosynthetic traffic to the plasma membrane in polarized epithelial cells. Traffic (Copenhagen, Denmark), 10, 972e981. Fo¨lsch, H., Ohno, H., Bonifacino, J. S., & Mellman, I. (1999). A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell, 99, 189e198. Fo¨lsch, H., Pypaert, M., Maday, S., Pelletier, L., & Mellman, I. (2003). The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains. The Journal of Cell Biology, 163, 351e362. Fo¨lsch, H., Pypaert, M., Schu, P., & Mellman, I. (2001). Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells. The Journal of Cell Biology, 152, 595e606. Gan, Y., McGraw, T. E., & Rodriguez-Boulan, E. (2002). The epithelial-specific adaptor AP1B mediates post-endocytic recycling to the basolateral membrane. Nature Cell Biology, 4, 605e609. Gephart, J. D., Singh, B., Higginbotham, J. N., Franklin, J. L., Gonzalez, A., Fo¨lsch, H., et al. (2011). Identification of a novel mono-leucine basolateral sorting motif within the cytoplasmic domain of amphiregulin. Traffic (Copenhagen, Denmark), 12, 1793e1804. Gravotta, D., Carvajal-Gonzalez, J. M., Mattera, R., Deborde, S., Banfelder, J. R., Bonifacino, J. S., et al. (2012). The clathrin adaptor AP-1A mediates basolateral polarity. Developmental Cell, 22, 811e823. Gravotta, D., Deora, A., Perret, E., Oyanadel, C., Soza, A., Schreiner, R., et al. (2007). AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells. Proceedings of the National Academy of Sciences of the United States of America, 104, 1564e1569. Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., Rodriguez-Boulan, E., Scheller, R. H., et al. (1998). Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells. Cell, 93, 731e740.

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Guo, X., Mattera, R., Ren, X., Chen, Y., Retamal, C., Gonzalez, A., et al. (2013). The adaptor protein-1 mu1B subunit expands the repertoire of basolateral sorting signal recognition in epithelial cells. Developmental Cell, 27, 353e366. Hunziker, W., Harter, C., Matter, K., & Mellman, I. (1991). Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell, 66, 907e920. Kang, R. S., & Fo¨lsch, H. (2011). ARH cooperates with AP-1B in the exocytosis of LDLR in polarized epithelial cells. The Journal of Cell Biology, 193, 51e60. Koivisto, U. M., Hubbard, A. L., & Mellman, I. (2001). A novel cellular phenotype for familial hypercholesterolemia due to a defect in polarized targeting of LDL receptor. Cell, 105, 575e585. Matter, K., Hunziker, W., & Mellman, I. (1992). Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell, 71, 741e753. Matter, K., & Mellman, I. (1994). Mechanisms of cell polarity: sorting and transport in epithelial cells. Current Opinion in Cell Biology, 6, 545e554. Matter, K., Whitney, J. A., Yamamoto, E. M., & Mellman, I. (1993). Common signals control low density lipoprotein receptor sorting in endosomes and the Golgi complex of MDCK cells. Cell, 74, 1053e1064. Mattera, R., Boehm, M., Chaudhuri, R., Prabhu, Y., & Bonifacino, J. S. (2011). Conservation and diversification of dileucine signal recognition by adaptor protein (AP) complex variants. The Journal of Biological Chemistry, 286, 2022e2030. Mellman, I., & Nelson, W. J. (2008). Coordinated protein sorting, targeting and distribution in polarized cells. Nature Reviews, 9, 833e845. Nabi, I. R., Le Bivic, A., Fambrough, D., & Rodriguez-Boulan, E. (1991). An endogenous MDCK lysosomal membrane glycoprotein is targeted basolaterally before delivery to lysosomes. The Journal of Cell Biology, 115, 1573e1584. Nokes, R. L., Fields, I. C., Collins, R. N., & Fo¨lsch, H. (2008). Rab13 regulates membrane trafficking between TGN and recycling endosomes in polarized epithelial cells. The Journal of Cell Biology, 182, 845e853. Ohno, H., Tomemori, T., Nakatsu, F., Okazaki, Y., Aguilar, R. C., Fo¨lsch, H., et al. (1999). Mu1B, a novel adaptor medium chain expressed in polarized epithelial cells. FEBS Letters, 449, 215e220. Pigati, L., Kang, R. S., & Fo¨lsch, H. (2013). Using replication defective viruses to analyze membrane trafficking in polarized epithelial cells. Methods in Cell Biology, 118, 125e137. Reales, E., Sharma, N., Low, S. H., Fo¨lsch, H., & Weimbs, T. (2011). Basolateral sorting of syntaxin 4 is dependent on its N-terminal domain and the AP1B clathrin adaptor, and required for the epithelial cell polarity. PloS One, 6, e21181. Rodriguez-Boulan, E., & Zurzolo, C. (1993). Polarity signals in epithelial cells. The Journal of Cell Science, (Suppl. 17), 9e12. Schreiner, R., Frindt, G., Diaz, F., Carvajal-Gonzalez, J. M., Perez Bay, A. E., Palmer, L. G., et al. (2010). The absence of a clathrin adapter confers unique polarity essential to proximal tubule function. Kidney International, 78, 382e388. Shteyn, E., Pigati, L., & Fo¨lsch, H. (2011). Arf6 regulates AP-1B-dependent sorting in polarized epithelial cells. The Journal of Cell Biology, 194, 873e887. Sugimoto, H., Sugahara, M., Fo¨lsch, H., Koide, Y., Nakatsu, F., Tanaka, N., et al. (2002). Differential recognition of tyrosine-based basolateral signals by AP-1B subunit micro1B in polarized epithelial cells. Molecular Biology of the Cell, 13, 2374e2382.

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CHAPTER

Recycling of the insulinresponsive glucose transporter Glut4 regulated by the small GTPase RalA and the exocyst complex

19

Chao Nie*, Xiao-Wei Chen*, x, 1 x

*Institute of Molecular Medicine, Peking University, Beijing, China PKU-THU Center for Life Sciences, Peking University, Beijing, China 1


CHAPTER OUTLINE Introduction ............................................................................................................ 308 1. Objective and Rationale...................................................................................... 309 2. Materials and Instruments .................................................................................. 310 2.1 Reagents ............................................................................................ 310 2.2 Buffers............................................................................................... 310 2.3 Plasmids and Cell Lines....................................................................... 311 2.4 Instruments ........................................................................................ 311 3. Methods and Results .......................................................................................... 311 3.1 Preparing GST-Ral Binding Domain as Affinity Probes to Detect Active RalA......................................................................................... 311 3.2 Maintenance and Differentiation of 3T3-L1 Preadipocytes ...................... 312 3.3 Pulldown Assay Using Immobilized GST-RBD to Determine Cellular RalA Activity........................................................................... 312 3.3.1 Results............................................................................................. 314 3.4 RNAi-Mediated Depletion of RalA in Mature 3T3-L1 Adipocytes.............. 314 3.4.1 Results............................................................................................. 315 3.5 Detection of Cell Surface Glut4 with the Myc7-Glut4-eGFP Reporter........ 315 3.5.1 Results............................................................................................. 316 Summary ................................................................................................................ 317 References ............................................................................................................. 317 Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.05.003 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 19 Recycling of the insulin-responsive glucose transporter

Abstract Endocytic recycling represents a major mechanism for continuous supply of molecules to the plasma membrane. Particularly, outbound trafficking of the recycling endosome (RE) or RE-derived vesicles can be upregulated by cellular signaling, through mobilization of specialized protein complexes acting as transport machineries. Therefore, biochemical and functional characterization of cell signaling molecules that operate multimeric protein complexes in membrane transport provides important insights to signaling-regulated trafficking events. In this chapter, we described biochemical approaches and reporter assays in differentiated adipocytes to determine the activity and function of the small GTPase RalA, which relays upstream insulin signaling to the exocyst complex that targets intracellular vesicles bearing the Glut4 transporter to the plasma membrane. The experimental design outlined in this chapter can be applied to other regulated transport events facilitated by the exocyst complex, as well as other GTPases that operate distinct transport complexes in specific physiological settings.

INTRODUCTION Endocytic recycling is a critical process that delivers specific receptors, channels, and transporters to the cell surface following prior rounds of internalization (Maxfield & McGraw, 2004). In particular, physiological signals can trigger rapid re-distribution of proteins to the plasma membrane (Maxfield & McGraw, 2004). One such scenario is insulin-stimulated translocation of the glucose transporter Glut4 to the plasma membrane in fat and muscle cells (Maxfield & McGraw, 2004; Saltiel & Kahn, 2001; Watson, Kanzaki, & Pessin, 2004). The event is central to efficient glucose clearance and utilization, the rate-limiting step in the anabolic actions of insulin (Saltiel & Kahn, 2001). In the basal state, Glut4 is retained in intracellular vesicles. Upon insulin stimulation, Glut4 vesicles undergo rapid and robust translocation toward the plasma membrane, and subsequently fuse with the target membrane to expose the Glut4 transporter to the extracellular environment. Plasma membrane-localized Glut4 will be primarily internalized via the classic clathrinmediated endocytosis and resorted for the next rounds of translocation events (Watson et al., 2004). Insulin-stimulated Glut4 trafficking is initiated upon activation of insulin signaling cascades, which consequently mobilize unique transport machinery to facilitate the physical movement of vesicles bearing Glut4 toward the cell surface (Huang & Czech, 2007; Watson & Pessin, 2006). The transition from cell signaling to vesicle trafficking can be mediated by members of Ras superfamily small GTPases (Leto & Saltiel, 2012), which cycle between their GDP-bound (inactive) states and GTP-bound (active) states (Takai, Sasaki, & Matozaki, 2001). Among these, the small GTPase RalA is activated by insulin through the canonical PI3Kinase/Akt signaling (Chen, Leto, Xiong, et al., 2011). Upon its activation, RalA can then interact with and mobilize the vesicle-tethering exocyst complex to target

1. Objective and rationale

Glut4 vesicles to the plasma membrane for the final fusion events, resulting in exposure of Glut4 to extracellular environment for glucose uptake (Chen, Leto, Chiang, Wang, & Saltiel, 2007; Chen, Leto, Xiao, et al., 2011; Chen, Leto, Xiong, et al., 2011). The exocyst complex is an evolutionarily conserved octameric complex that mediates the initial flexible contacts between the incoming vesicles and their target plasma membrane from relatively long range (He & Guo, 2009; Munson & Novick, 2006). This process is thought to increase efficiency and/or provide specificity in vesicle targeting and to enrich vesicles and bridge them to the SNARE fusion machinery (Cai, Reinisch, & Ferro-Novick, 2007). Loss of the exocyst in dividing cells often causes cell cycle defects, largely resulted from failure in membrane delivery for cytokinesis (Chen, Inoue, Hsu, & Saltiel, 2006; Gromley et al., 2005). However, disruption of the exocyst function in post-mitotic, differentiated eukaryotic cells leads to blockade in the endocytic recycling of cargos such as E-cadherin and Glut4 (EauClaire & Guo, 2003; Inoue, Chang, Hwang, Chiang, & Saltiel, 2003). Notably, the exocyst is able to interact with a number of small GTPases, including Rab, Rho, Arf, and Ral family GTPases, thus could specifically respond to signaling cues that turn on these so-called “molecular switches” to coordinate regulated trafficking events (Cai et al., 2007; Munson & Novick, 2006; Novick & Guo, 2002). In this chapter, we described biochemical assays to determine cellular activity of RalA, a key regulatory GTPase of the exocyst complex (Feig, 2003; Novick & Guo, 2002). We also described methods for electroporation-mediated delivery of RNAi reagents into post-mitotic, mature adipocytes to deplete RalA, and methods for measurement of cell surface Glut4 upon insulin stimulation following the endocytic recycling routes. Combination of these techniques allows the researchers to assess unique endocytic recycling events under the control of hormonal signaling and may shed light to other signaling-regulated membrane transport processes (Chieregatti & Meldolesi, 2005; Maxfield & McGraw, 2004).

1. OBJECTIVE AND RATIONALE The exocyst plays a key role in directing the outbound trafficking from the recycling endosome to plasma membrane, in a manner orchestrated by several small GTPases (He & Guo, 2009; Novick & Guo, 2002). In turn, cell signaling cascades could activate these small GTPases to mobilize the exocyst complex to facilitate specific transport events (He & Guo, 2009; Novick & Guo, 2002). Such regulatory scheme is employed by insulin in the process of relocating the glucose transporter Glut4 to the cell surface in differentiated fat and muscle cells (Chen & Saltiel, 2011; Leto & Saltiel, 2012). During this process, insulin activates the small GTPase RalA by repressing the Ral GAP complex, thus allows the engagement between RalA and the exocyst (Chen et al., 2007; Chen, Leto, Xiong, et al., 2011). Hence, tracking the activity of small GTPases, such as RalA, allows the assessment of the status quo of exocyst function, particularly in the context of signaling-regulated transport events.

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The insulin-responsive glucose transporter Glut4 contains 12-span transmembrane domains, and cycles between the plasma membrane and intracellular vesicles (Huang & Czech, 2007). The outbound trafficking of Glut4 toward plasma membrane relies on the exocyst (Chen et al., 2007; Inoue et al., 2003). Notably, this specialized transport program takes place in postmitotic cells, thus could bypass the cell cycle defects resulted from disruption of the exocyst in dividing cells (Chen et al., 2006; Gromley et al., 2005). Here, we also described procedures of monitoring plasma membrane-exposed Glut4 by the Myc7-Glut4-eGFP reporter, in which 7 tandem Myc epitopes have been inserted into the exofacial loops of the Glut4 transporter with eGFP fused in the cytosolic tail of Glut4 (Bogan, Hendon, McKee, Tsao, & Lodish, 2003; Chen et al., 2007). The combination of biochemical and cell biology analysis provides important insights into specific functions of the endocytic recycling process in physiological contexts.

2. MATERIALS AND INSTRUMENTS 2.1 REAGENTS LB medium, Ampicillin (Sigma), DNase I (Roche), Lysozyme (Roche), DTT (Sigma), IPTG (Life Technology), GSH beads (GE healthcare), NP-40 (Sigma), 10% Formalin (Sigma) Tissue culture supplies: Stealth RNA oligo (Life Technology), 0.25% and 0.05% Trypsin (Gibco), phosphate-buffered saline (Gibco), Penicillin/Streptomycin (Gibco), DMEM (Gibco), Fetal bovine serum (Sigma), newborn calf serum (Sigma), Dexamethasone (Sigma), Isobutyl-1-methylxanthine (Sigma), Insulin (sigma), Wortmannin (Sigma) Antibodies: Mouse anti-Myc antibody, clone 9E10 (Sigma), mouse anti-RalA antibody (BD Bioscience), mouse anti-Sec8 antibody (BD Bioscience), Rabbit anti-phospho-Akt (S473) antibody (Cell Signaling), HRP conjugated secondary antibodies (Perice), Alexa-Fluor 594 conjugated goat antimouse antibody (Molecular Probe)

2.2 BUFFERS TEN buffer: 10 mM Tris (pH 8.0), 1 mM EDTA, and 100 mM NaCl HNTG buffer: 20 mM HEPES buffer pH 7.5, 150 mM NaCl, 0.1% (w/v) Triton X-100, and 10% (w/v) glycerol. NaCl-Mg buffer: 1.5 M NaCl and 10 mM MgCl2 Cell lysis buffer: 50 mM Tris (pH 7.5), 1% (w/v) NP-40, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 10% (w/v) glycerol, 10 mM MgCl2, supplemented with protease inhibitor tablets (Roche). 4X SDS Sample buffer: 125 mM Tris (pH 6.8), 4% (w/v) SDS, 20% (w/v) Glycerol, 10% (w/v) b-Mercaptoethanol, 0.5 mg/mL Bromophenol Blue.

3. Methods and results

2.3 PLASMIDS AND CELL LINES pGEX 4T-1 GST-Sec5 RBD, or pGEX 4T-1 GST-RalBP1 RBD Wild-type 3T3-L1 preadipocytes and 3T3-L1 preadipocytes stably expressing the Myc7-Glut4-eGFP reporter are established in previous reports (Chang, Chiang, & Saltiel, 2007; Chen et al., 2007).

2.4 INSTRUMENTS Bio-Rad Gene Pulser II electroporator and 1 mL gene pulser cuvette from Bio-rad Olympus Fluoview 500 confocal microscope

3. METHODS AND RESULTS 3.1 PREPARING GST-RAL BINDING DOMAIN AS AFFINITY PROBES TO DETECT ACTIVE RalA 1. Day 1: Transform BL21 competent cells with the pGEX 4T-1 GST-RBD plasmid. Grow the transformants at 37 C overnight on agar plates containing 100 mg/mL Ampicillin. 2. Day 2: Inoculate a single colony of the GST-RBD transformants into a small LB culture (5e50 mL) containing 100 mg/mL Ampicillin. Grow the culture overnight at 37 C with constant shaking at 200 rpm. 3. Day 3: Dilute overnight culture 1:100 into 500 mL LB medium containing 100 mg/mL Ampicillin, and grow at 37 C with constant shaking at 200 rpm until OD600 is between 0.5 and 1.0 (in w2e3 h). 4. Induce GST-RBD protein expression by adding 250 mL 0.4M IPTG for 6 h at 30 C with constant shaking at 200 rpm. 5. Harvest the bacteria by pelleting at 4000 g at 4 C for 10 min. 6. Completely resuspend the bacteria pellet in 20 mL ice-cold TEN buffer. 7. Add lysozyme (0.2 mL of 100 mg/mL) and DTT (80 mL of 1 M) to the resuspended bacteria. Mix well and incubate on ice for 15 min. 8. Add 400 mL 10% NP-40 to resuspended bacteria and mix well. 9. Place the bacteria lysate at 80 C for overnight. 10. Day 4: Thaw the bacteria pellet in a 37 C water bath until the pellet has completely melted. 11. Add NaCl-Mg buffer (30 mL) and DNase I (200 mL of 1 mg/mL). 12. Incubate at 4 C for 2 h with constant stirring. Pass the lysate through a 26gauge needle every 30 min to completely break down bacteria cell wall and DNA. 13. Pellet insoluble proteins at 20,000 g at 4 C for 20 min, transfer the supernatant to a 50 mL conical tube. 14. Add 1 mL 50% GSH beads to the supernatant. Rotate at 4 C for 2e4 h.

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15. Wash GSH beads with 5 mL HNTG buffer for 4 times. 16. Wash GSH beads with 5 mL 50% Glycerol in PBS for 4 times. Store the beads in 1 volume of 50% glycerol in PBS (50% slurry) at 20 C for up to 3 months. The prep should generate w5 mg GST-RBD proteins sufficient for 400e500 RalA activation assays.

3.2 MAINTENANCE AND DIFFERENTIATION OF 3T3-L1 PREADIPOCYTES 1. 3T3-L1 preadipocytes are grown in 15 cm dishes in 30 mL DMEM containing 10% NCS (New born Calf Serum) and 1% Penicillin/Streptomycin. When the cells reach w70% confluence, split them using 0.05% Trypsin into seven 15 cm dishes at w10% confluence after replating (Day 1). 2. On Day 4, the previous passage of cells will reach w70% confluence again. Whereas some preadipocytes need to be passaged like step 1, the rest of them could be used for differentiation (adipogenesis). 3. Induction of differentiation (adipogenesis) starts at Day 7, or 2 days after preadipocytes reach 100% confluence (at Day 5). Thereafter, 3T3-L1 adipocytes are differentiated and maintained in 15 cm dishes in 30 mL DMEM containing 10% FBS (Fetal Bovine Serum) and 1% Penicillin/Streptomycin, referred as L1 medium. 4. At Day 7, aspirate previous culture medium, and the cells are switched to 30 mL L1 medium containing 1 mM dexamethasone (DEX), 0.5 mM isobutyl-1methylxanthine (MIX), and 1 mg/mL insulin. 5. At Day 10, the cells are switched to 30 mL L1 medium containing 1 mg/mL insulin. Lipid accumulation (as lipid droplets) will be evident around Day 12. 6. At Day 13, the cells are switched to 30 mL L1 medium. 7. During Day 15e17, adipocytes can be replated or subject to electroporation as described in later sections. Differentiated adipocytes can be maintained in culture up to 2 weeks, by feeding with fresh L1 medium every 3e4 days. Biochemical analysis that does not involve transfection could be performed in these 2 weeks.

3.3 PULLDOWN ASSAY USING IMMOBILIZED GST-RBD TO DETERMINE CELLULAR RalA ACTIVITY 1. Prepare four plates of adipocytes (in 100 mm dishes), switch cells from complete culture medium to 10 mL basal medium (DMEM þ0.5% FBS) for 4e16 h prior to insulin stimulation and further experiments. Prepare cell lysis buffer. 2. Two hours prior to insulin treatment, treat two plates of cells with 100 nM Wortmannin. Stimulate one plate of untreated adipocytes and one plate of Wortmannin-treated adipocytes with 1 mg/mL insulin for 10 min. Leave the other two plates adipocytes unstimulated as basal conditions.


3. Upon completion of insulin stimulation, quickly aspirate culture medium, and wash cells with 10 mL ice-cold PBS twice. 4. Lyze adipocytes with 1 mL ice-cold cell lysis buffer per 100 mm dish. Scrape cells off the dish with a rubber cell scraper, then transfer cell lysates to a 1.5 mL Eppendorf tube, keep on ice until all the plates are finished, then rotate cell lysates at 4 C for 15 min for complete cell lysis. 5. Centrifuge at 4 C in an Eppendorf benchtop centrifuger at top speed for 5 min. Then filter the supernatant with a 0.22 mm filter to remove lipids in cell lysates. 6. Use clean scissors to cut w3 mm off P200 pipette tips, then aliquot 20 mL immobilized GST-RBD (50% slurry, w10 mg GST fusion protein coupled to GSH beads, as prepared previously) into new Eppendorf tubes. Note: Addition of equal amount of immobilized GST-RBD proteins (w5e10 mg per reaction) to different cell lysates as affinity probes to capture active RalA is critical to the consistency of the RalA activation assay. 7. Transfer 900 mL cell lysates to tubes with GST-RBD proteins, incubate at 4 C for 45 min with head-to-end rotation. Take another 60 mL from the remaining cellular lysates, and add 20 mL 4X SDS sample buffer to generate Total Cell Lysates (TCL). 8. Upon completion of the 45 min incubation, quickly wash beads 3 times with 1 mL ice-cold cell lysis buffer. Use an insulin syringe to remove all the residual buffers without aspirating any beads. Then release all proteins bound to GSH beads by adding 40 mL 1X SDS sample and boiling at 95 C for 10 min. Note: As GTP hydrolysis of the GTPase persists during the pulldown assay, minimizing assay time to a total of w1 h would reduce loss of the GTPase activity during the experiment. 9. Proceed to SDS-PAGE and WB analysis of RalA captured by immobilized GSTRBD (active RalA) and present in TCL (total RalA). A representative result is shown in Figure 1.

FIGURE 1 Insulin activation of RalA is Wortmanin sensitive.

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3.3.1 Results Figure 1 illustrates insulin-stimulated activation of endogenous RalA, as shown by increased levels of RalA isolated by immobilized GST-RBD beads (top blot, lane 2 vs lane 1). Furthermore, activation of RalA by insulin is blocked upon inhibition of the PI3-kinase by Wortmannin (top blot, Lane 3 and 4). Total RalA present in TCL is constant in all four conditions. Phospho-Akt blot in TCL indicates activation of insulin signaling cascade (bottom blot, lane 2), and inhibition of PI3-kinase activity (bottom blot, lane 3 and 4). This figure is adopted from previously published work (Chen et al., 2007) with permission.

3.4 RNAi-MEDIATED DEPLETION OF RalA IN MATURE 3T3-L1 ADIPOCYTES 1. Differentiate 3T3-L1 preadipocytes or preadipocytes stably expressing the Myc7-Glut4-eGFP reporter (Chang et al., 2007; Chen et al., 2007) as described in previous sections and perform the following electroporation experiments between D15-17 (after switching to L1 medium for 2e4 days) 2. Wash one plate (15 cm) of 3T3-L1 adipocytes twice with 30 mL room temperature PBS. 3. Cover cells with 4 mL 0.25% Trypsin by gently swirling the plates, then aspirate w3 mL Trypsin prior to incubation at 37 C for 10 min. 4. Lift the cells from culture plates by pipetting 20 mL prewarmed (37 C) L1 medium onto the cells, and transfer the cell suspension to a 50 mL conical tube. 5. Spin down cells at a benchtop centrifuge at 750 g for 5 min at room temperature. 6. Aspirate the medium, wash cells with 50 mL room temperature PBS. 7. Resuspend cells by flipping the tube upside down till most of the adipocytes are in suspension. 8. Pellet cells again by centrifuging at 750 g for 5 min at room temperature. 9. Aspirate PBS, wash cells with room temperature PBS, repeat steps 7 and 8. 10. Aspirate PBS; gently resuspend the cell pellet with 1.0 mL PBS. 11. Aliquot 0.5 mL cell suspension each to two new Eppendorf tube, then add 20 mL control siRNA oligosolutions to one tube and 20 mL siRNA against RalA to the second tube. With a P1000 pipette, mix cell suspension with siRNA by gently pipetting 3e4 times, then transfer cell/siRNA mixture to a Bio-rad Gene Transducer cuvette. 12. Electroporate cells with the Bio-Rad Gene Pulser II electroporator, with voltage at 160 KV and resistance at 950 mF. The time constant after electroporation should be between 20 and 25 mS. 13. Immediately add 1 mL of prewarmed L1 medium into the electroporation cuvette. Transfer the cells to a 15 mL falcon tube with additional 5 mL L1 medium, then replate cells onto 2 glass coverslips seeded in a 6-well plate for


microscopy and one additional well in the 6-well plate for biochemical analysis. 14. Repeat steps 12 and 13 for other siRNA oligos. 15. Sixteen hours post replating, switch the electroporated cells to 2 mL of fresh L1 medium per well. 16. Protein loss may start 48 h postelectroporation, and will last up to 5 days. Typically, 72e96 h post electroporation, the adipocytes display best knockdown effects and insulin response. A representative knockdown experiment is shown in Figure 2.

3.4.1 Results Figure 2 illustrates acute depletion of endogenous RalA in mature adipocytes by electroporation-mediated RNAi delivery. Cell lysates were harvested 96 h post electroporation and were subjected to SDS-PAGE and western blotting with indicated antibodies. Three different oligos were used in depleting cellular RalA. The antiSec8 blot indicates equal loading of total cellular proteins. This figure is adopted from previously published work (Chen et al., 2007) with permission.

3.5 DETECTION OF CELL SURFACE Glut4 WITH THE Myc7-Glut4-eGFP REPORTER 1. Replate adipocytes expressing the Myc7-Glut4-eGFP reporter onto glass coverslips placed in 6-well plates. For RNAi experiments, the cells could be analyzed 72e96 h post electroporation. Four hours prior to insulin stimulation, switch cells from complete culture medium to 2 mL basal medium (DMEM þ0.5% FBS). 2. For two wells of adipocytes that received the same siRNA, treat one well of cells with 1 mg/mL of insulin for 20 min, and leave the other well of adipocytes untreated at basal conditions. 3. Upon completion of insulin stimulation, quickly aspirate medium, and wash cells with 2 mL PBS twice. 4. Fix cells with 10% formalin in PBS for 10 min at room temperature. 5. Wash cells three times with 2 mL PBS for 5 min each time at room temperature.

FIGURE 2 Knock down of RalA by using three different RalA siRNA oligos.

