Visualizing