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6. Without permeablizing cells, incubate the intact cells in blocking buffer (1% BSA þ1% Ovalbumin in PBS) for 30 min at room temperature. Note: Maintaining the integrity of adipocytes is critical for using the exofacial Myc tag as indicators of plasma membrane exposed Glut4. 7. Incubate cells in mouse anti-Myc antibody (1:500 diluted in 0.5 mL blocking buffer) for 4 h at room temperature or up to overnight at 4 C with agitation. 8. Wash cells with PBS for 4 times for 10 min each at room temperature with agitation. 9. Incubate cells with Alexa-Flur594 conjugated goat antimouse secondary (1:1000 diluted in 0.5 mL blocking buffer) for 2 h at room temperature under agitation. 10. Wash with PBS for 4 times for 10 min each time at room temperature with agitation. 11. Mount the coverslip with anti-fade reagent. Acquire images for both Myc (exposed Glut4) and GFP (total Glut4) using Olympus Fluoview 500 confocal microscope. A representative result is shown in Figure 3.

3.5.1 Results Figure 3 illustrates plasma membrane localization of the Myc7-Glut4-eGFP reporter in both basal and insulin-stimulated states. In adipocytes treated with control RNAi, insulin stimulation leads to a substantial increase in Myc staining on the plasma membrane, resulted from translocation and insertion of Glut4 into the plasma

FIGURE 3 Loss of RalA dramaticlly decreases the Myc/GFP ratio. (See color plate)

References

membrane (top two panels). In adipocytes treated with RNAi against RalA, this effect of insulin is largely attenuated (bottom two panels). In all conditions, GFP signal serves as surrogates of total pool of the Glut4 reporter. This figure is adopted from previously published work (Chen et al., 2007) with permission.

SUMMARY In this chapter, we have outlined experimental protocols for assessing the exocyst function in insulin-stimulated outbound Glut4 trafficking toward the plasma membrane, as a specialized transport event following the endocytic recycling route in insulin-responsive cells (Leto & Saltiel, 2012). The biochemical characterization of the RalA GTPase that operates multimeric protein complexes could be adapted to other trafficking processes/steps activated by cellular signaling (Chen & Saltiel, 2011; Leto & Saltiel, 2012). Furthermore, the Myc7-Glut4-eGFP reporter serves as a useful surrogate of the exocytic flow and its design could be adapted to other transmembrane proteins (Bogan et al., 2003). Our results highlight the importance of coordination between cell signaling and membrane trafficking and call future investigations into this integrative field.

REFERENCES Bogan, J. S., Hendon, N., McKee, A. E., Tsao, T. S., & Lodish, H. F. (2003). Functional cloning of TUG as a regulator of GLUT4 glucose transporter trafficking. Nature, 425, 727e733. Cai, H., Reinisch, K., & Ferro-Novick, S. (2007). Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. Developmental Cell, 12, 671e682. Chang, L., Chiang, S. H., & Saltiel, A. R. (2007). TC10alpha is required for insulin-stimulated glucose uptake in adipocytes. Endocrinology, 148, 27e33. Chen, X. W., Inoue, M., Hsu, S. C., & Saltiel, A. R. (2006). RalA-exocyst-dependent recycling endosome trafficking is required for the completion of cytokinesis. The Journal of Biological Chemistry, 281, 38609e38616. Chen, X. W., Leto, D., Chiang, S. H., Wang, Q., & Saltiel, A. R. (2007). Activation of RalA is required for insulin-stimulated Glut4 trafficking to the plasma membrane via the exocyst and the motor protein Myo1c. Developmental Cell, 13, 391e404. Chen, X. W., Leto, D., Xiao, J., Goss, J., Wang, Q., Shavit, J. A., et al. (2011). Exocyst function regulated by effector phosphorylation. Nature Cell Biology, 13, 580e588. Chen, X. W., Leto, D., Xiong, T., Yu, G., Cheng, A., Decker, S., et al. (2011). A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action. Molecular Biology of the Cell, 22, 141e152. Chen, X. W., & Saltiel, A. R. (2011). Ral’s engagement with the exocyst: breaking up is hard to do. Cell Cycle, 10, 2299e2304. Chieregatti, E., & Meldolesi, J. (2005). Regulated exocytosis: new organelles for nonsecretory purposes. Nature Reviews Molecular Cell Biology, 6, 181e187. EauClaire, S., & Guo, W. (2003). Conservation and specialization. The role of the exocyst in neuronal exocytosis (comment). Neuron, 37, 369e370

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Feig, L. A. (2003). Ral-GTPases: approaching their 15 minutes of fame. Trends in Cell Biology, 13, 419e425. Gromley, A., Yeaman, C., Rosa, J., Redick, S., Chen, C. T., Mirabelle, S., et al. (2005). Centriolin anchoring of exocyst and SNARE complexes at the midbody is required for secretory-vesicle-mediated abscission. Cell, 123, 75e87. He, B., & Guo, W. (2009). The exocyst complex in polarized exocytosis. Current Opinion in Cell Biology, 21, 537e542. Huang, S., & Czech, M. P. (2007). The GLUT4 glucose transporter. Cell Metabolism, 5, 237e252. Inoue, M., Chang, L., Hwang, J., Chiang, S. H., & Saltiel, A. R. (2003). The exocyst complex is required for targeting of Glut4 to the plasma membrane by insulin. Nature, 422, 629e633. Leto, D., & Saltiel, A. R. (2012). Regulation of glucose transport by insulin: traffic control of GLUT4. Nature Reviews Molecular Cell Biology, 13, 383e396. Maxfield, F. R., & McGraw, T. E. (2004). Endocytic recycling. Nature Reviews Molecular Cell Biology, 5, 121e132. Munson, M., & Novick, P. (2006). The exocyst defrocked, a framework of rods revealed. Nature Structural & Molecular Biology, 13, 577e581. Novick, P., & Guo, W. (2002). Ras family therapy: Rab, Rho and Ral talk to the exocyst. Trends in Cell Biology, 12, 247e249. Saltiel, A. R., & Kahn, C. R. (2001). Insulin signalling and the regulation of glucose and lipid metabolism. Nature, 414, 799e806. Takai, Y., Sasaki, T., & Matozaki, T. (2001). Small GTP-binding proteins. Physiological Review, 81, 153e208. Watson, R. T., Kanzaki, M., & Pessin, J. E. (2004). Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes. Endocrine Reviews, 25, 177e204. Watson, R. T., & Pessin, J. E. (2006). Bridging the GAP between insulin signaling and GLUT4 translocation. Trends in Biochemical Sciences, 31, 215e222.

CHAPTER

GPCR sorting at multivesicular endosomes

20

Michael Robert Dores, JoAnn Trejo1 Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, CA, USA 1


CHAPTER OUTLINE Introduction ............................................................................................................ 320 1. Objectives and Rationale .................................................................................... 321 2. Materials and Instruments .................................................................................. 322 2.1 Reagents ............................................................................................ 322 2.1.1 Cell culture, media, and general reagents.......................................... 322 2.1.2 Antibodies ........................................................................................ 322 2.1.3 Proteinase K protection assay reagents ............................................. 323 2.1.4 Confocal immunofluorescence microscopy assay reagents................. 323 2.2 Instrumentation .................................................................................. 323 3. Methods ............................................................................................................ 323 3.1 Cell Plating and Transfection ............................................................... 323 3.2 Proteinase-Protection Assay ................................................................. 324 3.2.1 Workflow of experiment .................................................................... 324 3.2.2 Considerations.................................................................................. 326 3.2.3 Results............................................................................................. 326 3.3 Quantification of GPCR Sorting at MVEs Expanded by Rab5-Q79L Expression.......................................................................................... 327 3.3.1 Workflow of experiment .................................................................... 327 3.3.2 Considerations and alternative approaches........................................ 328 3.3.3 Results and fluorescence intensity analysis ....................................... 330 Summary ................................................................................................................ 330 References ............................................................................................................. 330

Abstract The lysosomal degradation of G protein-coupled receptors (GPCRs) is essential for receptor signaling and down regulation. Once internalized, GPCRs are sorted within the endocytic pathway and packaged into intraluminal vesicles (ILVs) that bud inward to form the multivesicular endosome (MVE). The mechanisms that control GPCR sorting and ILV Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.05.006 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 20 GPCR sorting at multivesicular endosomes

formation are poorly understood. Quantitative strategies are important for evaluating the function of adaptor and scaffold proteins that regulate sorting of GPCRs at MVEs. In this chapter, we outline two strategies for the quantification and visualization of GPCR sorting into the lumen of MVEs. The first protocol utilizes a biochemical approach to assay the sorting of GPCRs in a population of cells, whereas the second strategy examines GPCR sorting in individual cells using immunofluorescence confocal microscopy. Combined, these assays can be used to establish the kinetics of activated GPCR lysosomal trafficking in response to specific ligands, as well as evaluate the contribution of endosomal adaptors to GPCR sorting at MVEs. The protocols presented in this chapter can be adapted to analyze GPCR sorting in a myriad of cell types and tissues, and expanded to analyze the mechanisms that regulate MVE sorting of other cargoes.

INTRODUCTION G protein-coupled receptors (GPCRs) comprise the largest family of mammalian transmembrane signaling receptors and are important drug targets for the treatment of multiple diseases, including cancer and cardiovascular disease. Endosomal sorting and lysosomal degradation are critical for regulating GPCR signaling, and defects in receptor degradation impact many pathophysiological conditions. At late endosomes, activated GPCRs are packaged into membrane invaginations that bud inward to form intraluminal vesicles (ILVs) (Marchese, Paing, Temple, & Trejo, 2008). Once formed, multivesicular endosomes (MVEs) fuse with lysosomes, facilitating receptor degradation. A diverse array of adaptor and scaffold proteins mediate GPCR sorting into ILVs. GPCRs targeted for degradation by ubiquitination are bound by ubiquitin-binding subunits of the ESCRT (endosomal sorting complexes required for transport) complexes (Hislop & von Zastrow, 2011; Marchese et al., 2003; Shenoy et al., 2008). The ESCRT complexes are evolutionarily conserved (Hurley & Emr, 2006) and facilitate the biogenesis of MVEs containing GPCRs and other transmembrane proteins (Babst, 2005) through a process that involves cargo binding at early endosomes by the ESCRT-0/I complexes. The ESCRT-I complex recruits members of the ESCRT-II complex, which mediates ILV formation (Im, Wollert, Boura, & Hurley, 2009) and encaptures ubiquitinated cargo (Wollert & Hurley, 2010). Receptors are then deubiquitinated and the ESCRT-III complex mediates the scission of ILVs (Hurley & Hanson, 2010). However, some GPCRs are sorted into ILVs without being ubiquitinated. The adaptor proteins GASP1 and ALIX facilitate the interaction between specific GPCRs and the ESCRT complexes. GASP1 binds to the C-terminal tail of multiple GPCRs and facilitates interaction with all four ESCRT complexes (Cho et al., 2013; Henry, White, Marsh, von Zastrow, & Hislop, 2011). In addition, the adaptor protein ALIX directs the sorting of protease-activated receptor 1 (PAR1), a GPCR for the coagulant protease thrombin. ALIX binds to a YPXnL motif within the second intracellular loop of PAR1, and facilitates interaction between PAR1 and the ESCRT-III complex, bypassing the ubiquitin-binding ESCRT complexes (Dores, Chen, et al., 2012). However, the molecular mechanisms that regulate these pathways are not


well understood. In addition, these pathways are defined by studies of a small number of human GPCRs, suggesting that many more multivesicular endosomal sorting pathways could exist. Robust and quantitative strategies to assay GPCR sorting into the lumen of MVEs are important tools for investigating the regulation and dynamics of receptor sorting at the late endosome. This chapter will outline two experimental strategies for quantifying and visualizing GPCR sorting into the lumen of MVEs. The first strategy surveys GPCR sorting in a population of cells by analyzing the amount of GPCRs that are incorporated into MVEs and protected from protease cleavage. The second strategy employs confocal immunofluorescence microscopy to visualize and quantify the sorting of GPCRs into the lumen of expanded endosomes in individual cells. Together, these strategies can be used to quantify defects in GPCR endosomal sorting following the genomic or posttranscriptional manipulation of target endocytic adaptor and scaffold proteins, and define the dynamics of GPCR sorting into MVEs in a variety of cell types.

1. OBJECTIVES AND RATIONALE There are multiple approaches to studying the sorting of GPCRs into MVEs. Traditionally, immune-electron microscopy (IEM) has served as the benchmark for imaging GPCRs in ILVs and at the limiting membrane of MVEs. IEM allows for high resolution at high magnification and reveals valuable qualitative information regarding the subcellular and subendosomal localization of GPCRs in relation to other endosomal markers. However, electron micrographs are difficult to quantify, and time-course experiments are resource intensive. In addition, antibodies for many GPCRs are unavailable or not sensitive enough for IEM. In the following sections, we will outline two strategies that complement IEM studies, providing quantitative biochemical and visual evidence of GPCR sorting into the lumen of MVEs. The proteinase-protection assay is a robust method for studying the trafficking of GPCRs at MVEs. The assay requires gentle permeabilization of the plasma membrane that leaves internal endosomal membranes intact. This process has been described for studying endosome acidification (Diaz & Stahl, 1989), and is a powerful tool for quantifying GPCR levels within protective endosomal compartments. Following permeabilization of the plasma membrane, GPCRs that are presented at the plasma membrane and at the limiting membranes of endosomes are susceptible to proteinase degradation, whereas receptors that have been internalized into MVEs remain protected from proteinase cleavage. The levels of GPCRs within protective endosomes are measured by immunoblot analysis and quantified using densitometry or other methods such as the Odyssey system (LI-COR). This strategy allows for the survey of GPCR sorting in an entire population of cells, and can be expanded to encompass multiple time points in a single experiment. Immunofluorescence confocal microscopy is a robust method for assaying the localization of GPCRs within the cell and is widely used to investigate the role of

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adaptor and scaffold proteins in GPCR endosomal trafficking. However, the relatively small size of endosomes prevents quantitative analysis of receptor sorting into ILVs of MVEs using fluorescence microscopy techniques. Research focused on the biogenesis and regulation of endosomes has produced a number of useful tools for labeling specific endosomal populations and manipulating endosome morphology. Rab GTPases regulate fusion of vesicles and endosomes (Rink, Ghigo, Kalaidzidis, & Zerial, 2005) and define discrete endosomal compartments, including early, late, and recycling endosomes (Simons & Zerial, 1993). Specifically, Rab5 mediates homotypic fusion of endocytic vesicles that form early endosomes (Stenmark et al., 1994), and a constitutive active form of Rab5 (Rab5-Q79L) induces the formation of enlarged endosomes through increased endosomal membrane fusion (Stenmark et al., 1994) that retains the ability to sort endocytic cargo. The expression of Rab5-Q79L fused to green fluorescence protein (GFP) labels expanded endosome limiting membrane with green fluorescence, facilitating the visualization of GPCR sorting into the endosomal lumen (Dores, Paing, et al., 2012; Hislop, Henry, & von Zastrow, 2011). This technique complements the proteinase-protection assay and provides direct visual evidence of GPCR sorting into endosomes.

2. MATERIALS AND INSTRUMENTS 2.1 REAGENTS 2.1.1 Cell culture, media, and general reagents HeLa cells Plasmid constructs encoding: FLAG-PAR1 in pBJ vector (Trejo, Altschuler, Fu, Mostov, & Coughlin, 2000), Rab5-Q79L-GFP in pcDNA (Gullapalli et al., 2004) Dulbecco’s Modification of Eagles Medium (DMEM) þ glucose, L-glutamine, pyruvate Fetal bovine serum Penicillin Streptomycin Fibronectin Polyethylenimine (PEI) HEPES buffer Bovine serum albumen (BSA) Phosphate-buffered salt (PBS) without calcium or magnesium

2.1.2 Antibodies Immunoblotting Antibodies: Rabbit polyclonal anti-FLAG (Rockland Immunochemicals, Cat. No. 600-401-383), mouse monoclonal anti-early endosomal antigen-1 (EEA1) (BD Biosciences, Cat. No. 610457).

3. Methods

Immunofluorescence Antibodies: Rabbit polyclonal anti-FLAG (Rockland Immunochemicals, Cat. No. 600-401-383), Alexa FluorÒ 594-conjugated goat anti-rabbit (Life Technologies, Cat. No. A-11012).

2.1.3 Proteinase K protection assay reagents Dipotassium phosphate (K2HPO4) Monopotassium phosphate (KH2PO4) 1 M magnesium chloride (MgCl2) Sucrose Digitonin Proteinase K Phenylmethylsulfonyl fluoride (PMSF) Triton X-100

2.1.4 Confocal immunofluorescence microscopy assay reagents Paraformaldehyde (PFA) Saponin Goat serum Methanol Nonfat dry milk 3 M sodium acetate FluorSaveÔ Reagent (Millipore)

2.2 INSTRUMENTATION Microscope: Olympus IX81, spinning-disk confocal imaging system (DSU), PlanApo 60x oil objective (1.4 NA), ORCA-ER digital camera (Hamamatsu Photonics). Software: SlideBook 5.0 software (Intelligent Imaging Innovations).

3. METHODS 3.1 CELL PLATING AND TRANSFECTION HeLa cells are cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin, and streptomycin at 37 C in a 5% CO2 incubator. For proteinaseprotection assays, cells are split into fibronectin-coated 6-well polystyrene tissue culture plates at 5.0 105 cells/well and grown for 48 h until 80e90% confluent. For immunofluorescence confocal microscopy experiments, circular glass coverslips (Fisherbrand Cat. No. 12-454-100, 18CIR-1) that have been ethanol-dipped and dried are placed in 12-well polystyrene tissue culture plates. Coverslips are then precoated with 300 mL fibronectin for 15 min at room temperature. Cells are diluted to 0.75 105 cells/well and grown 24 h until 30e40% confluent. Cells are then

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transfected with plasmid expressing Rab5-Q79L-GFP using PEI. Transfected cells are grown 24e48 h until cells are 70e80% confluent prior to processing.

3.2 PROTEINASE-PROTECTION ASSAY Cells are first incubated with DMEM supplemented with 20 mM HEPES, 1 mg/mL BSA, and 2 mM leupeptin to inhibit lysosomal degradation at 37 C. Prior to stimulation, cells are incubated in a water-bath incubator at 37 C for 1 h, then stimulated for the appropriate times with agonist. The duration of stimulation will vary depending upon the GPCR, but will coincide with wild-type (WT) receptor degradation. Following stimulation, cells are placed on ice to halt membrane trafficking and washed with ice-cold PBS without Mg2þ or Ca2þ. Cells are then gently lifted from the plates using a cell scraper and pelleted by centrifugation. Cells are resuspended in DMEM media supplemented with 6.5 mg/mL digitonin to gently permeabilize the plasma membrane, leaving endosomal membranes intact. Following digitonin treatment, membranes are pelleted by high-speed centrifugation, then resuspended in homogenization buffer which is split into three equal fractions. The first fraction is left untreated and represents the total amount of receptor in limiting membranes as well as inside protective endosomal compartments (Figure 1(A)). The second fraction is treated with 2.5 ng/mL proteinase K, which will degrade any receptors on limiting membranes of endosomes or the plasma membrane (Figure 1(A)). The third fraction is treated with 2.5 ng/mL proteinase K and 0.1% Triton X-100 detergent. This treatment will degrade all membranes and serve as a control for proteinase K activity (Figure 1(A)). Samples are then treated with 2X Laemmli sample buffer supplemented with 20 mM PMSF, then analyzed by immunoblotting (Figure 1(B)). As an additional control for digitonin treatment, samples are probed with antibodies targeted to early endosome antigen 1 (EEA1), a cytosolic protein that associates strongly with the cytoplasmic surface of endosomes (Figure 1(B)). Immunoblots are quantified by densitometry using ImageJ or appropriate software. Statistical analysis of GPCR levels by 2-way ANOVA requires a minimum of three replicate experiments.

3.2.1 Workflow of experiment 1. Incubate cells for 1 h in 1 mL 37 C DMEM supplemented with 2 mM HEPES, 1 mg/mL BSA, and 2 mM leupeptin 2. Stimulate cells with agonist diluted in 1 mL DMEM supplemented with 2 mM HEPES and 1 mg/mL BSA preheated to 37 C 3. Remove cells from 37 C water bath and place on ice 4. Wash 1 with 1 mL ice-cold PBS (Ca2þ/Mg2þ-free) 5. Incubate cells in 1 mL ice-cold PBS for 5 min 6. Scrape cells and collect into prelabeled 1.5 mL tubes 7. Pellet cells at 1500 rpm for 5 min at 4 C 8. Resuspend cell pellets in 1 mL DMEM supplemented with 20 mM HEPES, 1 mg/mL BSA, 6.5 mg/mL digitonin

3. Methods

(A) Untreated

Proteinase-K GPCR

Proteinase-K + Triton X-100 GPCR

ILV

ILV

Late Endosome/ Multivesicular Body

(B)

Late Endosome/ Multivesicular Body

+SF 15min

- + + - - +

- + + - - +

Ctrl

+SF 15 min

- + + - - +

- + + - - +

kDa

IB: anti-FLAG

PAR1

64

148

IB: anti-EEA1

EEA1

98

lane:

PAR1 Remaining (Fraction of Untreated)

ILV Late Endosome/ Multivesicular Body

PAR1 Y206A

PAR1 WT Ctrl

Proteinase K: Triton X-100:

GPCR

1.00

1

2

3

4

Untreated + Proteinase K PAR1 WT ***

5 6

7

8

9 10 11 12

PAR1 Y206A

0.75 0.50 0.25 0.00

Ctrl

+SF 15 min Ctrl

+SF 15 min

FIGURE 1 Proteinase-protection endosomal sorting assay. (A) A schematic of proteinase treatment assay with predicted results. Untreated samples represent the total amount of GPCR. Treatment with proteinase K degrades all GPCRs exposed on the plasma membrane or limiting membranes of internal endosomes (dashed lines), whereas GPCRs sorted into intraluminal vesicles (ILVs) are protected from proteinase activity (solid lines). Membranes treated with Triton X-100 detergent acts as a control for proteinase K activity, exposing all receptors to proteinase K degradation. (B) Agonist-stimulated PAR1 sorting into protective endosomal compartments is mediated by the YPXnL motif localized in the second intracellular loop. HeLa cells expressing FLAG-PAR1 WT or FLAG-PAR1 Y206A were stimulated with 100 mM SFLLRN for 15 min. Cells were harvested and treated with digitonin to permeabilize the plasma membrane, then treated with either H2O (untreated), proteinase K, or proteinase K with Triton X-100 detergent. The amount of PAR1 was analyzed by immunoblot and quantified using densitometry (***, p < 0.001, n ¼ 3, 2-way ANOVA).

a. Incubate for 5 min at room temperature b. Incubate for 30 min on ice 9. Centrifuge at 14,000 rpm for 5 min at 4 C to pellet membranes 10. Resuspend cell pellets in 300 mL homogenization buffer (See Table 1)

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Table 1 Homogenization Buffer (pH 6.7) 100 mM K2HPO4/KH2PO4 5 mM MgCl2 250 mM sucrose H 2O

0.87 g K2HPO4/0.68 g KH2PO4 500 mL (1 M stock) 8.6 g 100 mL

11. Combine 100 mL of sample with either of the following: a. 1 mL H2O treat (no enzyme) b. 2.5 ng/mL proteinase K (add 1 mL of 250 ng/mL stock proteinase K) c. 2.5 ng/mL proteinase K þ 0.1% Triton X-100 (add 1 mL of 250 ng/mL stock proteinase K and 1 mL 10% Triton X-100 stock) 12. Incubate at room temperature for 10 min 13. Stop reaction by adding 100 mL 2X Laemmli sample buffer containing 20 mM PMSF 14. Boil at 100 C for 3 min prior to loading on to an SDS-PAGE gel

3.2.2 Considerations We have observed that digitonin and proteinase K have a limited shelf life typically between 2 and 3 weeks following dilution when stored at 4 C. Enzymes in solution can be frozen and stored for 2e3 months at 20 C. Samples may require vortexing immediately following boiling due to the presence of genomic DNA.

3.2.3 Results We found that stimulation of WT PAR1 with a synthetic peptide agonist SFLLRN (SF) induces translocation into protective endosomal compartments. In the absence of agonist, stimulation of PAR1 is sensitive to proteinase K treatment (Figure 1(B), lane 2), indicating that the majority of PAR1 is localized to limiting membranes at steady state. In contrast, PAR1 stimulated with SFLLRN for 15 min is protected from proteinase K degradation (Figure 1(B), lane 5). PAR1 is degraded in samples treated with proteinase K and Triton X-100 detergent, confirming proteinase K activity (Figure 1(B), lanes 3 and 6). In comparison, PAR1 Y206A, a mutant that cannot bind to the adaptor protein ALIX and is not degraded (Dores, Chen, et al., 2012), fails to be protected from proteinase K degradation following agonist stimulation (Figure 1(B), lane 11). The degradation of EEA1 indicates that digitonin treatment permeabilized the plasma membrane, allowing proteinase K access to proteins anchored to the cytosolic side of endosomal limiting membranes. These data provide quantitative evidence that activated PAR1 is sorted into the lumen of endosomes and protected from protease cleavage, and suggest that mutation of the ALIX binding site on PAR1 blocks receptor sorting into the endosomal lumen.

3. Methods

3.3 QUANTIFICATION OF GPCR SORTING AT MVEs EXPANDED BY Rab5-Q79L EXPRESSION Cells should be covered or protected from direct light during incubations to prevent photo bleaching of the Rab5-Q79L-GFP. Cells transfected with Rab5Q79L-GFP were seeded onto 18 mm glass coverslips (Fisherbrand Cat. No. 12-454-100, 18CIR-1) and preincubated in DMEM supplemented with 20 mM HEPES, 1 mg/mL BSA, and 2 mM leupeptin to inhibit lysosomal degradation. Following pretreatment, cells are shifted to ice and chilled to halt membrane trafficking. Primary antibody against the GPCR is then added to each well to label the surface cohort of receptor while maintaining leupeptin treatment. Following primary antibody labeling for 1 h, cells are washed two times with ice-cold DMEM. Cells are then stimulated with prewarmed 37 C media containing agonist and shifted to a 37 C water bath. Unstimulated control plates are maintained on ice. Following stimulation, cells are shifted back to ice and washed once with ice-cold PBS. Cells are fixed with 4% PFA, washed with ice-cold PBS, then permeabilized with a short methanol treatment. Permeabilized cells are gently washed twice with ice-cold PBS. The coverslips are removed from the wells onto a flat surface coated with parafilm at room temperature. Cells are incubated in quench buffer that is pipetted directly on top of the coverslips. Cells are then incubated with wash buffer, followed by incubation with secondary antibody diluted in wash buffer for 1 h at room temperature. Following incubation with secondary antibody, coverslips are washed with room temperature PBS and then mounted onto glass slides using FluorSaveÔ reagent.

3.3.1 Workflow of experiment 1. Incubate cells for 1 h in 0.4 mL 37 C DMEM supplemented with 2 mM HEPES, 1 mg/mL BSA, and 2 mM leupeptin 2. Place cells on ice, incubate 10 min to chill cells 3. Dilute primary antibody in ice-cold DMEM supplemented with 2 mM HEPES and 1 mg/mL BSA and add to wells a. For a 1:1000 antibody dilution: 0.5 mL primary antibody (1.0 mg/mL)/well diluted in 100 mL DMEM (500 mL total volume/well) 4. Incubate on ice for 1 h to surface-label receptor expressed in cells 5. Wash 2 with 500 mL ice-cold DMEM supplemented with 2 mM HEPES and 1 mg/mL BSA 6. Stimulate cells with agonist diluted in 500 mL DMEM supplemented with 2 mM HEPES and 1 mg/mL BSA, preheated to 37 C 7. Remove cells from 37 C water bath and place on ice 8. Wash 1 with 500 mL ice-cold PBS (Ca2þ/Mg2þ-free) 9. Incubate on ice with 500 mL ice-cold 4% PFA (pH 7.2) for 5 min to fix cells 10. Wash 1 with 500 mL ice-cold PBS (Ca2þ/Mg2þ-free) 11. Add 500 mL ice-cold 100% methanol for 30 s to permeabilize cells 12. Wash 2 with 500 mL ice-cold PBS (Ca2þ/Mg2þ-free)

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Table 2 Quench and Wash Buffers 1% Nonfat dry milk 150 mM sodium acetate 1 PBS Total volume

Quench Buffer

Wash Buffer

0.15 g 750 mL (3 M stock) 14.25 mL 15 mL

0.3 g 30 mL 30 mL

13. Remove coverslips from wells and place onto a flat surface covered with parafilm at room temperature 14. Incubate 3 with 300 mL quench buffer (See Table 2) for 5 min at room temperature, bubbled on top of coverslips 15. Incubate 3 with 300 mL wash buffer (See Table 2) for 5 min 16. Incubate with 300 mL secondary antibody diluted in wash buffer for 1 h at room temperature 17. Wash 4 with 300 mL PBS (Ca2þ/Mg2þ-free) for 5 min at room temperature 18. Place 25 mL FluorSaveÔ reagent (Millipore) onto glass slide, then invert coverslip onto drop 19. Aspirate excess FluorSaveÔ reagent from edges of coverslip 20. Dry slides 1e24 h prior to collecting images

3.3.2 Considerations and alternative approaches Due to the short duration of methanol treatment, we recommend treating four or fewer samples at a time. Methanol permeabilization may not be optimal for labeling certain GPCRs and cytosolic proteins. We have also used saponin to permeabilize cells prior to antibody labeling with minimal effect on the morphology of Rab5Q79L-expanded endosomes. Following fixing, coverslips are removed from wells and placed on a flat surface covered with parafilm at room temperature. Samples are incubated for 30 min in blocking buffer (PBS supplemented with 1% goat serum and 0.03% saponin) to facilitate permeabilization. Samples are washed with PBS and then treated for 1 h with secondary antibody diluted in blocking buffer. Unbound antibody is washed away with PBS and coverslips are mounted to glass slides using FluorSaveÔ reagent.

3.3.2.1 Workflow of saponin permeabilization 1. Complete steps 1e10 above 2. Remove coverslips and place onto a flat surface covered with parafilm at room temperature, cell-side up 3. Incubate for 30 min at room temperature with 300 mL blocking buffer (PBS supplemented with 1% goat serum and 0.03% saponin) 4. Wash 2 with 300 mL PBS, no incubation 5. Label for 1 h at room temperature with 300 mL secondary antibody diluted in blocking buffer

3. Methods

6. 7. 8. 9.

Wash sequentially 3 with 300 mL PBS, no incubation Place 25 mL FluorSaveÔ reagent onto glass slide, then invert coverslip onto drop Aspirate excess FluorSaveÔ reagent from edges of coverslip Dry slides 1e24 h prior to collecting images

(A) PAR1 WT Rab5 Q79L

P1 WT

+SF Rab5 Q79L +SF MERGE 15 min 15 min

+SF 15 min

PAR1 Y206A Rab5 Q79L

P1 Y206A


+SF 15 min

(B)

(C)

PAR1 WT

PAR1 Endosomal Fluorescence (A.U.)

Rab5 Q79L Fluorescence (A.U.)

10.0 7.0 6.0 5.0 4.0 3.0 2.0 -1.0 -0.5 0.0

0.5

1.0

1.5

2.0

***

7.5 5.0 2.5 0.0

WT

Y206A

FIGURE 2 Visualization and quantification of GPCR sorting into expanded endosomes. (A) Agoniststimulated PAR1 sorting into the lumen of expanded endosomes is mediated by the YPXnL motif. HeLa cells expressing FLAG-PAR1 WT or FLAG-PAR1 Y206A were transfected with Rab5-Q79L-GFP, then surface-labeled with anti-FLAG antibody. Cells were stimulated for 15 min with 100 mM SFLLRN, then fixed and analyzed by confocal microscopy. (B) Line scan analysis of Rab5-Q79L-GFP endosomes. GFP-fluorescence intensity was quantified by line scan (dashed line) and plotted to determine the limiting edges of the endosome (black vertical lines). (C) The PAR1 Y206A mutant is defective for sorting into the lumen of endosomes. PAR1 fluorescence intensity was measured within the limits defined by Rab5Q79L-GFP (B) and averaged from multiple expanded endosomes (***, p < 0.001, n ¼ 6, Student’s t-test). (See color plate)

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3.3.3 Results and fluorescence intensity analysis Activated PAR1 sorts into ILVs of MVEs enlarged by Rab5-Q79L expression. The expression of Rab5-Q79L-GFP induces the expansion of endosomal compartments, but does not affect the surface expression of unstimulated PAR1 (Figure 2(A)). Treatment of cells with the PAR1 peptide agonist SFLLRN for 15 min induces WT receptor internalization and sorting to the limiting membrane and the lumen of Rab5-positive endosomes (Figure 2(A)). In comparison, agonist stimulation of the PAR1 Y206A mutant induces trafficking to the limiting membrane of Rab5positive endosomes, however, the Y206A mutant receptor is not sorted into the lumen of expanded endosomes (Figure 2(A)). To quantify the amount of PAR1 sorted into the lumen of endosomes expanded by Rab5-Q79L expression, we used a line scan tool available in the SlideBook 5.0 microscope software to measure fluorescence intensity. Alternatively, line scans can be performed using ImageJ. The fluorescence intensity of Rab5-Q79L-GFP defines the limiting membrane of the endosome (Figure 2(B)), and fluorescence intensity of PAR1 is represented as an average of intensity values measured between the Rab5-maxima. Consistent with our proteinase-protection assay, PAR1 WT fluorescence intensity is significantly higher in the lumen of expanded MVEs compared to PAR1 Y206A (Figure 2(C)). These results provide visual confirmation that activated PAR1 is sorted into the lumen of endosomes, and that ALIX binding to PAR1 is required for receptor sorting into ILVs.

SUMMARY In this chapter, we have described two techniques for the quantification and visualization of GPCR sorting into MVEs. We have outlined a biochemical strategy for the quantification of GPCR sorting into protective endosomal compartments, and we have also provided a protocol for visualizing GPCR sorting into MVEs using confocal immunofluorescence microscopy. These methods can be used to investigate the function of endocytic adaptor and scaffold proteins in the endolysosomal sorting of specific GPCRs. These strategies also provide temporal and spatial information about the sorting of GPCRs in cell populations and within individual cells. Our results demonstrate the strength of these techniques in defining the components and regulatory mechanisms of novel endosomal sorting pathways for GPCRs.

REFERENCES Babst, M. (2005). A protein’s final ESCRT. Traffic, 6(1), 2e9. Cho, D. I., Zheng, M., Min, C., Kwon, K. J., Shin, C. Y., Choi, H. K., et al. (2013). ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D(2) receptors mediated by GRK and PKC in transfected cells. British Journal of Pharmacology, 168(6), 1355e1374. http://dx.doi.org/10.1111/bph.12025.

References

Diaz, R., & Stahl, P. D. (1989). Chapter 2 Digitonin permeabilization procedures for the study of endosome acidification and function. In M. T. Alan (Ed.), Methods in cell biology (Vol. 31, pp. 25e43). Academic Press. Dores, M. R., Chen, B., Lin, H., Soh, U. J. K., Paing, M. M., Montagne, W. A., et al. (2012). ALIX binds a YPX3L motif of the GPCR PAR1 and mediates ubiquitin-independent ESCRT-III/MVB sorting. The Journal of Cell Biology, 197(3), 407e419. http:// dx.doi.org/10.1083/jcb.201110031. Dores, M. R., Paing, M. M., Lin, H., Montagne, W. A., Marchese, A., & Trejo, J. (2012). AP-3 regulates PAR1 ubiquitin-independent MVB/lysosomal sorting via an ALIX-mediated pathway. Molecular Biology of the Cell, 23(18), 3612e3623. http://dx.doi.org/10.1091/ mbc.E12-03-0251. Gullapalli, A., Garrett, T. A., Paing, M. M., Griffin, C. T., Yang, Y., & Trejo, J. (2004). A role for sorting nexin 2 in epidermal growth factor receptor down-regulation: evidence for distinct functions of sorting nexin 1 and 2 in protein trafficking. Molecular Biology of the Cell, 15(5), 2143e2155. http://dx.doi.org/10.1091/mbc.E03-09-0711. Henry, A. G., White, I. J., Marsh, M., von Zastrow, M., & Hislop, J. N. (2011). The role of ubiquitination in lysosomal trafficking of delta-opioid receptors. Traffic, 12(2), 170e184. http://dx.doi.org/10.1111/j.1600-0854.2010.01145.x. Hislop, J. N., Henry, A. G., & von Zastrow, M. (2011). Ubiquitination in the first cytoplasmic loop of m-opioid receptors reveals a hierarchical mechanism of lysosomal downregulation. The Journal of Biological Chemistry, 286(46), 40193e40204. http:// dx.doi.org/10.1074/jbc.M111.288555. Hislop, J. N., & von Zastrow, M. (2011). Role of ubiquitination in endocytic trafficking of G-protein-coupled receptors. Traffic, 12(2), 137e148. http://dx.doi.org/10.1111/j.16000854.2010.01121.x. Hurley, J. H., & Emr, S. D. (2006). The ESCRT complexes: structure and mechanism of a membrane-trafficking network. Annual Review of Biophysics and Biomolecular Structure, 35, 277e298. Hurley, J. H., & Hanson, P. I. (2010). Membrane budding and scission by the ESCRT machinery: it’s all in the neck. Nature Reviews Molecular Cell Biology, 11(8), 556e566. Im, Y. J., Wollert, T., Boura, E., & Hurley, J. H. (2009). Structure and function of the ESCRT-II-III interface in multivesicular body biogenesis. Developmental Cell, 17(2), 234e243. Marchese, A., Paing, M. M., Temple, B. R. S., & Trejo, J. (2008). G protein-coupled receptor sorting to endosomes and lysosomes. Annual Review of Pharmacology and Toxicology, 48(1), 601e629. http://dx.doi.org/10.1146/annurev.pharmtox.48.113006.094646. Marchese, A., Raiborg, C., Santini, F., Keen, J. H., Stenmark, H., & Benovic, J. L. (2003). The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4. Developmental Cell, 5(5), 709e722. Rink, J., Ghigo, E., Kalaidzidis, Y., & Zerial, M. (2005). Rab conversion as a mechanism of progression from early to late endosomes. Cell, 122(5), 735e749. Shenoy, S. K., Xiao, K., Venkataramanan, V., Snyder, P. M., Freedman, N. J., & Weissman, A. M. (2008). Nedd4 mediates agonist-dependent ubiquitination, lysosomal targeting, and degradation of the beta2-adrenergic receptor. The Journal of Biological Chemistry, 283(32), 22166e22176. http://dx.doi.org/10.1074/jbc.M709668200. Simons, K., & Zerial, M. (1993). Rab proteins and the road maps for intracellular transport. Neuron, 11, 789e799.

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Stenmark, H., Parton, R. G., Steele-Mortimer, O., Lutcke, A., Gruenberg, J., & Zerial, M. (1994). Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. The EMBO Journal, 13(6), 1287e1296. Trejo, J., Altschuler, Y., Fu, H.-W., Mostov, K. E., & Coughlin, S. R. (2000). Proteaseactivated Receptor-1 down-regulation. The Journal of Biological Chemistry, 275(40), 31255e31265. http://dx.doi.org/10.1074/jbc.M003770200. Wollert, T., & Hurley, J. H. (2010). Molecular mechanism of multivesicular body biogenesis by ESCRT complexes. Nature, 464(7290), 864e869.

CHAPTER

Visualizing and quantitating sequencedependent GPCR recycling

21

Shanna L. Bowman, Amanda L. Soohoo, Manojkumar A. Puthenveedu1 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA 1


CHAPTER OUTLINE Introduction ............................................................................................................ 334 1. Objectives and Rationale .................................................................................... 335 2. Materials and Instruments .................................................................................. 335 2.1 Reagents ............................................................................................ 335 2.2 Equipment ......................................................................................... 336 3. Methods ............................................................................................................ 336 3.1 Cell Culture, Plating, and Transfection .................................................. 336 3.2 Live Cell Imaging Conditions ................................................................ 336 3.3 Experimental Strategies ....................................................................... 337 3.3.1 Strategy 1: ensemble measurement of surface levels......................... 337 3.3.2 Strategy 2: quantifying recycling at single-event resolution ................. 339 3.3.3 Strategy 3: visualization of sorting and recycling in the endosome ...... 341 Summary ................................................................................................................ 344 References ............................................................................................................. 344

Abstract Recent advances in direct imaging have given us a new appreciation of the spatial and temporal dynamics of membrane trafficking processes, and have allowed us to ask questions that were difficult to address with traditional methods. A relevant example of this is protein sorting in the endosome, which serves as the primary sorting station for proteins internalized from the cell surface. In this chapter, we discuss fluorescence imaging protocols to directly visualize and quantitate the recycling of G protein-coupled receptors (GPCRs)da highly physiologically relevant family of signaling receptorsdin real time in living cells. The protocols allow direct visualization and quantitation of both GPCR exit from the endosome and GPCR delivery to the cell surface. The methods may be extended Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.05.007 © 2015 Elsevier Inc. All rights reserved.

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to study the endolysosomal sorting of many proteins that undergoes endocytic cycling, and may be adapted to other organelles and systems where proteins are sorted.

INTRODUCTION How organelles sort cargo proteins to various destinations while maintaining their own identity is a fundamental question in cell biology. The early endosome, the primary sorting station for internalized proteins, serves as a poster child for this question. The endosome sorts internalized membrane proteins to one of four main pathways. First, nutrient receptors are transported to the cell surface as part of bulk membrane flow by geometric sorting (Dunn, McGraw, & Maxfield, 1989; Maxfield & McGraw, 2004; Mayor, Presley, & Maxfield, 1993). Second, many signaling receptors are targeted to the lysosome to be degraded (Hislop, Henry, & von Zastrow, 2011; Huang et al., 2013; Marchese, Paing, Temple, & Trejo, 2008; Tanowitz & Von Zastrow, 2002; Trejo, 1999). Third, some proteins are transported in a retrograde manner to the Golgi apparatus (Cheng & Filardo, 2012; Chia, Gunn, & Gleeson, 2013; Johannes & Wunder, 2011; Zhao et al., 2013). Fourth, many members of the G protein-coupled receptor (GPCR) familydby far the largest and physiologically most diverse family of signaling receptorsdare recycled in a regulated sequence-dependent manner, distinct from bulk recycling (Hanyaloglu & von Zastrow, 2008). While the mechanisms directing proteins into the first three pathways (geometric sorting by tubule-forming proteins, involution by the endosomal sorting complexes required for transport proteins, and retrograde transport by the retromer complex) have been studied at structural and functional levels, we have only recently made inroads into understanding sequence-dependent recycling of GPCRs (Drake, Shenoy, & Lefkowitz, 2006; Hanyaloglu & von Zastrow, 2008; Marchese et al., 2008; Romero, von Zastrow, & Friedman, 2011). A major hurdle in understanding GPCR sorting had been the lack of assays to directly visualize and quantitate endosomal sorting in living cells in real time. Recent advances in fluorescence microscopy-based approaches have enabled us to overcome this hurdle and to resolve endosomal sorting with high spatial and temporal resolution (Puthenveedu et al., 2010; Vistein & Puthenveedu, 2013; Yu, Dhavan, Chevalier, Yudowski, & Zastrow, 2010; Yudowski, Puthenveedu, Henry, & von Zastrow, 2009). A relevant aspect of this is that small kinetic changes in recycling, as is seen with physiological regulation by signaling pathways, are often difficult to detect using traditional assays, but these might cause fairly significant cumulative changes at physiological timescales. Here we describe protocols for using fluorescence imaging to visualize and quantitate the recycling of internalized GPCRs, using two prototypic receptorsdthe beta 2 adrenergic receptor (B2AR) and the mu opioid receptor (MOR). We outline methods to image both the sorting of GPCRs into recycling domains on the endosome and their delivery to the cell surface, at ensemble (whole cell) scales as well as at single-event resolution. These assays, using live cell imaging in real time, allow for detection of even small changes in the rates of recycling.


1. OBJECTIVES AND RATIONALE The overall goal of this chapter is to define protocols for quantitating GPCR recycling using fluorescence imaging. We will outline protocols using confocal microscopy and total internal reflection fluorescence microscopy (TIRFM) to measure receptor delivery to the cell surface at ensemble and single-event spatial scales. We will also describe the use of confocal microscopy to visualize the sorting of fluorescently tagged receptors at the endosome, spatially resolved at the level of individual microdomains on endosomes that mediate GPCR recycling. To measure recycling at the whole cell level, receptors tagged to a pH-sensitive GFP (superecliptic pHluorin, SpH) will be used as a biosensor. This fluorophore is highly fluorescent at pH 7.1 or above. Reduction of pH below 7.1 causes a rapid single protonation of the GFP, and a practically instantaneous reduction in fluorescence by w50-fold (Miesenbo¨ck, De Angelis, & Rothman, 1998). When receptors are tagged with SpH on their extracellular N-termini, the total fluorescence is an accurate estimate of the surface receptor levels, because the pH of most intracellular organelles is less than 7, and because endocytic vesicles are rapidly acidified (pH < 7). This property makes this sensor ideal to measure dynamic changes in surface receptor levels in response to activation. Endocytosis following receptor activation causes a fluorescence decrease because the SpH is in the lumen of endosomes, and recycling causes an increase in fluorescence, as the SpH is exposed to the extracellular medium and is dequenched when receptors return to the plasma membrane. Individual recycling events can be measured by imaging receptors tagged with SpH, using TIRFM to visualize the surface of cells. As vesicles of SpH-tagged receptors fuse with the membrane, SpH is dequenched and individual recycling events can be detected as transient bursts of fluorescence. To measure the dynamics of GPCR endosomal sorting, receptors N-terminally tagged with a FLAG epitope are specifically labeled on the cell surface with fluorescent anti-FLAG. Labeled receptors are visualized in endosomes, following endocytosis, using live cell confocal fluorescence microscopy with high spatial and temporal resolution.

2. MATERIALS AND INSTRUMENTS 2.1 REAGENTS Human embryonic kidney (HEK 293) cells Plasmid constructs encoding signal sequence (ss)-superecliptic pHluorin (SpH)tagged GPCR or signal sequence (ss)-FLAG (DDDDK) epitope-tagged GPCR of choice (e.g., the MOR or B2AR) Dulbecco’s minimum essential medium (DMEM), high glucose Leibovitz’s L15, CO2-independent medium, without phenol red Fetal bovine serum (FBS) Effectene transfection reagent, Qiagen (or an equivalent transfection reagent)

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Geneticin (G418) #1.5 mm coverslip Specific agonist for the GPCRde.g., [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) for the MOR or isoproterenol for the B2AR. Immersion oil, type 37LDF (Cargille)

2.2 EQUIPMENT A microscopy system is capable of imaging in the confocal and TIRFM modes, with appropriate excitation modes. We use a Nikon Eclipse Ti-inverted microscope with a 60x or 100x 1.49 NA. TIRF objective, with an Andor Revolution spinning disk confocal system, using 50 mW 488 and 561 DPSS lasers as light sources with appropriate filter sets. The microscope stage is housed within a temperature- and environment-controlled chamber. Coverslip holder and chamber for live imaging. Alternatively, glass-bottomed dishes in which to plate cells.

3. METHODS 3.1 CELL CULTURE, PLATING, AND TRANSFECTION HEK 293 cells are grown in DMEM with 10% by volume FBS at 37 C with 5% CO2. Phenol red containing DMEM may be used during cell culture, but should be removed during any live cell microscopy assay as the phenol red contributes significantly to background fluorescence. Cells are plated onto #1.5 mm coverslip to reach 70% confluency after overnight incubation. The cells are transfected with SpH-tagged receptor (for the ensemble assay, Section 3.3.1, or the single-event recycling assay, Section 3.3.2) or Flag-tagged receptor (endosomal sorting assay, Section 3.3.3) using a lipid-based transfection reagent, such as Effectene (Qiagen). Cells should be imaged at 72e96 h after transfection. Alternatively, transfected cells may be placed under selection for 1e2 weeks to generate a stable cell line for the expressed plasmid. The stable cell lines are plated onto #1.5 coverslip for imaging at 50e90% confluency.

3.2 LIVE CELL IMAGING CONDITIONS The coverslips with HEK 293 cells are moved to a live cell imaging chamber, and imaged in Leibovitz’s L15, CO2-independent medium, without phenol red and with 5% FBS (imaging media). For live cell imaging of receptor recycling and endosomal sorting, it is essential that experiments are performed at 37 C, the physiological temperature for mammalian cells, because membrane trafficking events are very sensitive to temperature. Individual imaging protocols are described below for three separate experiments: ensemble measurement of surface levels, quantifying recycling at single-event resolution, and visualization of sorting and recycling in the endosome.

3. Methods

3.3 EXPERIMENTAL STRATEGIES 3.3.1 Strategy 1: ensemble measurement of surface levels 3.3.1.1 Background and objective A straightforward method to assay GPCR recycling is to measure the overall number of receptors at the cell surface across several cells. Here, we describe an ensemble assay to measure the surface levels of receptors over time. Cells expressing the SpHeN-terminally tagged MORs are used as an example, described in Section 1. SpH-MOR will fluoresce at the cell surface when SpH is exposed to the neutral environment of the extracellular medium. When SpH-MORs are activated by the synthetic opioid peptide, DAMGO (described in Materials), endocytosis will redistribute MOR to acidic endosomal compartments, and SpH-MOR fluorescence will decrease as SpH is quenched (Figure 1(A)). This decrease can be used to measure endocytosis of receptors in a time-resolved manner. Similarly, recycling can be measured as an increase in surface fluorescence of SpH-MOR upon removal of agonist (Figure 1(A)). Cells are imaged at 20x magnification with a relatively low (0.4e0.75) numerical aperture objective, for optimum depth of field and optimum number of cells within one field. Several fields can be imaged to gather a large sample size of cells. The effects of drugs and other manipulations in the cell can be measured across treatments by adding drugs with agonist or during the washout to test for an effect on receptor recycling.

FIGURE 1 Strategy 1: Ensemble measurement of surface levels. (A) Montage of fields visualized in the live cell ensemble SpH recycling assay. SpH-MOR is localized primarily to the surface of cells. Following DAMGO addition, surface fluorescence decreases over time as receptors endocytose and SpH is quenched in acidic endosomes. Following DAMGO washout, and addition of antagonist, SpH-MOR recycles, as seen by the increase of cell surface signal. (B) Example of SpH-MOR fluorescence intensity data in ensemble recycling assay. The mean fluorescence intensity was measured for a whole field of cells, and the graph shows the average fluorescence intensity across 20 fields. After DAMGO was added, SpH-MOR intensity decreased to around 50% of the baseline fluorescence after 20 min, indicative of receptor endocytosis. Following the washout, mean fluorescence increased to approximately 80% of the initial surface fluorescence after 20 min. Error bars are s.e.m.

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3.3.1.2 Flow of experiments 1. Plate SpH-tagged receptor, for example, SpH-MOR, on #1.5 coverslip. We generate stable cell lines with Geneticin selection, following transfection of SpH-MOR with the Effectene transfection reagent, according to the manufacturer’s protocol. 2. Mount coverslips in a live cell imaging chamber. Alternatively, glass bottom dishes can be used. 3. Replace growth medium (i.e., DMEM þ 10% FBS if using HEK 293 cells) with imaging medium (L15 with 5% FBS). 4. Use a lower magnification objective (i.e., 20x) in order to include several cells in one field. 5. Using confocal fluorescence microscopy and a 488 nm excitation laser, focus cells until membrane SpH fluorescence is crisp and concentrated at the periphery of the cell (Figure 1(A)). For a GPCR-like MOR that is expressed primarily at the cell surface, a strong surface SpH signal should be visible. 6. Choose 10 fields of cells with similar brightness and cell number. It is best if cells are 90e95% confluent and in a smooth monolayer. 7. Acquire a time-lapse image, acquiring every 1 min, with the lowest laser intensity that gives a reasonable dynamic range, to prevent phototoxicity and bleaching of SpH. 8. Acquire a 5 min baseline of surface SpH fluorescence. This will give an estimate of dynamic range and bleaching. 9. To measure recycling following a washout of receptor agonist, acquire a 15e 20 min time lapse following agonist addition, then wash out agonist media two times, replace it with antagonist media, and acquire a 20e30 min time lapse to measure recycling. 10. To quantitate endocytosis and recycling, analyze the mean fluorescence intensity of each field of cells, or of each individual cell using image segmentation, and plot the mean fluorescence intensity over time.

3.3.1.3 Considerations Persistent agonist exposure shifts the steady of receptors to an intracellular pool. Therefore, washing out agonist and replacing it with antagonist allow for detection of recycling without the confounding effects of continued endocytosis (Bowman et al., 2015; Hanyaloglu, McCullagh, & von Zastrow, 2005; Tanowitz & von Zastrow, 2003; Yu, Arttamangkul, Evans, Williams, & von Zastrow, 2009). However, rapid recycling begins shortly after agonist-induced endocytosis (Vistein & Puthenveedu, 2013; Yudowski et al., 2009). Therefore, it is important to consider that the SpH-tagged receptor levels in the presence of agonist are a combination of both recycling and endocytosis. A strategy to specifically measure recycling in the presence of persistent agonist is described below in Strategy 2dQuantifying recycling at single-event resolution.

3. Methods

3.3.1.4 Results The fluorescence intensity over time will measure endocytosis and recycling of receptors during the time lapse. As receptors endocytose, the mean fluorescence intensity decreases, and as the receptors recycle back to the cell surface, it will increase. For example, SpH-MOR mean intensity decreases during the 20 min of agonist exposure, consistent with receptor endocytosis to endosomes and quenching of the SpH sensor (Figure 1(B)). Following the agonist washout, SpH-MOR fluorescence intensity increased, indicative of MOR recycling and dequenching of SpH as vesicles fuse with the plasma membrane (Figure 1(B)). The average changes of fluorescence typically fit single exponential curves, as expected.

3.3.2 Strategy 2: quantifying recycling at single-event resolution 3.3.2.1 Background and objective The surface population of receptors measured in the presence of agonist reflects the balance between endocytosis and recycling. Therefore, ensemble assays rely on experimental paradigms such as agonist washout to estimate recycling. In the presence of agonist, independent measures of endocytosis, measured under conditions known to block recycling, are needed for ensemble assays to indirectly estimate recycling. Here we describe the use of TIRFM to directly visualize individual vesicle fusion events that mediate GPCR recycling in real time in live cells. TIRFM relies on finding the critical angle where the light source does not pass through the sample, but instead gets reflected back toward the source (Ambrose, 1956; Axelrod, 1981). This specifically illuminates the cell surface nearest to the coverslip (Figure 2(A)). This assay can be used to directly resolve recycling without the confounding effects of endocytosis, even in the persistent presence of agonist (Vistein & Puthenveedu, 2013; Yudowski et al., 2009). This assay is very efficient for quantitating the changes in the number of recycling events per unit area per time, as well as the amount of receptors in individual recycling events, in the same cells after acute manipulations such as drug treatments. This minimizes cell to cell variability, and allows collection of statistically significant data from low number of cells.

3.3.2.2 Flow of experiment 1. Plate cells expressing SpH-tagged receptor on #1.5 mm coverslip so they are 50e90% confluent 48 h after plating, on the day of imaging. 2. Mount the coverslip onto the live cell imaging chamber in L15 medium containing 5% FBS by volume. 3. Add a drop of immersion oil on top of the oil immersion 1.49 NA 60x TIRF objective. 4. Place the live cell imaging chamber on the stage and bring cells into focus. 5. Switch to TIRFM illumination mode. To do this, first, focus on the center of the cell. Since the SpH tag is fluorescent practically only on the surface, the plasma membrane should appear as a ring (Figure 2(A), images on the left). Then move the angle of the laser down until all fluorescence is lost. Then focus

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FIGURE 2 Strategy 2: Quantifying recycling at single-event resolution. (A) Schematic and corresponding example images for focusing a cell in TIRFM. When the excitation light passes straight through the sample, the center of the cell is readily visible. As the angle is moved down toward the critical angle, out of focus structures become more apparent in the “low-angle” illumination. Once past the critical angle, only the plasma membrane near the coverslip will be illuminated. The cell should appear bright, crisp, and uniform when focused down. (B) Example of a single recycling event. The whole cell is shown at the left. The inset focuses on a single recycling event. The recycling event first appears as a single bright spot whose fluorescence eventually diffuses out as vesicles fuse. Individual recycling events are very fast, each lasting less than a second. The scale bar is 5 mm. (See color plate)

6. 7. 8. 9. 10. 11. 12.

down toward the coverslip to see the plasma membrane in the TIRF field (Figure 2(A), images on the right). Select a bright (1000 counts or higher), large, flat cell that does not overlap with other cells. Nonoverlapping cells will be easier to analyze and quantify. Activate the receptors by addition of agonist and allow for internalization for 5 min. The time may be adjusted depending on the response of the receptor. Turn up the laser power to 60e70% to bleach the surface receptors until the signal events become evident. This will take about 10 s. Image the cell for 1 min at 10 frames per second. Recycling events appear as abrupt bursts of fluorescence that rapidly dissipate. Add secondary treatment to cells. This can be any acute manipulation, such as drugs. Take second image for 1 min at 10 frames per second. Save all image files. Have an unbiased third party or computer script scramble all file names before quantification.

3. Methods

13. Manually count the number of recycling events per cell by selecting a region of interest. Count each cell three times and take the average of these trials as the final recycling event count number. 14. Normalize the number of recycling events to the first series. If doing a nonpaired comparison (i.e., comparing different cells) normalize the areas to compare the number of recycling events per unit area.

3.3.2.3 Considerations Although the vast majority of events detected in the assay are recycling there is the potential to detect insertion of newly synthesized receptors from the biosynthetic pathway. Mock agonist treatment or cycloheximide pretreatment controls should be performed to verify that the fusion events are indeed recycling events, and not insertion of newly synthesized proteins. Recycling events require prior agonistmediated internalization, and will not be affected by cycloheximide. Due to the pH-sensitive nature of the SpH tag, the fluorescence should be restricted to the cell surface. Internal fluorescence may be caused by misfolded receptors or an imbalance of cellular pH. Unless there is constitutive recycling, there should be no clustering or puncta at the cell surface before agonist addition. Endocytosis receptor clusters are morphologically and temporally different. Recycling events are fast, on the order of about 1 s whereas endocytic clusters persist longer from 40 s to minutes long.

3.3.2.4 Results When a cell is properly focused in TIRFM, the plasma membrane should be the only plane in focus (Figure 2(A), far right). Background fluorescence from parts of the cell not at the plasma membrane should disappear, and raising the focus away from the plasma membrane will not be possible and only yield a blurry, faint image (Figure 2(A)). To find the critical angle to make focusing in TIRFM possible, begin by focusing at the center of the cell (outside the TIRFM field). When the critical angle is reached this part of the cell will no longer be in focus. Figure 2(B) shows a time course of a single recycling event, within the context and scale of an entire cell. Recycling events are characterized by a sharp immediate increase in fluorescence, when the vesicle fuses with the plasma membrane, followed by a fast spreading of this fluorescence as the receptors diffuse on the plasma membrane. These events are typically very fast, lasting around 1 s. The particular example shown lasts 1.2 s.

3.3.3 Strategy 3: visualization of sorting and recycling in the endosome 3.3.3.1 Background and objective GPCRs are sorted into cell surface recycling pathways at the early endosome. In addition to measuring the kinetics of GPCR recycling, recent advances in live cell imaging have made detection of GPCR sorting into subdomains of endosomes possible (Puthenveedu et al., 2010; Temkin et al., 2011; Vistein & Puthenveedu, 2013). Recycling of several GPCRs requires a sequence of amino acids in the

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C-terminal tail of the receptor, and these sequences sort the receptor into actinstabilized domains at the endosome, distinct from bulk recycling domains at the endosome (Lauffer et al., 2010; Puthenveedu et al., 2010). Recent work shows that signaling kinases can regulate GPCR recycling and resensitization by controlling entry of receptors into specific domains of the early endosome (Vistein & Puthenveedu, 2013). High magnification, live cell confocal fluorescence microscopy allows for visualization of rapid changes in GPCR sorting to these endosomal compartments.

3.3.3.2 Flow of experiment 1. Plate N-terminally FLAG-tagged receptor, for example, FLAG-B2AR, on #1.5 coverslips. We generate stable cell lines with antibiotic selection, following transfection of FLAG-B2AR with the Effectene transfection reagent, according to the manufacturer’s protocol. If other markers are required, for example, of actin-stabilized endosomal domains, fluorescent protein-tagged versions may be transfected into the FLAG-B2AR stable cell lines. Transfect stable receptor cell lines with actin or other markers 48e72 h prior to performing imaging experiments, and pass cells to coverslips 24e48 h prior to imaging. 2. Mount coverslips in a live cell imaging chamber. Alternatively, glass bottom dishes can be used. 3. Replace growth medium (i.e., DMEM þ 10% FBS if using HEK 293 cells) with imaging medium (L15 with 5% FBS). 4. Using confocal fluorescence microscopy with a 100x objective, find a cell that has crisp, even fluorescence of the FLAG-tagged receptor at the cell surface, with very little intracellular receptor, as in Figure 3(A), left image. 5. To acquire a time course of a large population of endosomes within a cell, capture images every 1 min, and take multiple z sections throughout the cell, with z sections of approximately 0.3 mm through the entire cell. Acquire 1e 2 min of baseline surface fluorescence of receptors, then add agonist to activate receptors and induce internalization, and acquire a 15e20 min movie. In the case of FLAG-B2AR, the majority of receptors will be localized to endosomes, as in Figure 3(A), right image. 6. To image the dynamics of receptor sorting within domains of a single endosome, acquire a fast time course, capturing at least every 1e2 s. Use 0.2 mm sections and capture a total of 1e2 mm of the cell. Because endosomes are mobile, this is necessary to follow endosome events, such as tubule fission and vesicle generation. For example, in Figure 3(C), a FLAG-B2AR tubule persists for approximately 10 s before vesicle fission.

3.3.3.3 Considerations Membrane trafficking events are very temperature sensitive, so it is necessary to image at 37 C to properly visualize receptor endocytosis and dynamics at the endosome. Also, endosomes move very fast within cells at 37 C. Therefore, when

3. Methods

FIGURE 3 Strategy 3: Visualization of sorting and recycling in the endosome. (A) Example image of FLAG-B2AR localized to the cell surface prior to agonist addition (left panel) and FLAG-B2AR in endosomes 10 min after iso addition (right panel). Endosomes are clearly visible after 10 min of agonist (arrows). Tubule domains are visible protruding from the endosome body (arrowheads). (B) Example image of FLAG-B2AR endosomes imaged with coronineGFP to mark endosomal actin. A coronineGFP spot localizes to the base of the FLAG-B2AR tubule, see inset in left corner. Inset scale bar is 2 mm. (C) Example of a FLAGB2AR tubule fission event imaged every 800 ms. An FLAG-B2AR tubule persists for w10 s before vesicle fission, and a vesicle is visible next to the endosome (see arrowheads). Scale bar is 1 mm. (See color plate)

acquiring z slices in multiple color time-lapse imaging experiments, it is important to minimize delay between channelsdi.e., acquire all channels at the same z section before moving to a different plane.

3.3.3.4 Results Prior to agonist addition, for example, isoproterenol (iso) for B2AR, receptors are primarily localized to the cell surface (Figure 3(A), left panel). Ten minutes after iso addition, the majority of FLAG-B2AR is localized to endosomes (Figure 3(A), right panel). B2AR localizes to tubules characterized by actin spots at the early endosome (Puthenveedu et al., 2010). An actin tubule, marked by coronineGFP, can be seen at the periphery of the endosome body, at the base of the FLAG-B2AR tubule (Figure 3(B)). Tubule fission events prior to vesicle formation and recycling can be visualized at the early endosome using rapid live cell imaging (Figure 3(C)). In Figure 3(C), a tubule persists for approximately 10 s before fission and vesicle formation occurs.

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SUMMARY In this chapter, we have described live cell imaging protocols for the detection and quantitation of GPCR recycling and endosomal sorting. These strategies can be used to determine kinetic changes in overall rates of GPCR recycling across several cells in response to different agonist and drug treatments. We have also described methods that allow for visualization of individual GPCR recycling events in real time. Additionally, we have explained methods for imaging the dynamics of GPCR sorting at the level of the endosome, as well as how to detect GPCR in different domains of endosomes. Together, these methods are powerful tools that can be used to understand the dynamics and regulation of GPCR trafficking in live cells.

REFERENCES Ambrose, E. J. (1956). A surface contact microscope for the study of cell movements. Nature, 178(4543), 1194. Axelrod, D. (1981). Cell-substrate contacts illuminated by total internal reflection fluorescence. Journal of Cell Biology, 89(1), 141e145. Bowman, S. L., Soohoo, A. L., Shiwarski, D. J., Schulz, S., Pradhan, A. A., & Puthenveedu, M. A. (2015). Cell-autonomous regulation of mu-opioid receptor recycling by substance P. Cell Reports, 10(11), 1925e1936. Cheng, S.-B., & Filardo, E. J. (2012). Trans-Golgi Network (TGN) as a regulatory node for b1-adrenergic receptor (b1AR) down-modulation and recycling. The Journal of Biological Chemistry, 287(17), 14178e14191. Chia, P. Z. C., Gunn, P., & Gleeson, P. A. (2013). Cargo trafficking between endosomes and the trans-Golgi network. Histochemistry and Cell Biology, 140(3), 307e315. Drake, M. T., Shenoy, S. K., & Lefkowitz, R. J. (2006). Trafficking of G protein-coupled receptors. Circulation Research, 99(6), 570e582. Dunn, K. W., McGraw, T. E., & Maxfield, F. R. (1989). Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome. The Journal of Cell Biology, 109(6 Pt 2), 3303e3314. Hanyaloglu, A. C., McCullagh, E., & von Zastrow, M. (2005). Essential role of Hrs in a recycling mechanism mediating functional resensitization of cell signaling. The EMBO Journal, 24(13), 2265e2283. Hanyaloglu, A. C., & von Zastrow, M. (2008). Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annual Review of Pharmacology and Toxicology, 48, 537e568. Hislop, J. N., Henry, A. G., & von Zastrow, M. (2011). Ubiquitination in the first cytoplasmic loop of m-opioid receptors reveals a hierarchical mechanism of lysosomal downregulation. The Journal of Biological Chemistry, 286(46), 40193e40204. Huang, F., Zeng, X., Kim, W., Balasubramani, M., Fortian, A., Gygi, S. P., et al. (2013). Lysine 63-linked polyubiquitination is required for EGF receptor degradation. Proceedings of the National Academy of Sciences of the United States of America, 110(39), 15722e15727. Johannes, L., & Wunder, C. (2011). Retrograde transport: two (or more) roads diverged in an endosomal tree? Traffic, 12(8), 956e962.

References

Lauffer, B., Melero, C., Temkin, P., Lei, C., Hong, W., Kortemme, T., et al. (2010). SNX27 mediates PDZ-directed sorting from endosomes to the plasma membrane. The Journal of Cell Biology, 190, 565e574. Marchese, A., Paing, M. M., Temple, B. R. S., & Trejo, J. (2008). G proteinecoupled receptor sorting to endosomes and lysosomes. Annual Review of Pharmacology and Toxicology, 48(1), 601e629. Maxfield, F. R., & McGraw, T. E. (2004). Endocytic recycling. Nature Reviews Molecular Cell Biology, 5(2), 121e132. Mayor, S., Presley, J. F., & Maxfield, F. R. (1993). Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process. The Journal of Cell Biology, 121(6), 1257e1269. Miesenbo¨ck, G., De Angelis, D. A., & Rothman, J. E. (1998). Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature, 394(6689), 192e195. Puthenveedu, M. A., Lauffer, B., Temkin, P., Vistein, R., Carlton, P., Thorn, K., et al. (2010). Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains. Cell, 143(5), 761e773. Romero, G., von Zastrow, M., & Friedman, P. A. (2011). Role of PDZ proteins in regulating trafficking, signaling, and function of GPCRs: means, motif, and opportunity. Advances in Pharmacology, 62, 279e314. Tanowitz, M., & Von Zastrow, M. (2002). Ubiquitination-independent trafficking of G protein-coupled receptors to lysosomes. The Journal of Biological Chemistry, 277(52), 50219e50222. Tanowitz, M., & von Zastrow, M. (2003). A novel endocytic recycling signal that distinguishes the membrane trafficking of naturally occurring opioid receptors. The Journal of Biological Chemistry, 278(46), 45978e45986. Temkin, P., Lauffer, Ja¨ger, S., Cimermancic, P., Krogan, N., & Zastrow, M. (2011). SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors. Nature Cell Biology, 13, 717e723. Trejo, J. (1999). The cytoplasmic tails of protease-activated receptor-1 and substance P receptor specify sorting to lysosomes versus recycling. The Journal of Biological Chemistry, 274(4), 2216e2224. Vistein, R., & Puthenveedu, M. (2013). Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch. Proceedings of the National Academy of Sciences, 110(38), 15289e15294. Yu, Y., Dhavan, R., Chevalier, M., Yudowski, G., & Zastrow, M. (2010). Rapid delivery of internalized signaling receptors to the somatodendritic surface by sequence-specific local insertion. The Journal of Neuroscience, 30, 11703e11714. Yu, Y. J., Arttamangkul, S., Evans, C. J., Williams, J. T., & von Zastrow, M. (2009). Neurokinin 1 receptors regulate morphine-induced endocytosis and desensitization of mu-opioid receptors in CNS neurons. The Journal of Neuroscience, 29(1), 222e233. Yudowski, G. A., Puthenveedu, M. A., Henry, A. G., & von Zastrow, M. (2009). Cargomediated regulation of a rapid Rab4-dependent recycling pathway. Molecular Biology of the Cell, 20(11), 2774e2784. Zhao, P., Canals, M., Murphy, J. E., Klingler, D., Eriksson, E. M., Pelayo, J. C., et al. (2013). Agonist-biased trafficking of somatostatin receptor 2A in enteric neurons. The Journal of Biological Chemistry, 288(36), 25689e25700.

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CHAPTER

Methods to study endocytic trafficking of the EGF receptor

22

Itziar Pinilla-Macua, Alexander Sorkin1 Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 1


CHAPTER OUTLINE Introduction ............................................................................................................ 348 1. Objectives and Rationale .................................................................................... 349 2. Materials........................................................................................................... 350 2.1 Reagents ............................................................................................ 350 2.2 Major Instruments ............................................................................... 351 3. Basic Techniques ............................................................................................... 351 3.1 Cell Culture ........................................................................................ 351 3.2 SDS-PAGE and Western Blot................................................................ 351 3.3 Fluorescence Microscopy ..................................................................... 352 4. Experimental Strategies...................................................................................... 352 4.1 Imaging EGFR Endocytosis in Living Cells by Fluorescence Microscopy, and the Effects of Dynamin Inhibitor on this Process.............................. 352 4.1.1 Theory ............................................................................................. 352 4.1.2 Experimental protocol ....................................................................... 352 4.1.3 Variation: examination of the effect of Dyngo-4a on EGFR endocytosis ............................................................................ 353 4.1.4 Results............................................................................................. 353 4.1.5 Considerations.................................................................................. 356 4.2 Measurement of EGFR Internalization Rates Using 125I-EGF................... 356 4.2.1 Theory ............................................................................................. 356 4.2.2 Experimental protocol ....................................................................... 356 4.2.3 Variation: effect of inhibitors on 125I-EGF endocytosis ........................ 357 4.2.4 Results............................................................................................. 357 4.2.5 Considerations.................................................................................. 358 4.3 Detection of EGFR Ubiquitination......................................................... 359 4.3.1 Theory ............................................................................................. 359 4.3.2 Experimental protocol ....................................................................... 359 Methods in Cell Biology, Volume 130, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.05.008 © 2015 Elsevier Inc. All rights reserved.

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CHAPTER 22 Methods to study endocytic trafficking of the EGF receptor

4.3.3 Results............................................................................................. 360 4.3.4 Considerations.................................................................................. 361 4.4 Ligand-Induced EGFR Degradation ....................................................... 361 4.4.1 Theory ............................................................................................. 361 4.4.2 Experimental protocol ....................................................................... 361 4.4.3 Results............................................................................................. 362 4.4.4 Considerations.................................................................................. 363 4.5 Measurements of the Rate of EGF:EGFR Complex Recycling Using 125I-EGF, and the Effect of PQ on This Process ............................ 363 4.5.1 Theory ............................................................................................. 363 4.5.2 Experimental protocol ....................................................................... 364 4.5.3 Variation: effect of PQ on recycling.................................................... 365 4.5.4 Results............................................................................................. 365 4.5.5 Considerations.................................................................................. 366 Summary ................................................................................................................ 366 References ............................................................................................................. 366

Abstract Endocytosis and postendocytic sorting of epidermal growth factor (EGF) receptor (EGFR) are the major regulators of EGFR signaling. EGFR endocytosis and ubiquitindependent lysosomal targeting are also considered to be the prototypic experimental system for studying the molecular mechanisms of stimulus-induced and constitutive endocytic trafficking. Therefore, elucidation of the mechanisms of EGFR endocytosis and its regulation of the signaling network is essential not only for better understanding of the EGFR biology but also for defining general regulatory principles in the endocytosis system. Comprehensive analysis of these mechanisms requires quantitative and physiologically relevant methodological approaches for measuring the rates of EGFR internalization, degradation, and recycling. Basic experimental protocols described in this chapter cover a combination of single-cell microscopy and biochemical methods that are used to follow EGF-induced endocytosis of EGFR in real time, measure the kinetic rate parameters of EGFR internalization and recycling, and analyze EGF-dependent ubiquitination and degradation of EGFR.

INTRODUCTION Epidermal growth factor (EGF) receptor (EGFR) plays an important role in the regulation of cell proliferation, differentiation, survival, and motility both in development and adulthood (Sibilia et al., 2007). At least six ligands for EGFR in addition to the best characterized EGF have been described (Henriksen, Grandal, Knudsen, van Deurs, & Grøvdal, 2013). Upon ligand binding to EGFR at the cell surface, receptors dimerize, which leads to activation of its intrinsic tyrosine kinase activity, and tyrosine phosphorylation of the cytoplasmic domain of the receptor as


well as other cytoplasmic substrates (Lemmon & Schlessinger, 2010). These phosphorylation events trigger several signal transduction cascades ultimately leading to altered gene expression. At the same time, activated EGFR is rapidly endocytosed through clathrindependent and clathrin-independent pathways. It is proposed that clathrin-mediated endocytosis of EGFR has limited capacity and is saturated by the excess of EGF: EGFR complexes at the cell surface (when high EGF concentrations are used) (Sorkin & Goh, 2009). Therefore, measurement of the EGFR internalization rates through clathrin pathway requires the use of low, physiological EGF concentrations. After internalization into early endosomes, EGF-receptor complexes are capable of recycling back to the plasma membrane but are also retained in endosomes and eventually sorted to late endosomes and lysosomes for degradation (Sorkin & Goh, 2009). EGFR ubiquitination by the E3 ligase Cbl is the key mechanism mediating lysosomal targeting of EGFR and many other endocytic cargos (Eden, Huang, Sorkin, & Futter, 2012; Hislop & von Zastrow, 2011; Weinberg & Drubin, 2014). The acceleration of internalization and lysosomal targeting of activated EGFR results in the reduction of EGFR protein levels and downregulation of EGFRdependent signaling as part of the negative feedback regulation loop (Sorkin & von Zastrow, 2009). The key role of EGFR trafficking in regulation of signaling processes underscores the importance of understanding the molecular mechanism of this trafficking. However, despite extensive studies for more than three decades, these mechanisms, in particular, those of the internalization step, remain elusive. Therefore, the use of standardized, universally accepted, and quantitative methodologies is vital for studying EGFR endocytosis in diverse experimental model systems. Analysis using a combination of such methodologies should allow careful reinterpretation and reconciliation of numerous contradictory experimental observations and proposed models of EGF endocytosis.

1. OBJECTIVES AND RATIONALE Internalization rates of EGF-occupied EGFR were traditionally measured by monitoring the uptake of radiolabeled EGF (125I-EGF) in the cell. 125I-EGF is also used to measure the rate of recycling of internalized 125I-EGF:EGFR complexes back to the cell surface, and the rate of 125I-EGF degradation. Because the bulk of endosomal EGF:EGFR complexes remain intact in endosomes, methods involving 125I-EGF indirectly measure EGFR recycling and degradation. While 125I-EGF-based methods remain most sensitive and quantitative, combining these methods with optical microscopy and direct EGFR protein quantification assays is the most desirable approach to conduct the comprehensive analysis of EGFR endocytosis. Availability of various biologically active labeled derivatives of EGF, numerous antibodies, and genetically encoded fluorescent fusion proteins of EGFR makes such analysis to be highly feasible. Importantly, unprecedented increase in the sensitivity of microscopy

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imaging systems provides an opportunity to follow endocytosis of EGFR activated by low, physiological concentrations of fluorescent EGF in living cells and in real time. Therefore, in this chapter we focus on the description of single-cell microscopy analysis of EGFR endocytosis, 125I-EGF-based methods of measuring internalization and recycling rates, and simple methods of measuring EGF-induced degradation of EGFR and EGFR ubiquitination using immunoprecipitation and western blotting. Additionally, we demonstrate the utility of these methods for examining the effects of two chemical compounds, Dyngo-4a and primaquine (PQ), on internalization and recycling of EGFR, respectively. Dyngo-4a is thought to inhibit the activity of the large GTPase dynamin that is necessary for the scission of vesicles during clathrin-mediated and several types of clathrin-independent endocytosis (Ferguson & De Camilli, 2012; McCluskey et al., 2013; Mettlen, Pucadyil, Ramachandran, & Schmid, 2009). PQ has been shown to inhibit recycling of the transferrin receptor by an unknown mechanism (Raub & Newton, 1991; van Weert, Geuze, Groothuis, & Stoorvogel, 2000).

2. MATERIALS 2.1 REAGENTS EGFR-expressing cells: •

UMSCC2 (University of Michigan squamous cell carcinoma of head and neck) • HeLa cells • Mouse NIH 3T3 cells stably expressing human EGFR (NIH3T3/EGFR) Dulbecco minimum essential medium (DMEM) Fetal bovine serum (FBS) Normal calf serum Bovine serum albumin (BSA) Binding medium (DMEM plus 0.1% (w/v) BSA) HEPES buffer Ca2þ, Mg2þ-free PBS (CMF-PBS) Tween-20 pH 4.3e4.5 acetate buffer (0.2 M sodium acetate buffer, 0.5 M NaCl) pH 2.5e2.8 acetate buffer (0.2 M acetic acid, 0.5 M NaCl) Trichloroacetic acid (TCA) Phosphotungstic acid (PTA) TCA/PTA (10%/2% w/v) in water Phosphate buffer saline (PBS) Tris buffer saline (TBS) Sample buffer 1x (62.5 mM Tris pH 6.8, 2% SDS, 5% b-mercaptoethanol, 10% glycerol, bromophenol blue) TGH buffer (1% (v/v) Triton X-100, 10% (v/v) glycerol, 50 mM HEPES pH 7.4)

3. Basic techniques

Lysis buffer (TGH buffer, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovandate,10 mM N-ethylmaleimide, 10 mg/mL aprotinin, 10 mg/mL leupeptin, and protease inhibitors) Recombinant human culture-grade EGF; receptor-grade mouse EGF (for labeling); 125I-EGF; EGF-Rhodamine (EGF-Rh); Alexa FluorÒ 647-labeled EGF complex (EGF-A647). Wheat germ agglutinin (WGA) Oregon Green Paraformaldehyde (16% solution; electron microscopy grade). Cycloheximide DMSO Dyngo-4a ProLongÒ Antifade mounting media Collagen-coated glass bottom 35 mm MatTek dishes 6-, 12-, and 24-well plastic cell culture plates 35 and 60 mm plastic cell culture dishes Mouse monoclonal antibody 528 to EGFR (ATTC) Rabbit polyclonal antibody 1005 to EGFR (Santa Cruz) Mouse monoclonal antibody P4D1 to ubiquitin (Santa Cruz)

2.2 MAJOR INSTRUMENTS •

• •

Zeiss Axio Observer Z1 inverted fluorescence microscope equipped with 63x Plan Apo PH NA 1.4 objective, computer-controlled spherical aberration correction unit, Yokogawa CSU-X1 spinning disk, Photometrics Evolve 16-bit EMCCD camera, environmental chamber, piezo stage controller and lasers (405, 445, 488, 515, 561, and 640 nm lines), all controlled by SlideBook software (Intelligent Imaging Innovation, Denver, CO). Odyssey Infrared Imaging system (LI-COR Biosciences, Lincoln, NE) Cobra Gamma-counter (Packard Instruments)

3. BASIC TECHNIQUES 3.1 CELL CULTURE Cells expressing EGFR are grown and maintained in DMEM supplemented with serum at 37 C, humidity and 5% CO2. 10% (v/v) and 5% (v/v) FBS is used to grow HeLa and UMSCC2, respectively. NIH 3T3/EGFR cells are grown in DMEM supplemented with 10% (v/v) NCF. Cells are plated at 40,000e 50,000 cells/cm2 and grown to a desired confluency. Prior to any experiment, cells are serum-starved to minimize basal EGFR activity. Starvation is typically performed in binding medium for 6e16 h.

3.2 SDS-PAGE AND WESTERN BLOT Protein are resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% acrylamide gels that are used to allow optimal resolution

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in the high-molecular weight range (150 kDa and higher) to clearly detect EGFR (170 KDa) and its slowly migrating, posttranslationally modified forms. Proteins are transferred to the nitrocellulose membrane at 500 mA for 3.5 h. The membrane is then blocked with TBS containing 0.02% (v/v) Tween-20 and 5% (w/v) nonfat milk (blocking solution) either 1 h at room temperature or overnight at 4 C with constant agitation. Immunoblotting with primary and secondary antibodies is performed in TBS/Tween buffer following manufacturer’s recommendations. The blots are washed with TBS/Tween after incubations with antibodies. Membranes are scanned using Odyssey LI-COR imager, and the data are analyzed using LI-COR software.

3.3 FLUORESCENCE MICROSCOPY MatTek dishes (living cells) or subject glass with mounted coverslips (fixed cells) are placed onto the microscope stage adaptor. Constant temperature (37 C), humidity, and 5% CO2 atmosphere are maintained throughout the duration of live-cell imaging. The capture integration time and other imaging parameters (number of optical section in the z-stack, stepsize between sections, time intervals between 3-D images during time-lapse imaging) are set to be identical during image acquisition of experimental variants and to allow nonsaturated fluorescence signals.

4. EXPERIMENTAL STRATEGIES 4.1 IMAGING EGFR ENDOCYTOSIS IN LIVING CELLS BY FLUORESCENCE MICROSCOPY, AND THE EFFECTS OF DYNAMIN INHIBITOR ON THIS PROCESS 4.1.1 Theory Fluorescently labeled EGFR ligands like EGF conjugated to Rhodamine (EGF-Rh) are used to visualize endocytosis of ligand-occupied EGFR in single cells. This type of experiments was first performed more than three decades ago (Haigler, Ash, Singer, & Cohen, 1978). However, only recently, new microscopy systems, such as spinning disk confocal microscope, that are capable of continuous 3-D time-lapse imaging of endocytosis of labeled EGF at physiological concentrations (1e4 ng/mL) in living cells without significant photobleaching and phototoxicity, have become available. Imaging the entire cell in 3-D, rather than a single confocal or optical plane is necessary to obtain truly quantitative information about the kinetics of EGF endocytosis in addition to the information about the morphology of various EGF-containing compartments in different parts of the cell. Certainly, endocytosis is a highly temperature-dependent process, and therefore, the assay is carried out at 37 C.

4.1.2 Experimental protocol 1. Cells are grown in 35 mm MatTek dishes until 50e60% confluency and serumstarved in binding medium.


2. The dish with 2 mL of medium is placed onto the microscopic stage in the environmental chamber. Time-lapse imaging is started by acquiring a z-stack of 10e20 xey confocal images (0.2e0.4 mm stepsize) through 561 nm (EGF-Rh) or 640 nm (EGF-A647) channels with the desired time intervals and image capture time of 100 ms or less. 3. After two to three time points, 200 mL of prewarmed binding medium containing fluorescent EGF and additional HEPES is rapidly added to the dish to ensure the final concentration of labeled EGF and HEPES buffer of 2e10 ng/mL and 10 mM respectively. 4. 3-D imaging is continued during EGF injection and further continued for the desired time, typically, 5e60 min.

4.1.3 Variation: examination of the effect of Dyngo-4a on EGFR endocytosis 1. Cells are grown in 35 mm MatTek dishes or on 12 mm round coverslips placed in 24-well dishes until w50e70% confluency and serum-starved. 2. Cells are washed with DMEM and preincubated with 30 mM of Dyngo-4a or vehicle (DMSO) in DMEM for 30 min at 37 C. 3. Cells in MatTek dishes are used for live-cell imaging of fluorescent EGF as in Steps 2e4 (Section 4.1.2) above. 4. Cells on coverslips are incubated in binding medium containing additional 10 mM HEPES and fluorescent EGF (2e10 ng/mL) in CO2 incubator or water bath. 5. At desired time points (5e60 min), EGF endocytosis is stopped by placing the plates firmly on ice and washing cells twice with ice-cold PBS. 6. Coverslips are fixed with freshly prepared paraformaldehyde (4%) in PBS for 30 min at 4 C and mounted on subject glass. 7. Optional: After fixation and three 5-min washes with PBS, the cells are incubated with 0.5 mg/mL WGA-Oregon Green in PBS for 10 min at room temperature to label the plasma membrane. 8. Coverslips are then washed with PBS and mounted. 9. 3-D confocal images through 488 nm (Oregon Green), 561 nm (EGF-Rh), or 640 nm (EGF-A647) channels are captured. Image acquisition times and other parameters of imaging depend on the cell type, EGFR levels and other experimental factors.

4.1.4 Results In the example presented in Figure 1, living UMSCC2 cells are imaged by capturing 20 xey images with 400 nm z-plane stepsize every 90 s for 15 min. Addition of EGF-Rh (4 ng/mL) to the cells resulted in rapid binding of EGF-Rh to surface receptors (1.5 min; Figure 1) and accumulation of EGF-Rh in endosomes (7.5 min; Figure 1). 3-D volume (xeyez) image (Figure 1(A)) that allows visualization of the entire image stack and the “3-D view” image (Figure 1(B)) that shows single xey, xez, and yez confocal planes are presented. These images demonstrate localization of EGF-Rh mostly at the cell surface at the 1.5-min

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FIGURE 1 Time-lapse imaging of UMSCC2 cells stimulated with EGF-Rhodamine (EGF-Rh). Cells were imaged before and after EGF-Rh (4 ng/mL) stimulation for 15 min. Z-stacks of 20 xey images (400 nm z-stepsize) of Rhodamine fluorescence were acquired every 1.5 min. 1.5 and 7.5 min images are shown. 3-D volume view image (A) and “3-D view” image (single confocal xey, yez, and xez sections) are presented (B). Scale bars, 10 mm. (See color plate)

time point (cell perimeter, filopodia, ruffles). Bright vesicular peripheral and perinuclear compartments containing EGF-Rh observed on the 7.5-min image are indicative of predominantly endosomal localization of EGF-Rh, although some diffusely distributed fluorescence in the cell perimeter (plasma membrane) is also observed. The strong inhibitory effect of Dyngo-4a on EGF-Rh internalization in UMSCC2 cells examined in living cells is demonstrated in Figure 2(A). The same effect is observed in fixed cells in which the cell surface was labeled with WGA-Oregon Green (Figure 2(B)). In these images of cells incubated with EGF-Rh for 15 min, most EGF-Rh containing endosomes do not overlap with WGA-Oregon Green staining in control cells and are practically absent in Dyngo-4a treated cells.


FIGURE 2 Effect of Dyngo-4a on EGF-Rhodamine (EGF-Rh) endocytosis in UMSCC2 cells. (A) Cells were incubated 30 min with Dyngo-4a 30 mM or vehicle (DMSO) and then imaged before and after EGF-Rh (4 ng/mL) stimulation as in Figure 1. 3-D volume view images of control and Dyngo-4a pretreated cells that were incubated with EGF-Rh for 5 min are shown. (B) Cells preincubated or not with Dyngo-4a were incubated with EGF-Rh for 15 min in the presence of inhibitor or vehicle, fixed and labeled with wheat germ agglutinin (WGA)-Oregon Green as described in the text. Insets show high magnification images of the peripheral cell regions marked by white rectangles. All image acquisition parameters and intensity scales are the same for corresponding images of control and Dyngo-4a-treated cells. Scale bars, 10 mm. EGF, epidermal growth factor. (See color plate)

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4.1.5 Considerations 1. Various preparations of fluorescently labeled EGF are commercially available. Direct conjugates of EGF with fluorochromes are preferable to use as compared to preparations consisting of the complex of biotinylated EGF with streptavidin, such as EGF-A647. Such complexes may be multivalent, and thus leading to receptor cross-linking. Actual concentration of EGF in these preparations could be difficult to determine. Among direct EGF conjugates, conjugates to fluorophores with high brightness and photostability, such as Rhodamine or TexasRed, are preferable compared with less stable FITC. 2. Endosomes and other compartments move rather rapidly, and therefore can move during an acquisition of a 3-D image. Therefore, shortest image capture times and minimal number of planes are recommended. 3. The activity of Dyngo-4a is dramatically reduced when albumin or serum is present ((McCluskey et al., 2013) and our observations). Therefore, BSA is omitted during preincubation of cells with this inhibitor. 4. We noticed that additional incubations of EGF-Rh labeled cells after fixation result in the substantial loss of EGF-Rh bound to the cell surface due to poor fixation of this conjugate by formaldehyde.

4.2 MEASUREMENT OF EGFR INTERNALIZATION RATES USING 125I-EGF 4.2.1 Theory The internalization assay is performed as a short time-course of 125I-EGF uptake (less than 5e6 min depending on cell type) to minimize the influence of recycling of 125I-EGF:EGFR complexes on the overall uptake. Because endocytosis is highly temperature-dependent, the assay is performed at 37 C. The key step of this assay is stripping of surface 125I-EGF at low pH to separate the surface and internalized pools of 125I-EGF. Either Naþ acetate or glycine/HCl buffers (pH 2.5e2.8), the latter combined with 3 M urea, are effective in rapid removal of 125I-EGF bound to surface receptors without affecting intracellular 125I-EGF (Haigler, Maxfield, Willingham, & Pastan, 1980; Wiley & Cunningham, 1982).

4.2.2 Experimental protocol 1. Cells are plated into 12-well dishes and grown for 2e3 days until confluency. 2. 125I-EGF-containing binding medium (0.5 mL per well) is prepared. Unlabeled EGF (50e100 M excess) is added to the half of the medium with 125I-EGFd this is the “nonspecific” medium. It is important to prepare some excess of the 125 I-EGF-containing medium to save an aliquot for measuring the specific activity of 125I-EGF. 3. The dish is placed in the water bath at 37 C, and two wells are quickly washed with warm (37 C) binding media (w1 mL). 0.5 mL of prewarmed (37 C) specific (not containing cold EGF) or nonspecific 125I-EGF containing media


4. 5.

6.

7.

8.

are added to two wells simultaneously, preferably, using two pipetteman, one in each hand. These cells correspond to the longest time point (3e6 min). The same procedure (Step 3) is repeated with other pairs of wells at 30 s or 1-min intervals. Rapid handling of all steps of this procedure is critical because of the short incubations of cells with 125I-EGF. After the last pair of wells has been incubated with 125I-EGF, the dish is placed on ice to stop endocytosis, and the radioactive media are rapidly aspirated. In a very short time-course experiments (0e3 min), the actual timing of 125I-EGF medium addition to cells are recorded because the fast pace of the assay often does not allow the precisely planned timing of incubations. The monolayers are rapidly washed three times with w1 mL/well ice-cold DMEM followed by vacuum aspiration to remove as much unbound 125I-EGF as possible after the last wash. This step is performed as quickly as possible to avoid significant dissociation of 125I-EGF from surface receptors during washes. All wells are then incubated for w5 min with the pH 2.8 Naþ acetate buffer (1 mL/well) at 4 C to strip surface-bound 125I-EGF followed by with another short acetate buffer wash (0.5 mL/well). Both acidic washes are combined and transferred to g-counter vials to determine the amount of surface-bound 125 I-EGF. The cells are lysed in 1 mL of 1 N NaOH for w1 h at 37 C to determine the amount of internalized 125I-EGF.

4.2.3 Variation: effect of inhibitors on 125I-EGF endocytosis To test whether endocytosis is dynamin-dependent, the cells are preincubated with Dyngo-4a in the medium without BSA as described in Section 4.1.3, and the protocol further follows Steps 2e8 above. Dyngo-4a is also present in the 125I-EGF-containing uptake medium.

4.2.4 Results Figure 3(A) shows an example of the data obtained in the 125I-EGF internalization experiment. The amounts of internalized (I) and surface (S) 125I-EGF, and the ratio I/S are calculated as follows. The specific radioactivity of the acid-sensitive (surface) and acid-insensitive (internalized) fractions is obtained by subtracting nonspecific counts from the corresponding counts of “specific” wells. Nonspecific radioactivity is typically not more than 5e10% of the total counts. Internalization is thought to be a first-order kinetics process where the specific rate of internalization depends on the concentration of EGF-receptor complexes at the cell surface. The calculation of the specific internalization rate constant ke can be performed if the 125 I-EGF concentration at the cell surface does not significantly change (or remains constant) during the time-course of 125I-EGF uptake. Under these conditions the I/S ratio displays a linear dependence on time, and therefore, ke corresponds to the linear regression coefficient of this dependence (Wiley & Cunningham, 1982). The calculation of the ke value yields 0.18 min1 (Figure 3(A)). Figure 3(B) demonstrates an

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FIGURE 3 Internalization of 125I-EGF. (A) NIH 3T3/EGFR cells were incubated with 1 ng/mL 125I-EGF for 1e6 min at 37 C, and the amount of surface and internalized radioactivity was determined as described in Section 4.2. (B) UMSCC2 cells were incubated with 30 mM Dyngo-4a in DMEM, and the internalization assay with 1 ng/mL 125I-EGF was performed as in (A).

example of 125I-EGF internalization experiment in which EGFR endocytosis is inhibited by Dyngo-4a. The ke value in the presence of Dyngo-4a is 0.04 min1.

4.2.5 Considerations 1. The assay is typically performed when cells are confluent to maximize specific 125 I-EGF binding and minimize binding of 125I-EGF to plastic surfaces. 2. These single 12-well dish experiments must be repeated multiple times to obtain statistically reliable values of ke. 3. When high concentrations of 125I-EGF are used, significant amount of surface EGFRs is rapidly internalized leading to a substantial decrease of empty EGFR levels at the cell surface, thus resulting in nonlinear I/S time dependence and


making it impossible to use the linear regression coefficient for calculation of ke values. In such cases, the I/S ratio plotted against incubation time is considered to be an approximate measure of an apparent internalization rate.

4.3 DETECTION OF EGFR UBIQUITINATION 4.3.1 Theory The EGFR ubiquitination assay is based on efficient immunoprecipitation of EGFR and detection of ubiquitin immunoreactivity in immunoprecipitated EGFR by western blotting. Typically, EGFR is significantly ubiquitinated only after activation by the ligand. In most cells, the maximal extent of EGFR ubiquitination is observed after 3e5 min of cell stimulation with EGF at 37 C.

4.3.2 Experimental protocol 1. Cells are grown in 60 mm dishes until w80e90% confluency, and serumstarved. 2. Cells are incubated with or without EGF (2e20 ng/mL) in binding medium for 0e60 min at 37 C. 3. The incubation is stopped by placing the dishes on ice and washing three times with ice-cold CMF-PBS. 4. 400 mL of ice-cold lysis buffer containing protease, phosphatase, and deubiquitination inhibitors is added to each dish; the cells are scraped with the rubber cell lifter, and transferred to 1.5 mL tubes. 5. The tubes are placed on the nutator for 10-min agitation at 4 C to complete cell lysis in, and the lysates are cleared by centrifugation at 16,000 x g for 10 min at 4 C. Supernatants are transferred to new tubes. 6. Aliquots (10%) of cleared lysates are saved, and EGFR monoclonal antibody 528 (10 mg) is added to the rest of the lysate. The lysates are incubated with the antibody on the nutator for 3e4 h of agitation at 4 C. 7. 75 mL of Protein A Sepharose (20% solution) are added to each tubes, and the incubation on the nutator continues for 1 h at 4 C. 8. Samples are centrifuged (16,000 x g; 2 min) to pellet the beads. Aliquots of supernatants (10% of total volume) are saved (for evaluation of the efficiency of immunoprecipitation) and the rest of supernatant is aspirated. 9. Sepharose beads are washed two times with 700 mL/tube of TGH buffer containing 1 mM sodium orthovandate and 10 mM NEM, and one time with the same buffer supplemented with 0.5 M NaCl. 10. After as much of the last wash as possible is aspirated using protein gel loading tips, 100 mL of 1x sample buffer is added, tubes are briefly vortexed and heated at 95 C for 5 min to release from beads and denature proteins. 11. Aliquots of lysates (Step 6) and supernatants (Step 8) are mixed with an equal volume of the 2x sample buffer, and heated at 95 C for 5 min. 12. Immunoprecipitates, lysates, and supernatants are resolved by SDS-PAGE.

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13. Proteins are transferred to nitrocellulose, and western blotting is performed with antiubiquitin antibody followed by secondary antimouse antibodies conjugated to far-red fluorescent dye (IRDye-680) and the detection of the fluorescence signal using the Odyssey LI-COR imager. Subsequently, blot is probed with anti-EGFR 1005 (total EGFR) followed by the secondary antibody (labeled with IRDye-800). A part of the blot corresponding to lanes loaded with lysates is probed with an antibody to a housekeeping protein to control for equal loading.

4.3.3 Results In the experiment presented in Figure 4, UMSCC2 cells were stimulated with 4 ng/ mL EGF for 5 min. Very little EGFR ubiquitination is detected in nonstimulated

FIGURE 4 Epidermal growth factor receptor (EGFR) ubiquitination. UMSCC2 cells were incubated with or without 4 ng/mL epidermal growth factor (EGF) for 5 min at 37 C. Cells were solubilized, and EGFR immunoprecipitated as described in Section 4.3.2. EGFR immunoprecipitates and lysates were resolved by electrophoresis, and western blotting was performed using antibodies to ubiquitin, EGFR, and Akt (loading control). The extent of EGFR ubiquitination is calculated as the amount of ubiquitin normalized to the amount of EGFR is shown under the image of the ubiquitin blot.


cells whereas a strong signal of high-molecular weight ubiquitin immunoreactivity is detected in EGF-treated cells. Several ubiquitinated EGFR species are resolved which reflects heterogeneity of EGFR ubiquitination by one to three short (mostly diubiquitin) ubiquitin chains. In our experience the appearance of ubiquitinated EGFR immunoreactivity varies from a single smear above 170 kDa to several high-molecular weight bands (as in Figure 4) in different experiments. Similar amounts of EGFR in immunoprecipitates from cells treated or not with EGF, and equal amount of protein in the lysates used for immunoprecipitation is evident based on the intensities of EGFR and Akt (that is used as loading control in this experiment) bands, respectively.

4.3.4 Considerations 1. Lysis buffer must be supplemented with NEM, inhibitor of deubiquitination enzymes to preserve ubiquitination of EGFR. 2. Antibodies to the EGFR extracellular domain (such as EGFR-528) tend to be more efficient in immunoprecipitating native EGFR than antibodies to the intracellular domain of EGFR. 3. EGFR ubiquitination can be detected in cells treated with EGF at 4 C. 4. Western blot detection of ubiquitinated and total EGFR can be performed simultaneously because different antibody species are used and two corresponding secondary antibodies labeled with different fluorophores can be detected simultaneously by two-channel LI-COR imaging.

4.4 LIGAND-INDUCED EGFR DEGRADATION 4.4.1 Theory The simplest method to examine degradation of the EGFR protein is using western blot detection of the amount of EGFR in cell lysates. Since under steady-state cell growth conditions degradation of a protein is compensated by its synthesis, measurements of the degradation rates are carried out in cells incubated with cycloheximide, that inhibits protein synthesis. Because EGF stimulation dramatically accelerates EGFR degradation, protein synthesis does not significantly contribute in the total concentration of EGFR in EGF-stimulated cells, and therefore, cycloheximide treatment is not essential for measurement of the rate of EGF-induced EGFR degradation.

4.4.2 Experimental protocol 1. Cells are grown in 35 or 60 mm dishes, until they reach w80e90% confluency, and serum-starved. 2. Cells are treated with cycloheximide (50 mg/mL) for 2 h to block de novo synthesis of EGFR. 3. 2e100 ng/mL EGF is added to several dishes (depending on the desired timecourse) whereas other dishes are continued to be incubated in the cycloheximide-containing binding medium without EGF.

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4. At the desired time points (0e24 h) the incubation is stopped by placing the plates firmly on ice and washing three times with ice-cold CMF-PBS. Aspirate as much as CMF-PBS as possible, and store dishes at 80 C until the completion of the longest incubation. 5. The cells are lysed in the lysis buffer and the lysates are cleared as described in Section 4.3.2 with the exception that the lysis buffer does not include sodium orthovanadate and NEM. 6. Protein concentration is measured in each sample using Bradford method. 7. Aliquots of lysates with equal protein amounts are resolved on SDS-PAGE, proteins transferred to nitrocellulose, and EGFR is detected by western blotting as described in Section 4.3.2 above.

4.4.3 Results As shown in Figure 5, treatment of UMSCC2 cells with EGF results in rapid decrease in the amount of EGFR. Quantitation of the immunoreactive signal

FIGURE 5 Epidermal growth factor (EGF)-induced EGF receptor (EGFR) degradation. UMSCC2 cells were incubated with 80 ng/mL EGF for 0e24 h. (A) EGFR was detected by western blotting in cell lysates using antibodies 1005. a-actinin is as loading control. (B) Quantification of EGFR immunoreactivity from two experiments including the data presented in (A).


intensities of EGFR and a-actinin (loading control) was performed using LI-COR software. The normalized amounts of EGFR (EGFR/a-actinin signals) are expressed as percent of this amount at time zero (Figure 5(B)). These calculations yielded the half-life of EGF-activated EGFR protein of w5 h.

4.4.4 Considerations 1. Because most EGFR antibodies that are used in western blotting target EGFR intracellular domain, these antibodies do not efficiently recognize phosphorylated and ubiquitinated EGFR. Therefore, the lysis buffer used in this protocol does not include sodium orthovandate and NEM, so that EGFR is dephosphorylated and deubiquitinated upon cell lysis. Alternatively, complete lysis buffer can be used, but the immunoblotting should be performed with an antibody to the extracellular EGFR domain, for instance, monoclonal antiEGFR 05-104 (Millipore). 2. Time-course should be designed based on the predicted half-life of the EGFR protein. For native, endogenous EGFR, the lesser the amount of the receptor, the shorter its half-life. For instance, in contrast to UMSCC2 cells, HeLa cells expressing low level of EGFR degrade activated EGFR with the half-life of 45e60 min.

4.5 MEASUREMENTS OF THE RATE OF EGF:EGFR COMPLEX RECYCLING USING 125I-EGF, AND THE EFFECT OF PQ ON THIS PROCESS 4.5.1 Theory Monitoring 125I-EGF recycling as a method to measure recycling of EGFeEGFR complexes is based on the observation that EGF does not significantly dissociate from EGFR in endosomes until it reaches the lysosome (Sorkin, Teslenko, & Nikolsky, 1988). The schematics of this protocol is presented in Figure 6(A). The recycling assay includes loading early endosomes with 125I-EGF-receptor complexes by allowing cells to endocytose 125I-EGF for short times at 37 C, followed by stripping the nonendocytosed, surface-bound 125I-EGF by the mild acidic buffer treatment at 4 C. When “125I-EGF-loaded cells” are returned to 37 C, a pool of 125 I-EGF-receptor complexes are recycled back to the cell surface, where 125IEGF is released from the receptor and incapable of rebinding to cells due to the excess of unlabeled EGF in the medium and full occupancy of surface EGFR with unlabeled EGF. During the 37 C chase incubation a pool of internalized 125 I-EGF-receptor complexes are also sorted from early endosomes to late endosomes and lysosomes where 125I-EGF-receptor complexes are degraded by proteolytic enzymes. This degradation results in the release of mono- and di-125iodotyrosines, which rapidly pass through the membranes and accumulate in the extracellular media. These low-molecular weight products are not precipitable by TCA and can be biochemically separated from intact 125I-EGF. Only negligible

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FIGURE 6 Recycling of 125I-EGF. (A) Incubation steps in the experimental protocol to measure recycling of 125I-EGF. (B) NIH 3T3/EGFR cells were incubated with 5 ng/mL 125I-EGF for 5 min at 37 C, surface 125I-EGF was removed by the mild acidic buffer wash, and the recycling assay was performed as described in Section 4.5. The chase incubation medium was supplemented or not with 300 mM primaquine.

amounts of TCA-soluble 125I can be detected inside the cells, and therefore the amount of TCA-soluble 125I in the medium reflects EGF degradation. The sum of intact 125I-EGF (TCA-precipitable) detected in the medium and at the cell surface (acid wash stripped) after the 37 C chase incubation of 125I-EGF-loaded cells corresponds to 125I-EGF-receptor complexes that were recycled from endosomes to the plasma membrane.

4.5.2 Experimental protocol 1. The cells grown in 35 mm culture dishes are incubated with 1e20 ng/mL 125IEGF in binding medium for 5e10 min at 37 C to allow for endocytosis of 125IEGF-receptor complexes into early endosomes. Control dishes are incubated with the same 125I-EGF-containing media supplemented with a 50-fold excess of unlabeled EGF to determine nonspecific radioactivity. 2. The dishes are placed on ice to stop endocytosis, and then rapidly washed three times with 1 mL of ice-cold media. The cells are incubated with 1 mL of pH 4.5 acetate buffer for 2.5 min and then additionally rinsed with 1 mL of the same


3. 4.

5. 6. 7. 8.

buffer to remove cell-surface 125I-EGF. The resulting cells contain 125I-EGF only in endosomes and are referred to as “125I-EGF-loaded cells.” The 125I-EGF-loaded cells are incubated in binding medium containing 200 ng/mL unlabeled EGF for 40 min at 4 C to occupy surface EGFR. The media are aspirated, and the cells further incubated with fresh prewarmed (37 C) media containing 200 ng/mL EGF (1 mL/dish) at 37 C (in the water bath for short times (less than 15 min) or CO2 incubator for longer times) to allow traffic of endosomal 125I-EGF-receptor complexes. The length of 37 C chase incubations of EGF-loaded cells is discussed below. Three dishes (two incubated with “specific” media and one with “nonspecific” media) are kept on ice. These dishes represent a “zero” time point. While other dishes are incubated at 37 C, the prewarmed media are added to “zero-time point” dishes and immediately collected into g-counter vials. At the end of the chase incubations, the cells are placed on ice, and the media are collected into g-counter vials. Surface-bound 125I-EGF is stripped by the pH 2.8 acetate buffer wash (as in the internalization assay Section 4.2.2) and collected into g-counter vials. The cells are then solubilized in 1N NaOH to measure the amount of intracellular 125 I-EGF as described in Section 4.2.2. The media collected at Step 5 (1 mL) is mixed with 0.3 mL of 10% TCA/2% PTA. The mixture is kept at 4 C for 1 h or longer and centrifuged at 5000 x g. The supernatants are transferred to new g-counter tubes to determine the amount of degraded 125I-EGF. The pellets are dissolved in 1 mL of 1 N NaOH to determine the amount of nondegraded, intact 125I-EGF.

4.5.3 Variation: effect of PQ on recycling 1. After Steps 1e3, Step 4 (chase incubation at 37 C) is performed using the same media but containing 300 mM PQ. 2. Follow Steps 5e8 in Protocol Section 4.5.2.

4.5.4 Results The example of an 125I-EGF recycling experiment in NIH3T3/EGFR cells is presented in Figure 6(B). The recycled 125I-EGF is expressed as percent of total (cells plus medium) 125I-EGF. For calculation, first, specific counts are obtained by subtracting nonspecific radioactivity of all fractions from the counts of the same fractions of “specific” dishes. The total amount of 125I-EGF associated with cells and media is calculated for each dish as the sum of second (pH 2.8) acid wash (surface 125 I-EGF, Step 6), intracellular 125I-EGF (Step 7), intact medium 125I-EGF (TCAprecipitated radioactivity, Step 8), and degraded medium 125I-EGF (Step 8). The amount of degraded 125I-EGF was negligible during the first 15-min chase, and therefore, is not plotted in Figure 6(B). The amount of recycled 125I-EGF is calculated by summing the surface 125I-EGF and intact medium 125I-EGF. The percent of each 125I-EGF pool (intracellular, recycled, and degraded) relative to the total

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cell/medium-associated 125I-EGF is calculated for each time point. Figure 6(B) shows that PQ strongly inhibits recycling of 125I-EGF.

4.5.5 Considerations 1. Because handling of multiple dishes in this type of experiments is difficult, it is recommended to use not more than two “specific” and one “nonspecific” dishes for each time point of the chase incubation (Step 5 below). The entire timecourse experiment is repeated two to three times to ensure the statistical significance of the data. 2. Confluent cells offer better signal/noise ratio because nonspecific binding of 125 I-EGF to plastic is minimized. 3. Because of its small size, EGF polypeptide is not efficiently precipitated by TCA alone. Therefore, addition of PTA is important for complete precipitation of intact 125I-EGF. 4. Recycling and degradation are considered to be first-order kinetics processes. This means that their specific rate constants are proportional to the concentration of 125I-EGF in early/sorting endosomes. However, the calculation of specific rate constants as the linear regression coefficients is difficult due to the decrease in the concentration of 125I-EGF in early/sorting endosomes during the 37 C chase incubation. Also, presence of unlabeled EGF and its contribution to overall EGF concentrations in various compartments makes calculations of specific rate constants challenging. Therefore, it is important to interpret the results of the recycling/degradation assay as providing values of the apparent rates. These apparent rates should be compared between experimental variants under conditions of the same starting concentration of 125I-EGF in endosomes, and preferably, the same total EGFR numbers per cell.

SUMMARY In this chapter, we describe several basic experimental procedures that are designed to perform quantitative analysis of EGFR internalization, ubiquitination, degradation, and recycling. These assays take advantage of the availability of biologically active radiolabeled EGF and fluorescent conjugates of EGF, and antibodies that efficiently immunoprecipitate native receptor and detect denatured EGFR by immunoblotting. The specific details in the design of these methods are based on the experience of using these assays to study EGFR traffic in the variety of cell types expressing a wide range of EGFR levels including various mutants of EGFR.

REFERENCES Eden, E. R., Huang, F., Sorkin, A., & Futter, C. E. (2012). The role of EGF receptor ubiquitination in regulating its intracellular traffic. Traffic, 13(2), 329e337.

References

Ferguson, S. M., & De Camilli, P. (2012). Dynamin, a membrane-remodelling GTPase. Nature Reviews Molecular Cell Biology, 13(2), 75e88. Haigler, H., Ash, J. F., Singer, S. J., & Cohen, S. (1978). Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proceedings of the National Academy of Sciences of the United States of America, 75(7), 3317e3321. Haigler, H. T., Maxfield, F. R., Willingham, M. C., & Pastan, I. (1980). Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. The Journal of Biological Chemistry, 255(4), 1239e1241. Henriksen, L., Grandal, M. V., Knudsen, S. L., van Deurs, B., & Grøvdal, L. M. (2013). Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands. PLoS One, 8(3), e58148. Hislop, J. N., & von Zastrow, M. (2011). Role of ubiquitination in endocytic trafficking of Gprotein-coupled receptors. Traffic, 12(2), 137e148. Lemmon, M. A., & Schlessinger, J. (2010). Cell signaling by receptor tyrosine kinases. Cell, 141(7), 1117e1134. McCluskey, A., Daniel, J. A., Hadzic, G., Chau, N., Clayton, E. L., Mariana, A., et al. (2013). Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. Traffic, 14(12), 1272e1289. Mettlen, M., Pucadyil, T., Ramachandran, R., & Schmid, S. L. (2009). Dissecting dynamin’s role in clathrin-mediated endocytosis. Biochemical Society Transactions, 37(Pt 5), 1022e 1026. Raub, T. J., & Newton, C. R. (1991). Recycling kinetics and transcytosis of transferrin in primary cultures of bovine brain microvessel endothelial cells. The Journal of Cellular Physiology, 149(1), 141e151. Sibilia, M., Kroismayr, R., Lichtenberger, B. M., Natarajan, A., Hecking, M., & Holcmann, M. (2007). The epidermal growth factor receptor: from development to tumorigenesis. Differentiation, 75(9), 770e787. Sorkin, A., & Goh, L. K. (2009). Endocytosis and intracellular trafficking of ErbBs. Experimental Cell Research, 315(4), 683e696. Sorkin, A., Teslenko, L., & Nikolsky, N. N. (1988). The endocytosis of epidermal growth factor in A431 cells: a pH of microenvironment and the dynamics of receptor complexes dissociation. Experimental Cell Research, 175, 192e205. Sorkin, A., & von Zastrow, M. (2009). Endocytosis and signalling: intertwining molecular networks. Nature Reviews Molecular Cell Biology, 10(9), 609e622. van Weert, A. W., Geuze, H. J., Groothuis, B., & Stoorvogel, W. (2000). Primaquine interferes with membrane recycling from endosomes to the plasma membrane through a direct interaction with endosomes which does not involve neutralisation of endosomal pH nor osmotic swelling of endosomes. The European Journal of Cell Biology, 79(6), 394e399. Weinberg, J. S., & Drubin, D. G. (2014). Regulation of clathrin-mediated endocytosis by dynamic ubiquitination and deubiquitination. Current Biology, 24(9), 951e959. Wiley, H. S., & Cunningham, D. D. (1982). The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. The Journal of Biological Chemistry, 257(8), 4222e4229.

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Volume 84 (2007) Biophysical Tools for Biologists, Volume One: In Vitro Techniques Edited by John J. Correia and H. William Detrich, III Volume 85 (2008) Fluorescent Proteins Edited by Kevin F. Sullivan Volume 86 (2008) Stem Cell Culture Edited by Dr. Jennie P. Mather Volume 87 (2008) Avian Embryology, 2nd Edition Edited by Dr. Marianne Bronner-Fraser Volume 88 (2008) Introduction to Electron Microscopy for Biologists Edited by Prof. Terence D. Allen Volume 89 (2008) Biophysical Tools for Biologists, Volume Two: In Vivo Techniques Edited by Dr. John J. Correia and Dr. H. William Detrich, III Volume 90 (2008) Methods in Nano Cell Biology Edited by Bhanu P. Jena Volume 91 (2009) Cilia: Structure and Motility Edited by Stephen M. King and Gregory J. Pazour Volume 92 (2009) Cilia: Motors and Regulation Edited by Stephen M. King and Gregory J. Pazour Volume 93 (2009) Cilia: Model Organisms and Intraflagellar Transport Edited by Stephen M. King and Gregory J. Pazour Volume 94 (2009) Primary Cilia Edited by Roger D. Sloboda Volume 95 (2010) Microtubules, in vitro Edited by Leslie Wilson and John J. Correia

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Volumes in Series

Volume 96 (2010) Electron Microscopy of Model Systems Edited by Thomas Mu¨eller-Reichert Volume 97 (2010) Microtubules: In Vivo Edited by Lynne Cassimeris and Phong Tran Volume 98 (2010) Nuclear Mechanics & Genome Regulation Edited by G.V. Shivashankar Volume 99 (2010) Calcium in Living Cells Edited by Michael Whitaker Volume 100 (2010) The Zebrafish: Cellular and Developmental Biology, Part A Edited by: H. William Detrich III, Monte Westerfield and Leonard I. Zon Volume 101 (2011) The Zebrafish: Cellular and Developmental Biology, Part B Edited by: H. William Detrich III, Monte Westerfield and Leonard I. Zon Volume 102 (2011) Recent Advances in Cytometry, Part A: Instrumentation, Methods Edited by Zbigniew Darzynkiewicz, Elena Holden, Alberto Orfao, William Telford and Donald Wlodkowic Volume 103 (2011) Recent Advances in Cytometry, Part B: Advances in Applications Edited by Zbigniew Darzynkiewicz, Elena Holden, Alberto Orfao, Alberto Orfao and Donald Wlodkowic Volume 104 (2011) The Zebrafish: Genetics, Genomics and Informatics 3rd Edition Edited by H. William Detrich III, Monte Westerfield, and Leonard I. Zon

Volumes in Series

Volume 105 (2011) The Zebrafish: Disease Models and Chemical Screens 3rd Edition Edited by H. William Detrich III, Monte Westerfield, and Leonard I. Zon Volume 106 (2011) Caenorhabditis elegans: Molecular Genetics and Development 2nd Edition Edited by Joel H. Rothman and Andrew Singson Volume 107 (2011) Caenorhabditis elegans: Cell Biology and Physiology 2nd Edition Edited by Joel H. Rothman and Andrew Singson Volume 108 (2012) Lipids Edited by Gilbert Di Paolo and Markus R Wenk Volume 109 (2012) Tetrahymena thermophila Edited by Kathleen Collins Volume 110 (2012) Methods in Cell Biology Edited by Anand R. Asthagiri and Adam P. Arkin Volume 111 (2012) Methods in Cell Biology Edited by Thomas Mu¨ler Reichart and Paul Verkade Volume 112 (2012) Laboratory Methods in Cell Biology Edited by P. Michael Conn Volume 113 (2013) Laboratory Methods in Cell Biology Edited by P. Michael Conn Volume 114 (2013) Digital Microscopy, 4th Edition Edited by Greenfield Sluder and David E. Wolf Volume 115 (2013) Microtubules, in Vitro, 2nd Edition Edited by John J. Correia and Leslie Wilson

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Volumes in Series

Volume 116 (2013) Lipid Droplets Edited by H. Robert Yang and Peng Li Volume 117 (2013) Receptor-Receptor Interactions Edited by P. Michael Conn Volume 118 (2013) Methods for Analysis of Golgi Complex Function Edited by Franck Perez and David J. Stephens Volume 119 (2014) Micropatterning in Cell Biology Part A Edited by Matthieu Piel and Manuel The´ry Volume 120 (2014) Micropatterning in Cell Biology Part B Edited by Matthieu Piel and Manuel The´ry Volume 121 (2014) Micropatterning in Cell Biology Part C Edited by Matthieu Piel and Manuel The´ry Volume 122 (2014) Nuclear Pore Complexes and Nucleocytoplasmic Transport - Methods Edited by Vale´rie Doye Volume 123 (2014) Quantitative Imaging in Cell Biology Edited by Jennifer C. Waters and Torsten Wittmann Volume 124 (2014) Correlative Light and Electron Microscopy II Edited by Thomas Mu¨ller-Reichert and Paul Verkade Volume 125 (2015) Biophysical Methods in Cell Biology Edited by Ewa K. Paluch Volume 126 (2015) Lysosomes and Lysosomal Diseases Edited by Frances Platt and Nick Platt

Volumes in Series

Volume 127 (2015) Methods in Cilia & Flagella Edited by Renata Basto and Wallace F. Marshall Volume 128 (2015) Building a Cell from its Component Parts Edited by Jennifer Ross and Wallace F. Marshall Volume 129 (2015) Centrosome & Centriole Edited by Renata Basto and Karen Oegema

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Index Note: Page numbers followed by “f” and “t” indicate figures and tables respectively.

A A280 protocol, 124 Abscission analyzing establishment, 25e26 data interpretation, 26e27 procedure, 26 asymmetric and symmetric models, 25f measuring delay, 24 data interpretation, 24 procedure, 24 mechanisms mediating, 21f ACAP1-based coat complex, 82e83 antibodies, 84 cargo binding, 85e87 cargo sequences recognizing, 90fe91f, 91 cells and reagents, 83e84 coat complex formation by ACAP1 and clathrin, 87e88, 89f endocytic recycling assays, 91e92 Glut4 recycling, 95, 96f integrin recycling, 94, 95f TfR recycling, 92, 93f plasmids and transfections, 84e85 ACAP1-based complex, 82 Actin dynamics, time-lapse analysis of, 27 analyzing actin dynamics, 29e30 and apical lumen formation in 3D cultures, 30e32 RAB11/FIP3-endosome transport analysis, 27e29 Actin filaments, 29 Additives, 116 DiR, 116 nickel, 116 Adenosine diphosphate ribosylation factor (ARF), 82e83 ADP ribosylation factor (Arf), 103 GEF activity measurement, 117e121 materials chemical reagents, 105e106 equipment, 104 software, 106 proteineArf1 interactions identification, 121e123 purification of required proteins, 106 protein buffers, 107t Sec7, 106e112

SMALL GTPases, 112e115 supporting protocol, 123e125 ADP-ribosylation factors (Arfs), 70 effects of, 70 Anion exchange, 110e112 Anti-early endosomal antigen-1 (anti-EEA1), 322 anti-EEA1, see Anti-early endosomal antigen-1 (anti-EEA1) Antibody/antibodies, 310, 322e323 internalization assays cell imaging, 133e134 cell plating, 132 internalization assays, 133 Anticellubrevin (Cbv), 84 AP-1B heterotetrameric clathrin adapter complex, 290 during endocytic recycling, biosynthetic delivery, or both, 299e303 instruments, 293 materials cell lines, 292 reagents and materials, 292e293 objectives and rationale, 291e292 Apical lumen formation in 3D cultures, 30 midbody-associated, 31f procedure, 30e32 Apical recycling endosome (ARE), 279e280 Apical sorting endosomes (ASE), 272e273 background and objective, 276 buffers composition, 276e277 experimental protocol, 277 apo-Tf loading with iron, 257e258 ARE, see Apical recycling endosome (ARE) ARF, see Adenosine diphosphate ribosylation factor (ARF) Arf, see ADP ribosylation factor (Arf) Arf G proteins in endosomal trafficking, 129f ARF-6-GFP, 83, 190 ARF6, 83 Arfs, see ADP-ribosylation factors (Arfs) ARH protein, see Autosomal recessive hypercholesterolemia protein (ARH protein) ASAP2, see Myc-tagged PAP ASE, see Apical sorting endosomes (ASE) Autosomal recessive hypercholesterolemia protein (ARH protein), 290

383

384

Index

B B2AR, see Beta 2 adrenergic receptor (B2AR) Baby hamster kidney cells (BHK cells), 37 Bacmid preparation, 107 Basolateral early endosomes (BEEs), 250 Basolateral sorting at steady state, see also AP-1B heterotetrameric clathrin adapter complex cargo proteins immunoprecipitation alternative approaches, 298 background and objective, 296 considerations, 297 expected results, 297e298 flow of experiment, 296e297 LLC-PK1 cells, protein sorting analysis in, 295f alternative approaches, 295 background and objective, 293 considerations, 294 expected results, 294e295 flow of experiment, 293e294 Basolateral sorting endosomes (BSE), 272e273, 278 Basolateral TfReholo-Tf complex, 250 Batch nickel affinity purification, 110e111 BBC, see Bovine brain cytoplasm (BBC) BEEs, see Basolateral early endosomes (BEEs) BEL, see Bromoenol lactone (BEL) Beta 2 adrenergic receptor (B2AR), 334 BFA, see Brefeldin A (BFA) BHK cells, see Baby hamster kidney cells (BHK cells) BHK-21 cells transfection, 40 Biochemical-based assay, 283e285, see also Microscopy-based trafficking assays Bioluminescence resonance energy transfer technology (BRET technology), 60, 61f analyzing conformational change induced by ERK1/2, 64e65 ERK2 purification, 65 materials, 65 quantification of Rabin8 conformational change, 66 autoinhibitory conformation of Rabin8, 62f Bovine brain cytoplasm (BBC), 163 Bovine serum albumin (BSA), 160, 254, 278, 292, 322, 350 Bradford semiquantitative analysis, 123e124 Brefeldin A (BFA), 163 stimulation of endosome membrane tubules formation, 167e168 BRET technology, see Bioluminescence resonance energy transfer technology (BRET technology)

Bromoenol lactone (BEL), 175 BSA, see Bovine serum albumin (BSA) BSE, see Basolateral sorting endosomes (BSE)

C C-terminal regions, 88 Caenorhabditis elegans (C. elegans), 182 general mechanisms of membrane traffic in, 182 materials and instruments reagents, 184 and software, 184 methods endocytic tracer analysis in intestinal epithelia, 186e188 endosomal dynamics, 194e195 living intact animals, morphometric analysis of endosomal markers in, 188e191 transmembrane recycling cargo assays, 191e194 yolk protein YP170 uptake assay, 184e186 MIG-14, 192 objectives and rationale, 183 polarized epithelia, 182e183 Caenorhabditis Genetics Center (CGC), 190e191 Canine holo-Tf, reagents to iodinate, 254 Cargo binding by ACAP1, 85, 86f ARF6 GAPs, 86e87 cytoplasmic domains of cargoes, 85e86 HeLa cells, 85 pulldown studies, 87 Cargo movement through RE, 8 experimental considerations, 11 flow of experiment, 9e10 results, 11e14 VAMP3 recruitment, 13f Cargo proteins immunoprecipitation alternative approaches, 298 background and objective, 296 considerations, 297 expected results, 297e298 flow of experiment, 296e297 Cbv, see Anticellubrevin (Cbv) CDE, see Clathrin-dependent endocytosis (CDE) Cell imaging, 133e134 Cell plating, 132, 204 Cell-free, endosome membrane tubules in vitro formation, 163 equipment, 164 microfuge tubes, 165 negative-stained endosomes, 166f reaction, 165

Index

reagents, 163e164 solutions and reagents, 164 Centrosome isolation and identification, 53 at interphase, methods to, 53e55 reconstituting GST-Rab11, methods for, 56 staining isolated centrosomes, methods for, 55 methods to isolate centrosome at interphase, 53e55 CGC, see Caenorhabditis Genetics Center (CGC) CHC, see Clathrin heavy chain (CHC) CI-M6PR, 201e203 antibody labeling to monitor retrograde trafficking, 204e205 CIE, see Clathrin-independent endocytosis (CIE) Clathrin heavy chain (CHC), 84 Clathrin-dependent endocytosis (CDE), 140e141 Clathrin-independent endocytosis (CIE), 128, 140e141 Clathrin-mediated endocytosis (CME), 128 Cloning, reagents for, 37 CM, see Complete medium (CM) CME, see Clathrin-mediated endocytosis (CME) Coat protein II complex (COPII complex), 82e83 Coat proteins, 82 Colloidal gold particles, 160 Colloidal gold-loaded endosomes, 161 equipment, 161 procedure, 161e163 reagents, 161 Column volumes (CV), 110 Common recycling endosome (CRE), 250 Complete medium (CM), 38 Confocal fluorescence microscopy localization by, 42 materials and reagents for, 38 Confocal immunofluorescence microscopy assay reagents, 323 Confocal microscopy, 168e169, 335 procedure, 169 Fiji macro script, 173e174 Sholl analysis, 172e173 step-by-step endosome membrane tubule “tracing”, 171e173 semiautomated endosome membrane tubule segmentation, 170f Constitutively active ERK2 (ERK2-CA), 65 Coomassie gel analysis, 124e125 COPII complex, see Coat protein II complex (COPII complex) CRE, see Common recycling endosome (CRE)

CRETfR, see TfR-rich perinuclear common recycling endosomes (CRETfR) Culture MDCK cells, reagents to, 256 Curve comparison, 120 extraction, 120 fitting, 120 CV, see Column volumes (CV) Cytokinesis, 20 methods for analyzing proteins regulating, 22 multinucleation analysis, 22e24 time-lapse analysis of endosome and actin dynamics during, 27 analyzing actin dynamics, 29e30 and apical lumen formation in 3D cultures, 30e32 RAB11/FIP3-endosome transport analysis, 27e29 Cytoskeleton, 216e217

D Data interpretation, 23e24, 26e27 DEAE cellulose, see Diethylaminoethyl cellulose (DEAE cellulose) DEPC, see Diethylpyrocarbonate (DEPC) Desferoxamine B, see Iron chelator desferrioxamine DEX, see Dexamethasone (DEX) Dexamethasone (DEX), 312 Diethylaminoethyl cellulose (DEAE cellulose), 106 Diethylpyrocarbonate (DEPC), 37 Dimethyl sulfoxide (DMSO), 239, 256, 284, 292 DiR die, 116 Discontinuous sucrose gradient flotation, 122 3,3’-Dithiobis(sulfosuccinimidylpropionate) (DTSSP), 292 Divalent metal transporter (DMT1), 250 DMEM, see Dulbecco’s modified eagle medium (DMEM) DMSO, see Dimethyl sulfoxide (DMSO) DMT1, see Divalent metal transporter (DMT1) Dominant negative forms of G proteins, 130e131 DTSSP, see 3,3’-Dithiobis(sulfosuccinimidylpropionate) (DTSSP) Dulbecco’s minimum essential medium, see Dulbecco’s modified eagle medium (DMEM) Dulbecco’s modified eagle medium (DMEM), 84, 209, 322, 335, 350

385

386

Index

Dulbecco’s modified essential medium, see Dulbecco’s modified eagle medium (DMEM) Dynamin inhibitor, 352 considerations, 356 experimental protocol, 352e353 results, 353e354 variation, 353 Dyngo-4a, 349e350 on EGFR endocytosis, 353, 355f

E Early endosomes maturation, biochemical analysis of, 245 Early/sorting endosome, 140e141 EDTA, see Ethylenediamminetetraacetate (EDTA) EGF, see Epidermal growth factor (EGF) EGF-Rhodamine (EGF-Rh), 351e352 EGF:EGFR complex recycling using 125I-EGF considerations, 366 experimental protocol, 364e365 results, 365 theory, 363e364 variation, 365 EGFR, see Epidermal growth factor receptor (EGFR) EHD proteins, see Eps15 homology domain proteins (EHD proteins) Electron microscopy (EM), 239e240 EM, see Electron microscopy (EM) Endocytic compartment, 70 Endocytic pathway, 141f, 216, 236, see also Rab GTPasesekinesin interactions Endocytic recycling, 308 assays, 91e92 Glut4 recycling, 95, 96f integrin recycling, 94, 95f TfR recycling, 92, 93f buffers, 143 cells, 142 equipment, 142 materials, 142 MHC I antibody-receptor complex recycling quantification, 152e154 qualitative assessment, 143e149 quantitative assessment, 149e152 reagents, 143 Endocytic recycling compartment (ERC), 140e141 Endocytic tracers, 182e183 analysis in intestinal epithelia, 186e188

Endocytosis, 128, 352 Endoplasmic reticulum (ER), 36 Endosomal compartments, 128 in epithelial cells, 272, 273f, 275 Endosomal dynamics experimental strategies and considerations, 194 principles, 194 time-lapse imaging, 194e195 Endosomal sorting complexes required for transport (ESCRT), 148e149, 320e321 complexes, 20 ESCRT-III complex, 20 Endosomal trafficking materials and instruments, 131e132 methods, 132 antibody internalization assays, 132e134 expected results, 136e137 expression of wild type and mutant Rab and Arf proteins and effects, 134 flow of experiment, 134e136 objectives and rationale, 130e131 Rab and Arf G proteins, 129f Endosome dynamics, time-lapse analysis of, 27 analyzing actin dynamics, 29e30 and apical lumen formation in 3D cultures, 30e32 RAB11/FIP3-endosome transport analysis, 27e29 Endosome tubulation imaging, 218, see also Wash microdomains live imaging considerations, 220e222 inducing by WASH inhibition, 221f microscope, 219 procedure cell plating and Tf uptake, 220 cell transfection, 219 imaging and results, 220 reagents, 218e219 Endosome(s), 128, see also Liposome(s); Recycling endosome (RE) membrane tubule formation, 158e160 from Golgi complex, 160 PLA2 enzymes, 174e178 in vitro, 160e165 in vivo, 167e174 membrane tubule tracing, 171e172 visualization of sorting and recycling in, 341e342, 343f considerations, 342e343 flow of experiments, 342 results, 343

Index

Ensemble measurement of surface levels, 337, 337f considerations, 338 flow of experiments, 338 results, 339 Epidermal growth factor (EGF), 142, 158e160, 239e240, 348e349 Epidermal growth factor receptor (EGFR), 239e240, 272, 348e349 cell culture, 351 degradation, 362f dynamin inhibitor, 352e356 EGF:EGFR complex, 349e350 recycling using 125I-EGF, 363e366 fluorescence microscopy, 352 imaging EGFR endocytosis in living cells, 352e356 instruments, 351 internalization rates measurement, 356 considerations, 358e359 experimental protocol, 356e357 results, 357e358 variation, 357 ligand-induced EGFR degradation, 361e363 PQ effect, 363e366 reagents, 350e351 SDS-page, 351e352 trafficking, 349 ubiquitination, 360f considerations, 361 experimental protocol, 359e360 results, 360e361 theory, 359 western blot, 351e352 Epithelial cells, 248 Epithelial tissues, 248 Eps15 homology domain proteins (EHD proteins), 141e142 ER, see Endoplasmic reticulum (ER) ERC, see Endocytic recycling compartment (ERC) ERK1/2, BRET assay to analyzing conformational change, 64e65 ERK2 purification, 65 materials, 65 quantification of Rabin8 conformational change, 66 ERK2-CA, see Constitutively active ERK2 (ERK2-CA) ERK2-KD, see Kinase-dead ERK2 (ERK2-KD) ESCRT, see Endosomal sorting complexes required for transport (ESCRT)

Ethanol (EtOH), 241e242 Ethylenediaminetetraacetic acid, see Ethylenediamminetetraacetate (EDTA) Ethylenediamminetetraacetate (EDTA), 87, 256 Evi5 centrosome-targeted protein, 49 Exocyst complex, 309 directing outbound trafficking, 309 Glut4 outbound trafficking, 310 RalA cellular activity, 309 Extrusion, 116e117

F FACS, see Fluorescence-activated cell sorting (FACS) Fast recycling route, 242 FBS, see Fetal bovine serum (FBS) FCS, see Fetal calf serum (FCS) Fetal bovine serum (FBS), 37, 84, 167, 240, 256, 292, 312, 335, 350 Fetal calf serum (FCS), 84 Fiji macro script, 173e174 Fix cells, 135 Fluorescence intensity analysis, 330 Fluorescence microscopy, 352 imaging EGFR endocytosis in living cells by, 352e356 Fluorescence-activated cell sorting (FACS), 208 stripping buffer, 208e209 Fluorescent protein (FP), 237 Fluorescent TF uptake, labeling tubular endocytic compartments by procedure, 167e168 reagents and supplies, 167 FP, see Fluorescent protein (FP) Fractional conversion calculation, 76e78 Functional assays, 239e240 biochemical analysis of early endosomes maturation, 245 kinesin depletion, 240e241 slow Tf recycling route analysis, 242e244 ultrastructural localization of internalized Tf, 241e242

G G protein-coupled receptor (GPCR), 320e321, 334 sorting at MVEs, 321 cell plating and transfection, 323e324 GFP, 321e322 IEM, 321 instrumentation, 323

387

388

Index

G protein-coupled receptor (GPCR) (Continued) proteinase-protection assay, 324e326 by Rab5-Q79L expression, 327e330 reagents, 322e323 visualizing and quantitating GPCR recycling, 334 cell culture, plating, and transfection, 336 confocal microscopy, 335 endocytosis, 335 ensemble measurement of surface levels, 337e339 equipment, 336 live cell imaging conditions, 336 quantifying recycling at single-event resolution, 339e341 reagents, 335e336 visualization of sorting and recycling in endosome, 341e343 G proteins, see GTP-binding proteins (G proteins) GAPs, see GTPase-activating proteins (GAPs) GDP, see Guanosine diphosphate (GDP) GEFs, see Guanine nucleotide exchange factors (GEFs) GFP, see Green fluorescent protein (GFP) GGA, see Golgi-localized, gamma-adaptin (GGA) Glucose transporter type 4 (Glut4), 83, 88 detection of cell surface, 315e317 recycling, 95, 96f 3T3-L1 preadipocytes maintenance and differentiation, 312 buffers, 310 detection of cell surface Glut4, 315e317 exocyst complex, 309 GST-RAL binding domain preparation, 311e312 instruments, 311 plasmids and cell lines, 311 pulldown assay, 312e314 reagents, 310 RNAi-mediated depletion of RalA, 314e315 Glutathione-S-transferase (GST), 84 Gold stabilized with bovine serum albumin (Gold-BSA particles), 160 Gold-BSA particles, see Gold stabilized with bovine serum albumin (Gold-BSA particles) Golgi-localized, gamma-adaptin (GGA), 82 GPCR, see G protein-coupled receptor (GPCR) Green fluorescent protein (GFP), 48e49, 84, 130, 134, 182, 321e322 GFP-RAB-10 fusion protein, 190 GFP-RAB-11.1 marker, 190 GFP-VAMP3 mirrors, 16

GFP::RME-1 fusion protein, 188e190 GST, see Glutathione-S-transferase (GST) GST-RAL binding domain preparation, 311e312 GTP-binding proteins (G proteins), 128 GTPase-activating proteins (GAPs), 70, 82e83, 130 background, 74 exchanging GXP for [a32P]GTP, 75 materials, 74e75 preparing large unilamellar vesicles, 75 results and analysis, 76e78 GTPases, see Guanosine triphosphatases (GTPases) Guanine nucleotide exchange factors (GEFs), 36, 60, 103, 130 activity measurement native tryptophan fluorescence of Arf1, 117e120 small GTPase crosstalk variation, 121 Guanosine diphosphate (GDP), 103 Guanosine triphosphatases (GTPases), 82e83, 103, 272

H HaloTag protein, 61 Hanks balanced salt solution (HBSS), 276 HBSS, see Hanks balanced salt solution (HBSS) HEK cells, see Human embryonic kidney cells (HEK cells) HeLa cells, 84, 203, 238e239, 243e245, 323e324 Holo-Tf iodination, 258e259 Horseradish peroxidase (HRP), 51e53, 251 Horseradish peroxide, see Horseradish peroxidase (HRP) HRP, see Horseradish peroxidase (HRP) hTAC, 192 hTfR, see human transferrin receptor (hTfR) Human embryonic kidney cells (HEK cells), 335 human transferrin receptor (hTfR), 192 Humidity chamber, 145f 125 I-EGF, 349e350 125 I-EGF-loaded cells, 364e365 EGF:EGFR complex recycling using, 363e366 EGFR internalization rates measurement using, 356 considerations, 358e359 experimental protocol, 356e357 results, 357e358 variation, 357 internalization, 358f recycling, 364f 125 I-Tf recycling, reagents to, 256

Index

I IC, see Intermediate compartment (IC) ICB, see Intracellular bridge (ICB) ICl method, see Iodine monochloride method (ICl method) IEM, see Immune-electron microscopy (IEM) IF, see Immunofluorescence (IF) ILVs, see Intraluminal vesicles (ILVs) Immune-electron microscopy (IEM), 321 Immunoblotting antibodies, 322 Immunofluorescence (IF), 205, 237 antibodies, 323 confocal microscopy, 321e322 In vitro endosome membrane tubule formation, 160 BBC, 163 cell-free, 163e165 colloidal gold particles preparation, 160 colloidal gold-loaded endosomes preparation, 161e163 In vivo endosome membrane tubule formation, 167 confocal microscopy, 168e174 labeling tubular endocytic compartments, 167e168 quantitative image analysis, 168e174 Insect cell transfection, 107e109 Insulin responsive glucose transporter, see Glucose transporter type 4 (Glut4) Insulin-stimulated Glut4 trafficking, 308e309 Integrin recycling analysis, 94, 95f, 208, see also Retrograde trafficking analysis experimental analysis, 210 experimental strategy equipment, 208 FACS stripping buffer, 208e209 reagents, 208 software, 208 methods antibody labeling of cells, 209 cell plating, RNAi transfection, and harvesting of cells, 209 controls, 210 recycling and acid-stripping of antibodylabeled cells, 209e210 Intermediate compartment (IC), 36 Internalization assays, 133, 135 process, 140e141 of receptor, 252 Internalized cargo proteins trafficking, 281e282 Intracellular bridge (ICB), 20

Intraluminal vesicles (ILVs), 320e321 Iodine monochloride method (ICl method), 251 IPTG, see Isopropyl b-D-galactopyranoside (IPTG) IRE, see Iron response/regulatory element (IRE) Iron chelator desferrioxamine, 252 Iron load canine apo-Tf, reagents to, 253e254 Iron response/regulatory element (IRE), 248e250 Isobutyl-1-methylzanthine (MIX), 312 Isopropyl b-D-galactopyranoside (IPTG), 105

J JNK-interacting protein 4 (JIP4), 130

K Kinase-dead ERK2 (ERK2-KD), 65 Kinesin, 236 depletion, 240e241 live cell imaging on cells coexpressing, 238f microtubule cytoskeleton, 239 material, 239 methods, 239 results, 239 Rab GTPasesekinesin interactions, screen for, 236 methods, 236e237 results, 237 spatiotemporal distribution, 237 material, 237 results, 238

L Labeling surface receptors, 251e252 Large unilamellar vesicles (LUVs), 74 Last eukaryotic common ancestor (LECA), 37 Late endosomes (LE), 272 Late-endosomal/lysosomal pathway (LE/Lys pathway), 140e141 LDLR, see Low-density lipoprotein receptor (LDLR) LE, see Late endosomes (LE) LE/Lys pathway, see Late-endosomal/lysosomal pathway (LE/Lys pathway) LECA, see Last eukaryotic common ancestor (LECA) Ligand-induced EGFR degradation considerations, 363 experimental protocol, 361e362 results, 362e363 theory, 361 Lilly Laboratories cell porcine kidney model cell line (LLC-PK1 model cell line), 292

389

390

Index

Lipid mixes, 116 Liposome(s), see also ADP ribosylation factor (Arf); Endosome(s) binding, 122 composition, 115 additives, 116 flotation, 122e123, 123f analysis, 122e123 discontinuous sucrose gradient flotation, 122 nucleotide exchange, 122 quantification of recovery, 122 production lipid mixes, 116 solubilization and extrusion, 116e117 size, 116 Live cell imaging, 5 Living intact animals, morphometric analysis of endosomal markers in ARF-6-GFP, 190 broadspectrum autofluorescence, 191 experimental strategies and considerations, 190 GFP emission, 191 GFP-RAB-10 fusion protein, 190 GFP-RAB-11.1 marker, 190 GFP::RME-1 fusion protein, 188e190 intestinal cell, 191 organelle markers, 190e191 principles, 188 LLC-PK1 cells alternative approaches, 295 background and objective, 293 considerations, 294 expected results, 294e295 flow of experiment, 293e294 protein sorting analysis, 295f LLC-PK1 model cell line, see Lilly Laboratories cell porcine kidney model cell line (LLC-PK1 model cell line) Low-density lipoprotein receptor (LDLR), 272, 294 LROs, see Lysosome-related organelles (LROs) LUVs, see Large unilamellar vesicles (LUVs) Lysate clearing, 109e111 Lysis clearing, 109e111 Lysosome-related organelles (LROs), 191

M mAb, see Monoclonal antibody (mAb) Macrophages, 2e3, see also Recycling endosomes (REs) localization of recycling endosome-associated Rab GTPases, 7f

recycling endosome cargo and trafficking in, 12f MadineDarby canine kidney cell lines (MDCK cell lines), 248, 292, see also Polarized MDCK cells Magnaporthe oryzae (M. oryzae), 37, see also Rabs recycling Major histocompatibility complex class I protein (MHC I protein), 133, 149e151, 158e160 antibody-receptor complex recycling quantification, 152e154 MDCK cell lines, see MadineDarby canine kidney cell lines (MDCK cell lines) Mean intensity fluorescence (MIF), 283 MEM, see Minimum essential medium (MEM) Membrane receptors, 295 trafficking events, 342e343 MHC I protein, see Major histocompatibility complex class I protein (MHC I protein) MICAL-L1, see Molecule interacting with CasLlike1 (MICAL-L1) Microscopy-based apical transcytosis assay, 282e283 Microscopy-based protocols, 275 ARE, 279e280 ASE, 276e277 BSE, 278 LE/Lys, 280e281 ligands and endosomal markers, 276t TfR-rich perinuclear common recycling endosomes, 279 Microscopy-based trafficking assays, 281 internalized cargo proteins trafficking, 281e282 microscopy-based apical transcytosis assay, 282e283 Microtubule dynamics analysis during cytokinesis, 29e30 Midbody, 29 Middle loop regions, 88 MIF, see Mean intensity fluorescence (MIF) MIG-14-GFP, 193e194 MIG-14, 192 Minimum essential medium (MEM), 161, 256, 292 MIX, see Isobutyl-1-methylzanthine (MIX) Molecular switches, 309 Molecule interacting with CasL-like1 (MICAL-L1), 141e142 Monoclonal antibody (mAb), 38 MOR, see mu opioid receptor (MOR) MoRab11, MoRab8, and MoRab1 cloning

Index

amplifying cDNAs, 39e40 total RNA extraction, 38e39 colocalization in BHK cells, 43f expression and intracellular localization BHK-21 cells transfection, 40 localization by confocal fluorescence microscopy, 42 protein expression immunoblot analysis, 40e42 mu opioid receptor (MOR), 334 Multinucleation analysis, 22e23 data interpretation, 23e24 procedure, 23 Multivesicular bodies (MVB), 200 Multivesicular endosomes (MVEs), 320e321, see also G protein-coupled receptor (GPCR) MVB, see Multivesicular bodies (MVB) MVEs, see Multivesicular endosomes (MVEs) Myc-tagged PAP, 84 Myc7-Glut4-eGFP reporter, cell surface Glut4 detection with, 315e317 Myristoylated Arf1 (myrArf1), 73 expression, 72e73 GAP assay, 75e76 background, 74 exchanging GXP for [a32P]GTP, 75 materials, 74e75 preparing large unilamellar vesicles, 75 results and analysis, 76e78 preparation background, 71e72 buffers, 72 materials, 72 purification, 73e74, 112e114 protein expression, 113 protein purification, 113e114 Rab GTPases, 115

N N-terminal regions, 88 NanoLuc, 61 Native tryptophan fluorescence of Arf1, 117 data analysis curve comparison, 120 curve extraction, 120 curve fitting, 120 data collection notes, 117e119 protein fluorescence calibration, 119 protocol, 119 before starting, 119

nucleotide exchange kinetics calculation, 118f working stocks, 117 Neuron-glia cell adhesion molecule (Ng-CAM), 301 Nickel, 116 Nocodazole (Noco), 239, 251e252 Nucleotide exchange, 122

O Object-based colocalization method, 226 Open reading frame (ORF), 62 Optical axis integration scan function (OAI scan function), 5 Optical density (OD), 72e73

P PAF, see Platelet-activating factor (PAF) PAR1, see Protease-activated receptor 1 (PAR1) Paraformaldehyde (PFA), 38, 203, 292, 323 pBI-Tet vector amplifying cDNAs of MoRab11, MoRab8, and MoRab1, 39e40 eGFP-labeled MoRabs bidirectional expression vector, 41f PBS, see Phosphate buffered saline (PBS) PC, see Phosphatidylcholine (PC) PE, see Phosphatidylethanolamine (PE) PEI, see Polyethylenimine (PEI) Personal Deltavision, 5 PFA, see Paraformaldehyde (PFA) Phagosomes, RE delivery to, 14 experimental considerations, 15 flow of experiment, 15 results, 16 Phenylmethylsulfonyl fluoride (PMSF), 63, 105, 323 Phosphate buffered saline (PBS), 23, 26, 37, 53, 85e86, 132, 203, 277, 296, 322, 350 Phosphate-buffered salt, see Phosphate buffered saline (PBS) Phosphatidylcholine (PC), 74 Phosphatidylethanolamine (PE), 74 Phosphatidylinositiol 3,4,5-trisphosphate (PIP3), 74 Phosphatidylinositol (PI), 74 Phosphatidylinositol 4,5-bisphosphate (PIP2), 74 Phosphatidylserine (PS), 74 Phosphoinositides (PIPs), 128 Phospholipase A2 (PLA2), 158e160 antagonists, 175 inhibitors use in in vitro assays, 178 reversible inhibitors, 175e177

391

392

Index

Phospholipase A2 (PLA2) (Continued) treatment of living cells, 175 endocytic organelles and steps, 159f in endosome membrane tubule formation, 174 inhibitors, 176te177t Phosphotungstic acid (PTA), 164, 350 PI, see Phosphatidylinositol (PI) pIgR, see Polymeric IgA receptor (pIgR) PIP2, see Phosphatidylinositol 4,5-bisphosphate (PIP2) PIP3, see Phosphatidylinositiol 3,4,5trisphosphate (PIP3) PIPs, see Phosphoinositides (PIPs) PLA2, see Phospholipase A2 (PLA2) Plasma membrane (PM), 140, 272 Plasmids, 37, 84e85 Platelet-activating factor (PAF), 174 PM, see Plasma membrane (PM) PMSF, see Phenylmethylsulfonyl fluoride (PMSF) PNS, see Postnuclear supernatant (PNS) Polarized epithelial cells microscopy-based protocols for endosomal labeling, 275 ARE, 279e280 ASE, 276e277 BSE, 278 LE/Lys, 280e281 ligands and endosomal markers, 276t TfR-rich perinuclear common recycling endosomes, 279 microscopy-based trafficking assays, 281 internalized cargo proteins trafficking, 281e282 microscopy-based apical transcytosis assay, 282e283 Polarized MDCK cells culture, 255f endocytic pathways in, 249f materials and instruments, 253 culture MDCK cells, reagents to, 256 equipment, 257 125 I-Tf recycling, reagents to, 256 iodinate canine holo-Tf, reagents to, 254 iron load canine apo-Tf, reagents to, 253e254 tricholoracetic acid precipitation, reagents to, 256e257 methods culturing MDCK cells on 12-MM transwells, 259e260 experimental strategy to receptor recycling measurement, 260e266 iodinating holo-Tf, 258e259

loading apo-Tf with iron, 257e258 objectives and rationale, 250e251 internalization of receptor, 252 labeling surface receptors, 251e252 receptor recycling measurement, 252e253 Polyethylenimine (PEI), 322 Polymeric IgA receptor (pIgR), 272e273 Postnuclear supernatant (PNS), 161 PQ, see Primaquine (PQ) Primaquine (PQ), 349e350 effect, 363e366 Protease-activated receptor 1 (PAR1), 320e321 Protein buffers, 107t concentration quantification A280, 124 Bradford semiquantitative analysis, 123e124 Coomassie gel analysis, 124e125 quick analysis of elution fractions, 123 expression, 110e111 approximately 4e5 days beforehand, 109 day 1, 109 day 3, 109 immunoblot analysis, 40e42 one day beforehand, 109 reagents for protein expression and analysis, 37e38 fluorescence calibration, 119 purification, 108f, 109 anion exchange, 110 batch nickel affinity purification, 110 lysis and lysate clearing, 109e110 ProteineArf1 interactions identification liposome flotation, 122e123, 123f analysis, 122e123 discontinuous sucrose gradient flotation, 122 liposome binding, 122 nucleotide exchange, 122 quantification of recovery, 122 notes, 121 Proteinase K protection assay reagents, 323 Proteinase-protection assay, 321, 324 considerations, 326 proteinase-protection endosomal sorting assay, 325f results, 326 workflow of experiment, 324e326 PS, see Phosphatidylserine (PS) PTA, see Phosphotungstic acid (PTA) Pulldown assay, 312e314

Index

Q Qualitative assessment of endocytic recycling experimental strategies, 143e144 humidity chamber, 145f imaging, 147e148 immunfluorescence, 144e147 postimage processing, 148 remarks, 148e149 Tf and EGF treatment, 144 Quantitative assessment of endocytic recycling experimental strategies, 149e151 flow cytometry analysis of fluorescently labeled Tf, 151e152 remarks, 152 Quantitative image analysis, 168e169 Quick analysis of elution fractions, 123

R Rab GTPases, 36, 115, 236 live cell imaging on cells coexpressing, 238f microtubule cytoskeleton in, 239 material, 239 methods, 239 results, 239 screen for Rab GTPasesekinesin interactions, 236 methods, 236e237 results, 237 spatiotemporal distribution, 237 material, 237 results, 238 Rab proteins, 60, 128e129, 141 in endosomal trafficking, 129f Rab11-effector proteins, 22 RAB11/FIP3-endosome transport analysis, 27e28 during cytokinesis, 28f data interpretation, 29 procedure, 28e29 Rab5 protein, 128e129 Rab5-Q79L expression, GPCR sorting quantification at MVEs by, 327 considerations and approaches, 328e329 fluorescence intensity analysis, 330 quench and wash buffers, 328t results, 330 workflow of experiment, 327e328 Rab5Q79L, 222e223 Rab8 protein, 36 Rabin8, 60 autoinhibitory conformation, 61 BRET assay setup, 64 detecting Rabin8 conformational change, 64 BRET technology, 61fe62f

materials, 62e63 NanoLuc-HaloTag fusion proteins expression and purification, 63 Rabs 4 protein, 128e129 Rabs 7 protein, 128e129 Rabs 9 protein, 128e129 Rabs 11 protein, 128e129 Rabs 22 protein, 128e129 Rabs 35 protein, 128e129 Rabs recycling, 37 equipment, 38 materials and reagents for confocal fluorescence microscopy, 38 MoRab11, MoRab8, and MoRab1 cloning, 38e40 expression and intracellular localization, 40e42 plasmid construction, 37 RE subcompartments by, 5e8 reagents for cloning, 37 reagents for protein expression and analysis, 37e38 Radioactive pulse-chase experiments, see also Polarized MDCK cells alternative approaches, 302e303 background and objective, 299 considerations, 301 expected results, 301 flow of experiment, 299e301 RalA cellular activity, 309 preparing GST-RAL binding domain, 311e312 pulldown assay using immobilized GST-RBD to determining, 312e314 RNAi-mediated depletion of RalA, 314e315 Ras-MEK-ERK signal pathway, 64e65 RAW264. 7 macrophages, 5 RE, see Recycling endosome (RE) Receptor recycling measurement, 252e253 background and objective, 260 flow of experiment considerations, 264e266 data analysis, 263e264 labeling cohort of TfR with 125I-Tf, and ensuring internalization, 260e261 perform recycling assay, 261e263 removal of cell-surface bound 125I-Tf, 261, 262f results, 266 TCA precipitation, 263 Reconstituting GST-Rab11, methods for, 56 Recycling, 248 recycling/transcytosis assay, 284e285

393

394

Index

Recycling endosome (RE), 2e3, 20, 48e49, see also Endosome(s) cargo movement, 8 experimental considerations, 11 flow of experiment, 9e10 results, 11e14 VAMP3 recruitment, 13f cell culture and transfection, 4 centrosome isolation and identification, 53 centrosomes isolation at interphase, methods to, 53e55 reconstituting GST-Rab11, methods for, 56 staining isolated centrosomes, methods for, 55 centrosome localization by electron microscopy, 51e53 delivery to phagosomes, 14 experimental considerations, 15 flow of experiment, 15 results, 16 instruments, 5 live cell imaging, 50 in interphase cells, 50e51 in mitotic cells, 51 live cell imaging, 5 in macrophages, 3 RE-associated proteins and lipids, 3 reagents, 4 SNARE protein family, 3 subcompartments by Rabs, 5e6 flow of experiment, 6 results, 6e8 Tfn-HRP-loaded endosomes, 52f Region of interest (ROI), 10 Retinal pigment epithelium (RPE), 274e275 Retrograde trafficking analysis, see also Integrin recycling analysis CI-M6PR, 201e203, 202f experimental analysis, 206e207 experimental strategy reagents and materials, 203e204 integrin a5b1 cartoon depicting recycling, 202f methods antibody labeling to monitor retrograde trafficking of CI-M6PR, 204e205 cell plating, 204 fixing and blocking of cells, 205 prepare and stain with primary antibody, 205e206 prepare and stain with secondary fluorescentconjugated antibodies, 206 RNAiMAX transfection on coverslips, 204

Ribonucleic acid (RNA), 53 rme-1, 186e188, 187f RNA, see Ribonucleic acid (RNA) RNA interference (RNAi), 201 RNAi, see RNA interference (RNAi) RNAi-mediated depletion of RalA, 314e315 RNAiMAX transfection on coverslips, 204 ROI, see Region of interest (ROI) Room temperature (RT), 219, 240 RPE, see Retinal pigment epithelium (RPE) RT, see Room temperature (RT)

S Saponin permeabilization workflow, 328e329 sb-Tf, see Sulfotag and biotin-Tf (sb-Tf) SDS, see Sodium dodecyl sulfate (SDS) SDS-PAGE, see Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) SE, see Sorting endosome (SE) Sec7, 106 protein expression, 110e111 protein purification, 111e112 purification of Sec7(203e2009), 106 protein expression, 109 protein purification, 109e110 virus preparation, 107e109 Semiautomated endosome membrane tubule segmentation, 170f Fiji macro script for, 173e174 SEP, see Super-ecliptic pHluorin (SEP) Sholl analysis, 172e173 signal sequence (ss), 335 Signal to noise ratio (SNR), 220 Single-event resolution, quantifying recycling at, 339 considerations, 341 flow of experiments, 339e341 results, 341 siRNA, see Small interfering RNA (siRNA) Slow recycling endosomes, 36 Small GTPase crosstalk variation, 121 SMALL GTPases myristoylated Arf1 purification, 112e114 soluble constructs purification, 112 Small interfering RNA (siRNA), 203 SNARE protein family, 3 SNR, see Signal to noise ratio (SNR) SNX, see Sorting nexin (SNX) Sodium dodecyl sulfate (SDS), 292

Index

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 85, 351e352 Solubilization, 116e117 Soluble constructs purification, 112 Sorting endosome (SE), 140e141, 272 Sorting nexin (SNX), 82 Sorting signals, 82 SpH, see Superecliptic pHluorin (SpH) ss, see signal sequence (ss) Stain cells, 135e136 Staining isolated centrosomes, methods for, 55 Step-by-step endosome membrane tubule “tracing”, 171e173 STX4, see Syntaxin-4 (STX4) Sulfotag and biotin-Tf (sb-Tf), 283 recycling, 283e285 Super-ecliptic pHluorin (SEP), 14e15 Superecliptic pHluorin (SpH), 335 Syntaxin-4 (STX4), 61

T TBS, see Tris buffer saline (TBS) TCA, see Trichloroacetic acid (TCA) TCL, see Total cell lysates (TCL) TEM, see Transmission electron microscope (TEM) Tf, see Transferrin (Tf) Tfn-568, loading endosomes with, 48f Tfn-HRP-loaded endosomes, 52f TfR, see Transferrin receptor (TfR) TfR-rich perinuclear common recycling endosomes (CRETfR), 272e273 TGN, see trans-Golgi network (TGN) Thin layer chromatography (TLC), 76 3T3-L1 cells, 85 3T3-L1 preadipocytes maintenance and differentiation, 312 RNAi-mediated depletion of RalA in mature, 314e315 Time-lapse analysis, 29 of endosome and actin dynamics during, 27 analyzing actin dynamics, 29e30 and apical lumen formation in 3D cultures, 30e32 RAB11/FIP3-endosome transport analysis, 27e29 Time-lapse imaging, 194e195 TIRFM, see Total internal reflection fluorescence microscopy (TIRFM) Tissue culture supplies, 310 TLC, see Thin layer chromatography (TLC)

TM proteins, see Transmembrane proteins (TM proteins) Total cell lysates (TCL), 313 Total internal reflection fluorescence microscopy (TIRFM), 335 Trafficking of receptors, 200 trans-Golgi network (TGN), 16, 103, 128, 201e203, 274 Transcytotic assay, 283e285 Transfections, 84e85, 134 Transferrin (Tf), 11, 92, 142, 158e160, 218, 239 recycling, 92, 93f recycling through endocytic compartments, 168 slow Tf recycling route analysis, 242 material, 242 methods, 243e244 results, 244 ultrastructural localization, 241e242 uptake into endocytic compartments, 168 Transferrin receptor (TfR), 82, 128, 143e144, 239e240, 272 TfR-1, 248 Transmembrane proteins (TM proteins), 3 recycling cargo assays experimental strategies and considerations, 192e193 hTAC, 192 MIG-14-GFP, 193e194 principles, 191e192 Transmission electron microscope (TEM), 53, 160 TRE, see Tubular recycling endosomes (TRE) Trichloroacetic acid (TCA), 260, 350 precipitation, 263 reagents to, 256e257 Tris buffer saline (TBS), 350 Tubular recycling endosomes (TRE), 141e142 Tubulation factors, 163

V Virus preparation, 107 bacmid preparation, 107 insect cell transfection, 107e109 viral amplification, 109

W WASH, see Wiskott-Aldrich syndrome protein and SCAR Homologue (WASH) WASH domain localization in fixed cells cell plating, Tf uptake and anti-WASH immunofluorescence, 227 considerations, 231 image analysis, 228e230, 229f

395

396

Index

WASH domain localization in fixed cells (Continued) imaging, 227 object-based colocalization method, 226 quantitative 3D analysis, 225e226 reagents and instruments, 226e227 results, 230e231 WASH microdomains live imaging, 222e223, 224f, see also Endosome tubulation imaging cell imaging, 223 cell plating and Tf uptake, 223 considerations, 225 image analysis and results, 223e225 reagents and instrument, 223

Western blot analysis, 245 Wheat germ agglutinin (WGA), 275e276, 351 Wild-type receptor (WT receptor), 324 Wiskott-Aldrich syndrome protein and SCAR Homologue (WASH), 200e201, 216e217 complex, 201 function, 201 WASH-mediated actin polymerization, 217 WT receptor, see Wild-type receptor (WT receptor)

Y Yolk protein (YP170), 182 uptake assay in C. elegans oocyte, 184e186 YP170::GFP transport assay sensitivity, 186

ADAM A. WALL ET AL., FIGURE 1 Localization of recycling endosome-associated Rab GTPases in macrophages. RAW264.7 macrophages coexpressing GFP-Rab11a and td-Tomato-Rab8a. (A) Fixed cells show perinculear concentrations of active Rabs11a and 8a on REs and scattered RE puncta throughout the cells. Rab8a is additionally found on larger peripheral macropinosomes. The inset shows a selection of punctate REs highlighting examples of complete overlap of both Rabs, their side-by-side juxtaposition on some REs and separately labeled REs, together representing the dynamic flux of membranes containing these Rabs on intersecting subcompartments. (B) Live cell imaging of cotransfected macrophages. The REs in live cells appear much more tubular, often ringlike and their dynamic nature can be appreciated in movies. Frames from a movie show Rab8a positive tubular membranes forming macropinosomes from surface ruffles and coursing back into the REs from the surface. These tubules skirt around a vacuole and intersect with Rab11a REs (arrowheads). Scale bar ¼ 10 mm.

ADAM A. WALL ET AL., FIGURE 2 Recycling endosome cargo and trafficking in macrophages. RAW264.7 macrophages trafficking the well-known recycling endosome cargo Transferrin. (A) Macrophages pulsed with Alexa647-transferrin (pseudo colored green) for 10 min and chased into REs for 20 min were fixed and F-actin labeled with Alexa594 phalloidin (red), the nuclei are labeled with DAPI (blue). The presence of internalized transferrin in REs throughout the cytoplasm is evident. (B) Macrophages transiently transfected with GFPRab11a (green) and pulsed with Alexa647-transferrin (red) both label REs, the plasma membrane is labeled with Alexa350-wheat germ agglutinin (blue). Rab11a and transferrin overlap in some REs but are not identical, highlighting the dynamic and differential distribution of elements within this network. (C) Macrophages pulsed with Alexa647transferrin (pseudo-colored blue) to label REs, Alexa488-dextran labeling macropinosomes (pseudo-colored red) and fluorescently tagged Alexa594-LDL (pseudo-colored green) showing receptor-mediated uptake over 20 min chase. Inset shows tri-labeled RE (arrows) containing transferrin, dextran, and LDL. (D) Quantification of colocalization between fluorescently labeled transferrin (Tfn), dextran (dex), and LDL over time. Manders overlap coefficient is represented as % colocalization. (E) Live cell image acquisition of macrophages pulsed with fluorescently labeled Alexa647-transferrin, with frames showing exit of transferrin over acquisition period. (F) Model for exit quantification of transferrin. Xin (total intracellular transferrin), Xout (extracellular transferrin), Kexit (arrow, rate of loss of transferrin). (G) Graphical comparison of experimental data (blue) of transferrin exit over time, and mathematically modeled decay (red) of transferrin exit, with a predicted rate constant of 0.0023116, which equates to a transferrin half-life of 300 s.

ADAM A. WALL ET AL., FIGURE 3 Recruitment of VAMP3 during early stage phagocytosis. (A) Schematic diagram of VAMP3 and C-terminal position of fluorescent tags. VAMP3 contains a cytoplasmic helical coiled-coiled domain that interacts with other SNARES, a TM domain. (Right panel) RAW264.7 macrophages transiently expressing GFP-VAMP3 phagocytosing an IgG-opsonized bead. Box indicates GFP-VAMP3 on surface membrane ruffles. Inset shows the localization of GFP-VAMP3 to the nascent phagocytic cup and on the phagosome after internalization (arrows). Another internalized IgG-bead is seen in the vicinity with GFP-VAMP3 enrichment on the phagosome which gradually dissociates from the maturing phagosome (asterisks). (B) Schematic diagram of fluorescence dynamics of SEP-VAMP3 showing quenching of SEP-VAMP3 in acidic endosomal compartments. SEP-VAMP3 fluoresces brightly upon exposure to the external environment (pH 7.4) when recycling endosomes fuse at the plasma membrane. (Right panel) RAW264.7 macrophages transiently expressing SEP-VAMP3 show SEP-VAMP3 on peripheral compartments, on surface ruffles (arrow) and on early phagosomes (box). The phagocytic membrane around an IgG-opsonized bead shows fluorescence “bursts” of SEP-VAMP3 denoting RE fusion at the phagocytic cup (arrowhead). More mature phagosomes in the inner part of the cytoplasm are not fluorescing, having lost VAMP3 or having SEP-VAMP3 quenched by the acidic lumens. Scale bar ¼ 10 mm.

YAOYAO QI ET AL., FIGURE 2 Colocalization of MoRab1, MoRab8, and MoRab11 with hRab1A/B, hRab8, and hRab11 in BHK cells. (A) Shown are confocal fluorescence images of intracellular structures in BHK cells labeled by eGFP-MoRabs (green) and tdTomato-hRabs (red), as indicated. The early endosomal hRab5 here serves as a negative control versus recycling endosomes. Nuclei are identified with DAPI staining (blue). The results are reproducible in three independent experiments. Bar ¼ 10 mm. (B) Quantification of colocalization between the eGFP-MoRabs and the coexpressed tdTomato-hRabs in the same cells. The graph shows Pearson’s correlation coefficient between each eGFP-MoRab and the corresponding tdTomato-hRabs, which is calculated using the confocal fluorescent images from four cells and the colocalization tool in the Velocity software. Shown are the mean and calculated SEM.

Plasma Membrane Localized

Early Endosome Localized

Recycling Endosome Localized

Addition of Tfn-568 (time point 0 min)



Tfn-568 Centrosome Nucleus

HUI-FANG HUNG ET AL., FIGURE 1 Loading endosomes with Tfn-568. Hela cells grown on glass coverslips are incubated with Tfn-568 on ice for 5 min to inhibit the endocytic pathway. Tfn will associate with its receptor at the plasma membrane. Cells are washed and placed into 37 C media for 5e30 min. At 5 min, Tfn-568 localizes mostly to peripheral early endosomes. By 30 min, Tfn-568 localizes to the pericentrosomal RE compartment.

HaloTag NanoLuc 460 1

Rabin8 Rabin8

GEF 144

245

NanoLuc

HaloTa HaloTag

Rabin8 1

GEF 144

245

460

BIN WU ET AL., FIGURE 1 Schematic diagram showing the bioluminescence resonance energy transfer (BRET) strategy to study Rabin8 conformational change. NanoLuc (donor) and HaloTag (acceptor) are fused to the N- and C-terminus of Rabin8, respectively. BRET will occur when Rabin8 is in its autoinhibitory conformation and the N- and C-terminus are in close proximity. During the activation of the Rabin8, Rabin8 switches to an “open” conformation, and the energy transfer between NanoLuc and HaloTag is significantly reduced. GEF, the catalytic domain of Rabin8 that mediates guanine nucleotide exchange of Rab8.

NanoLuc-STX4 NanoLuc-STX4-HaloTag

450 400 350 300 250 200 150 100

50 0 580 590 600 610 620 630 640 650 660

(B)


Wavelength (nm)

1.2 1.0



(A) 1400 1200

NanoLuc-Rabin8 NanoLuc-Rabin8-HaloTag

1000 800 600 400 200 0 580 590 600 610 620 630 640 650 660

Wavelength (nm)

*

0.8 0.6 0.4 0.2 0 Rabin8 Rabin8 (300-305)

BIN WU ET AL., FIGURE 2 Examine the autoinhibitory conformation of Rabin8 using bioluminescence resonance energy transfer (BRET). (A) Intramolecular BRET signals are detected for STX4 and Rabin8. STX4 and Rabin8 in pF-NLHT vector are expressed and purified from Escherichia coli. STX4 with a closed conformation is used as a positive control to test the BRET system. STX4 and Rabin8 with NanoLuc only are used as negative controls. Both STX4 and Rabin8 show intramolecular BRET signals with peak emission at 620 nm. (B) Rabin8(300e305D) loses the BRET signal. The luminescence/fluorescence intensities at 460 and 620 nm are recorded, respectively. Signals from mixtures without NanoBRET ligands are used as background. The calculation of BRET ratio is shown in Section 1.1.5 (5).

JIAN LI AND VICTOR W. HSU FIGURE 2 Elucidating how ACAP1 and clathrin form a coat complex. (A) Localization of different proteins in adipocytes. The 3T3-L1 cells were stably transfected with HAeGlut4eGFP and then differentiated into adipocytes. Confocal microscopy was then performed in the basal condition to assess for the colocalization of: ACAP1 (green) and CHC (red) (Top row), HA-Glut4-GFP (green) and CHC (red) (middle row), HA-Glut4-GFP (green) and ACAP1 (pseudored) (bottom row); bar, 10 mm. (B) ACAP1 and CHC form a complex with endosomal glut4 in adipocytes. Cell lysates were generated from either nonstimulated (basal) or insulin stimulated 3T3-L1 adipocytes that stably expressed myc-tagged glut4. Coprecipitation was then performed using an anti-Myc antibody to isolate glut4 and then immunoblotting for proteins as indicated. (C) The middle cytoplasmic loop of glut4 interacts directly with ACAP1. The different cytoplasmic domains of glut4 as indicated were bound to the beads as GST fusion proteins and then incubated with either soluble ACAP1 or clathrin. Bound components were detected by immunoblotting. The level of GST proteins on beads was assessed by Coomassie blue staining. (D) ACAP1 bridges the interaction between clathrin and glut4. The same pulldown experiment was performed as described above (C), except soluble proteins were incubated sequentially as indicated. Unbound soluble components after the first incubation were removed prior to the second incubation.

JIAN LI AND VICTOR W. HSU FIGURE 4 Sequences in TfR recognized by ACAP1 act as recycling sorting signals. (A, B) Tracking of endocytic Tf by live cell imaging. Wild-type (A) and mutant human TfR (B) were stably transfected into the TRVb cells. Alexa 594-labeled Tf was then bound to the cell surface and then allowed to internalize. Endocytic TfR was then tracked for appearance in the perinuclear region at times indicated; bar, 10 mm. (C) Mutant TfR reaches the recycling endosome. TRVb cells that stably expressed mutant TfR were transiently transfected with either myc-tagged ACAP1 or GFP-tagged Rab11. Alexa 594-labeled Tf was then bound to cells and allowed to internalize for 60 min. The cells were then analyzed by confocal microscopy to assess the colocalization of either Tf (red) and ACAP1 (green), or Tf (red) and Rab11 (green); bar, 10 mm. (D) Quantitation of endocytic Tf at the pericentriolar recycling endosome. At times indicated, the relative level of Tf at the perinuclear region of TRVb cells was quantified relative to the maximal level. The mean with standard error from three independent experiments is shown.

JIAN LI AND VICTOR W. HSU FIGURE 6 Sequences in glut4 recognized by ACAP1 act as recycling sorting signals. (A) 3T3-L1 adipocytes that stably express HAeGlut4eGFP were analyzed for the surface versus total glut4 in either basal or stimulated condition, which involves tracking the HA epitope (red) or GFP (green) signal; bar, 10 mm. (B) Mutation of sequence in glut4 recognized by ACAP1 inhibits glut4 recycling. The glut4 recycling assay was performed on adipocytes that expressed either wild type or mutant glut4. The mean with standard error from examining 10 randomly selected cells is shown.

DIPANNITA DUTTA AND JULIE G. DONALDSON FIGURE 1 Rab and Arf G proteins in endosomal trafficking. (A) Cartoon depicting entry of proteins via clathrin-mediated endocytosis (CME) (blue cargo) and clathrin-independent endocytosis (CIE) (red cargo). All incoming cargo reaches an early endosome that is associated with Rab5 and its effector EEA1 (early endosomal antigen 1). From there cargo moves on toward late endosomes and lysosomes for degradation, to the Golgi complex (TGN) or to recycling back to the cell surface. Approximate location and site of action for some of the different Rab proteins and Arf6 is indicated. (B) GTP-binding and GTPhydrolysis cycle for Rab (and Arf) G proteins is shown along with the GEFs and GAPs in promoting these two activities, respectively. Expression of the GTP-binding-defective (dominant negative) mutant forms can lock the Rab in the inactive, GDP-bound state; this state may also be observed upon siRNA depletion of the Rab in question. By contrast, expression of the GTP-hydrolysis-resistant (constitutive active) mutant forms can lock the Rab in the active, GTP-bound state. The active state can promote activities (i.e., Effector function) but in many cases, expression can impair function since the G protein functions best when it cycles between the two states.

Workflow and expected results for Tf-568 and EGF- 488 pulse-chase Pulse cells for 7 minutes with labeled ligand(s) ee times ti Wash cells three with PBS

Pulse only: Fix to visualize both cargos localized to SE with Tf localized to the tubulo-vesicular compartment and EGF localized to the vacuolar compartment.

Pulse + chase: Incubate cells in complete media containing unlabeled ligand (DMEM+FBS) and chase for 20 minutes to follow trafficking into the ERC (Tf) or late endosomes (EGF), or either 45 minutes (Tf) or 120 minutes (EGF) to visualize recycling or degradation, respectively.

Expected results 7 min. pulse only

20 min. pulse-chase 120 or 45 min. pulse-chase 120’

EGF-488

45’

Tf-568

Merge (inset)

JAMES B. REINEKE ET AL., FIGURE 3 Schematic workflow for qualitative monitoring of internalization and recycling of transferrin (Tf) and epidermal growth factor (EGF) and their respective receptors. Anticipated results are depicted in the micrographs showing Tf and EGF internalized for 7 min, and then “chased” for 20 min (both cargos), 45 min (Tf), or 120 min (EGF).

Workflow for measuring TfR or MHC I recycling (1) Pulse cells for 15 minutes with Tf-488 Wash cells three times with PBS Fix to measure the internalized Tf-488 (Tube A)

In Incubate cells in complete DMEM and chase for 30 minutes Fix to measure easu the amount of remaining Tf-488 in the cell after a 30 minute chase (Tube B)

(2) Pulse cells for 30 m minutes with anti-MHC I antibody

= Tf-488 = MHC I/antibody complex

Acid strip for 1 minute

= goat anti-mouse 568 antibody

Wash cells three times with PBS Fix and stain with goat anti-mouse 568 in the presence of saponin to measure the amount of internalized MHC I (Tube A)

Fix and d stain stain with goat anti-mouse 568 in the absence of saponin to measure the amount of internalized MHC I (Tube B)

Incubate cells in In DMEM for 3 hours

complete

Wash cells three e time times with PBS Fix and stain with h goa goat anti-mouse 568 in the absence of saponin to measure the amount of MHC I that has recycled back to plasma membrane (Tube C)

JAMES B. REINEKE ET AL., FIGURE 4 Schematic workflow for quantitatively measuring recycling of Tf and its receptor or MHC I by flow cytometry.

DANIELLE N. KALKOFEN ET AL., FIGURE 1 Endocytic organelles and steps utilizing membrane tubules and cytoplasmic phospholipase A2 (PLA2) enzymes. The inhibitory symbol (T) indicates membrane tubule-mediated trafficking steps that are inhibited by PLA2 antagonists. Previous studies have shown that endocytic trafficking of transferrin receptors (TfR), low-density lipoprotein (LDL), and epidermal growth factor receptors (EGFR) via clathrin-dependent endocytosis (CDE) involves PLA2-mediated membrane tubule formation at several steps. The cytoplasmic PLA2 PAFAHIb catalyzes the formation of membrane tubules and facilitates export of TfRs from early endosomes (EE) and the endocytic recycling compartment (ERC). Trafficking of EGFRs and LDL from late endosomes/multivesicular bodies (LE/MVB) is inhibited by PLA2 antagonists. Following clathrin-independent endocytosis (CIE), proteins such as MHC class I, CD55, and CD59 utilize cytoplasmic cPLA2ε at the CIE recycling compartment (CIE RE) to generate membrane tubules for recycling back to the plasma membrane. TGN, trans-Golgi network.

(A)

(B)

DANIELLE N. KALKOFEN ET AL., FIGURE 2 Setup for in vitro endosome membrane tubulation reactions and negative staining with multiple EM grids. (A) Multiple self-closing forceps can be organized to facilitate rapid application of tubulation reactions to EM grids. (B) Side view of a single self-closing forceps with sample on an EM grid.

DANIELLE N. KALKOFEN ET AL., FIGURE 4 Semi-automated endosome membrane tubule segmentation and subsequent Sholl analysis. Confocal z-projections were processed by a custom macro script that segmented the membrane tubules and yielded binary images ready for Sholl analysis. (A) An example original confocal z-projection micrograph. Both images are the same, the right image was contrast enhanced to visualize the membrane tubules. (B) The original unenhanced image is processed by the “Tubeness” plugin in Fiji to enhance the tubular features of the image. The image is then segmented using the Yen thresholding algorithm, which essentially traces all elements in the image. (C) The objects from the resulting binary image in (B) are processed by the “Analyze Particles” feature in Fiji. Tubular objects with a circularity between 0 and 0.4 are transferred to a new binary image resulting in a binary reproduction of the membrane tubules seen in the original image. (D) and (E) The filtered binary membrane tubule image can then processed with the Sholl analysis plugin. The Sholl plugin counts the number of intersections at specified step intervals (indicated by the different colors) along the distance from the center to the edge of the cell that is denoted by the white line. Given that endosomal tubules do not originate from a single point as the Golgi membrane tubules do, the Sholl analysis indicates the number of tubules found from the center to the edge of the cell.

(A)

(B)

(C)

(D)

ANBING SHI AND BARTH D. GRANT FIGURE 2 Loss of rme-1 causes recycling defects in the Caenorhabditis elegans intestine. (AeB) Nomarski images of the intestines in wild-type and rme-1(b1045) animals. Arrows indicate the accumulated transparent vacuoles in the intestines of rme-1(b1045) animals. (C) The C. elegans epithelial intestine is consisted of 20 individual epithelia forming nine intestinal rings (Int I-IX). Int I consists of four epithelial cells, all other rings are formed by two epithelial cells as illustrated here. (D) Working model of the fluid-phase basolateral recycling markers in the wild-type and rme-1(b1045) recycling mutant animals. In wild-type animals, no fluid-phase markers accumulation observed due to efficient recycling back to the pseudocoelom. In mutant animals, recycling defective vacuoles will be filled with basolaterally endocytosed fluid-phase markers. As scavenger cells, coelomocytes also endocytose fluid-phase markers from the body cavity.

DOUGLAS G. OSBORNE ET AL., FIGURE 1 Cartoon depicting recycling of integrin a5b1 and retrograde trafficking of CI-M6PR. Integrin a5b1 is internalized from the cell surface and enters the endosomal system. Subsequently it accumulates in the retromer-enriched region of the endosome, where WASH-generated F-actin corrals it into a sorting domain. Finally, a vesicle containing the accumulated integrin undergoes scission and the cargo-laden vesicle is transported back to the plasma membrane. In contrast, the retrograde-trafficked receptor CI-M6PR normally accumulates in the TGN where it picks up cargo and delivers it to the endosome. Like the integrin, CI-M6PR is corralled into retromer-enriched sorting domains, where it accumulates in vesicles and undergoes retrograde trafficking back to the Golgi. CI-M6PR can also be found on the plasma membrane. This plasma membrane-localized CI-M6PR also undergoes retrograde trafficking following internalization from the cell surface, a feature that can be used to monitor WASH and retromer function.

DOUGLAS G. OSBORNE ET AL., FIGURE 2 Loss of WASH or retromer impacts CI-M6PR retrograde trafficking. HeLa cells were transfected with siControl, siWASH, or siVPS35 oligos. Sixty hours posttransfection, cells were stained with anti-CI-M6PR antibody and allowed to internalize the antibody-receptor conjugates for 60 min. Coverslips were subsequently processed for immunofluorescence imaging as described in the text. (A) Wide field images of transfected cells stained for CI-M6PR (red), TGN46 (green), and DNA (blue) are shown. (B) CI-M6PR was scored as focal (localized at the TGN) or dispersed as described in the text. (C) Wide field images of transfected cells stained for CI-M6PR (red), EEA1 (green), and DNA (blue) are shown. (D) CI-M6PR and EEA1 overlap was measured using the Zen2009 software as described in the text.

EMMANUEL DERIVERY AND ALEXIS GAUTREAU FIGURE 2 Direct observation of WASH localization in endosome domains in live cells. 3T3 cells stably expressing GFP-WASH were loaded with Alexa-555-Tf until equilibrium, then imaged by spinning disk confocal microscopy. Both channels were acquired sequentially (no z-stack) and overall frame rate was two frames per second. The behavior of two moving endosomes is displayed as still frames extracted from the movie (A) or as a kymograph along the trajectory of the endosome. (B) WASH and Tf are continuously associated, but do not fully overlap. (C) Automated tracking of the fluorescence centroids in both GFP-WASH and Tf channels of the endosomes displayed in B shows that the distance between the two spots is always below the apparent radius of the endosome (0.4 mm). (D) Left panel: proposed organization of WASH and Tf-containing endosomes. On the one hand, WASH localizes to diffraction-limited subdomains at the surface of endosomes, and on the other hand, Tf uniformly localizes to the endosome inner membrane. Right panel: when the proposed domain organization is convolved by the PSF of the microscope, the distance between the fluorescence centroids of Tf and WASH is not zero.

EMMANUEL DERIVERY AND ALEXIS GAUTREAU FIGURE 3 Image analysis pipeline for the automated analysis of WASH microdomains in fixed cells. (A) Image processing pipeline allowing automatic 3D particle detection in both Tf and WASH channels, followed by assignation of each WASH domain to its proper endosome. (B) Details of the procedure of the 3D assignation. A common pixel stack is generated and segmented. Each WASH domain is then associated to its respective endosome using the property of the fluorescence centroid of the common pixel region to be contained within both the correct WASH domain and the correct endosome.

CE´DRIC DELEVOYE AND BRUNO GOUD FIGURE 1 Live cell imaging on cells coexpressing KIF13A and Rab11a. (A) Spinning-disk confocal microscopy was used to capture concomitant movements of KIF13A-YFP and mCherry-Rab11A in HeLa cells. KIF13A generates long tubular structures decorated by Rab11a that extend toward the cell periphery. (B) Magnified insets (of boxed area) of consecutive time-lapse single and merged images are presented as a gallery (one image/7 s) and show that KIF13A is enriched at the tip of the growing recycling tubules (arrows). (C) KIF13A deleted from its motor domain (KIF13A-ST) codistributes with Rab11a positive vesicles. (D) KIF13A positive endosomal tubules do not contain Rab5, demonstrating the specific localization of KIF13A to recycling endosomes. Two-colors stacks of 2e4 z-slices (0.2 mm) were acquired using 150 ms exposure for 50 or 100 time points under Metamorph. Scale Bars: 10 mm.

CE´DRIC DELEVOYE AND BRUNO GOUD FIGURE 2 Microtubule depolymerization inhibits the formation of recycling tubules by KIF13A but not its endosomal localization. KIF13A-YFP expressing HeLa cells that internalized TfA647 were incubated for 90 min at 4 C and treated or not (dimethyl sulfoxide (DMSO)) with 10 mM of nocodazole (Noco) to depolymerize microtubules. Cells were processed for immunofluorescence and labeled with an antibody against transferring receptor (TfR). Nocodazole treatment prevented the KIF13A-dependent formation of recycling endosome tubules without impacting on the endosomal localization of KIF13A. Single, merged, and magnified insets of boxed areas were shown. Scale Bars: 10 mm.

LUCIANA GALLO AND GERARD APODACA FIGURE 1 Endocytic pathways in polarized MDCK cells. General pathways for endocytosed cargo: Proteins, lipids, and fluid are internalized from the apical (step 1a) or basolateral (step 1b) surfaces, and delivered to apical early endosomes (AEEs) or basolateral early endosomes (BEEs). Cargoes can be recycled to the cell surface (steps 2a/b), be delivered to late endosomes (steps 3a/b), from which they will be delivered to lysosomes (not shown), or they can be delivered to the common recycling endosome (CRE; steps 4a/b). There may be a direct pathway from the AEE to the apical recycling endosome (ARE, step 4c). CRE cargoes are shuttled to the basolateral cell surface (step 5), or delivered to the ARE (step 6), from where they gain access to the apical cell surface (not shown). Pathways for transferrin receptor (TfR) recycling in polarized MDCK cells (marked with red arrows): The TfR, bound to holo-Tf, is internalized at the basolateral surface of the cell and then delivered to BEEs (step 1b). In response to a decrease in pH, which results from the activity of the Hþ-ATPase, Fe3þ is released from Tf. The Fe3þ is reduced by the activity of STEAP3, and the resulting Fe2þ is transported into the cytosol by the action of DMT1. While some TfR with bound apo-Tf rapidly recycles from the BEE (step 2b), a significant fraction is delivered to the CRE, where further dissociation and recovery of iron occurs. The TfR, with bound apo-Tf, is then recycled to the basolateral cell surface (step 5). At the basolateral cell surface, apo-Tf dissociates from the TfR, which is now primed to bind holo-Tf anew and resume additional rounds of recycling.

LUCIANA GALLO AND GERARD APODACA FIGURE 2 Culture of polarized MDCK cells. (A) MDCK cells cultured on 10-cm dish. (B) MDCK cells cultured on 12-mm Transwell. (C) Formation of a polarized epithelial sheet is confirmed by labeling the cell nuclei with ToPro-3 (green) and an antibody that recognizes the tight junction-associated protein ZO-1. The upper panel is a projection of a confocal Z-series. The bottom panel shows an XeZ section taken from the same stack. Bar ¼ 10 mm.

LUCIANA GALLO AND GERARD APODACA FIGURE 3 Assay to measure recycling of 125I-Tf. (A) Circulating water bath containing elevated aluminum plate and 12-well dish containing filter-grown MDCK cells labeled with 125I-Tf. (B) Sampling the apical medium of MDCK cells pulse-labeled with 125I-Tf. (C) Apical medium, containing 125I-Tf, is transferred into collection tube.

LUCIANA GALLO AND GERARD APODACA FIGURE 5 Endocytic fate of 125I-Tf in polarized MDCK cells. (A) The average data, obtained from the amount of 125I-Tf that was recycled or transcytosed in Figure 4, are plotted versus time. The line shows the best fit of the data to a single exponential curve. (B) MDCK cells were incubated in serum-free medium for 4 h, and then pulse-labeled with unlabeled Tf (25 mg/mL, using the same protocol described in this chapter). At t ¼ 0, 15, or 120 min, the cells were fixed and then immunolabeled with a polyclonal anti-Tf antibody and a rat monoclonal antibody against ZO-1. Upper panel: Single XeY confocal section at the level of the tight junction showing Tf in CRE. Bottom panel: XeZ section through entire cell layer.

ANDRES E. PEREZ BAY ET AL., FIGURE 1 Model. This cartoon displays the various endosomal compartments and endosomal trafficking pathways in nonepithelial (A) and polarized epithelial (B) cells. The basolateral pathways and mechanisms of polarized epithelial cells are shown in red and the apical in blue.

ANDRES E. PEREZ BAY ET AL., FIGURE 2 (A) ASE and ARERab11 labeling. Polarized WT MDCK cells were incubated for 5 min with Alexa488-WGA (green) from the apical surface, washed with N-methyl-D-glucosamine (NMDG) and immunostained for Rab11. (B) BSE and CRETfR labeling. Polarized WT MDCK cells were incubated for 15 min from the basolateral surface with Alexa633-Tf (green) followed by a 5 min chase, with CF594-Tf (red). Orthogonal view (top) and two confocal sections at the supranuclear (middle) and nuclear (bottom) regions are shown. Scale: 10 mm.

ANDRES E. PEREZ BAY ET AL., FIGURE 3 Trafficking of basolaterally internalized TfR to ARERab11. Polarized WT and AP-1B KD MDCK cells were incubated for 10 min with CF594-Tf (red) from the basolateral surface and immunostained with anti Rab11 antibody (green). Orthogonal view (top) and two confocal sections at the supranuclear (middle) and nuclear (bottom) regions are shown. Scale: 10 mm.

CHAO NIE AND XIAO-WEI CHEN FIGURE 3 Loss of RalA dramaticlly decreases the Myc/GFP ratio.

(A) PAR1 WT Rab5 Q79L

P1 WT


+SF 15 min

PAR1 Y206A Rab5 Q79L

P1 Y206A


+SF 15 min

(B)

(C)

PAR1 WT

PAR1 Endosomal Fluorescence (A.U.)

Rab5 Q79L Fluorescence (A.U.)

10.0 7.0 6.0 5.0 4.0 3.0 2.0 -1.0 -0.5 0.0

0.5

1.0

1.5

2.0

***

7.5 5.0 2.5 0.0

WT

Y206A

MICHAEL ROBERT DORES AND JOANN TREJO FIGURE 2 Visualization and quantification of GPCR sorting into expanded endosomes. (A) Agoniststimulated PAR1 sorting into the lumen of expanded endosomes is mediated by the YPXnL motif. HeLa cells expressing FLAG-PAR1 WT or FLAG-PAR1 Y206A were transfected with Rab5-Q79L-GFP, then surface-labeled with anti-FLAG antibody. Cells were stimulated for 15 min with 100 mM SFLLRN, then fixed and analyzed by confocal microscopy. (B) Line scan analysis of Rab5-Q79L-GFP endosomes. GFP-fluorescence intensity was quantified by line scan (dashed line) and plotted to determine the limiting edges of the endosome (black vertical lines). (C) The PAR1 Y206A mutant is defective for sorting into the lumen of endosomes. PAR1 fluorescence intensity was measured within the limits defined by Rab5-Q79L-GFP (B) and averaged from multiple expanded endosomes (***, p < 0.001, n ¼ 6, Student’s t-test).

SHANNA L. BOWMAN ET AL., FIGURE 2 Strategy 2: Quantifying recycling at single-event resolution. (A) Schematic and corresponding example images for focusing a cell in TIRFM. When the excitation light passes straight through the sample, the center of the cell is readily visible. As the angle is moved down toward the critical angle, out of focus structures become more apparent in the “low-angle” illumination. Once past the critical angle, only the plasma membrane near the coverslip will be illuminated. The cell should appear bright, crisp, and uniform when focused down. (B) Example of a single recycling event. The whole cell is shown at the left. The inset focuses on a single recycling event. The recycling event first appears as a single bright spot whose fluorescence eventually diffuses out as vesicles fuse. Individual recycling events are very fast, each lasting less than a second. The scale bar is 5 mm.

SHANNA L. BOWMAN ET AL., FIGURE 3 Strategy 3: Visualization of sorting and recycling in the endosome. (A) Example image of FLAG-B2AR localized to the cell surface prior to agonist addition (left panel) and FLAG-B2AR in endosomes 10 min after iso addition (right panel). Endosomes are clearly visible after 10 min of agonist (arrows). Tubule domains are visible protruding from the endosome body (arrowheads). (B) Example image of FLAG-B2AR endosomes imaged with coronineGFP to mark endosomal actin. A coronineGFP spot localizes to the base of the FLAG-B2AR tubule, see inset in left corner. Inset scale bar is 2 mm. (C) Example of a FLAG-B2AR tubule fission event imaged every 800 ms. An FLAG-B2AR tubule persists for w10 s before vesicle fission, and a vesicle is visible next to the endosome (see arrowheads). Scale bar is 1 mm.

ITZIAR PINILLA-MACUA AND ALEXANDER SORKIN FIGURE 1 Time-lapse imaging of UMSCC2 cells stimulated with EGF-Rhodamine (EGF-Rh). Cells were imaged before and after EGF-Rh (4 ng/mL) stimulation for 15 min. Z-stacks of 20 xey images (400 nm z-stepsize) of Rhodamine fluorescence were acquired every 1.5 min. 1.5 and 7.5 min images are shown. 3-D volume view image (A) and “3-D view” image (single confocal xey, yez, and xez sections) are presented (B). Scale bars, 10 mm.

ITZIAR PINILLA-MACUA AND ALEXANDER SORKIN FIGURE 2 Effect of Dyngo-4a on EGF-Rhodamine (EGF-Rh) endocytosis in UMSCC2 cells. (A) Cells were incubated 30 min with Dyngo-4a 30 mM or vehicle (DMSO) and then imaged before and after EGF-Rh (4 ng/mL) stimulation as in Figure 1. 3-D volume view images of control and Dyngo-4a pretreated cells that were incubated with EGF-Rh for 5 min are shown. (B) Cells preincubated or not with Dyngo-4a were incubated with EGF-Rh for 15 min in the presence of inhibitor or vehicle, fixed and labeled with wheat germ agglutinin (WGA)-Oregon Green as described in the text. Insets show high magnification images of the peripheral cell regions marked by white rectangles. All image acquisition parameters and intensity scales are the same for corresponding images of control and Dyngo-4a-treated cells. Scale bars, 10 mm. EGF, epidermal growth factor.
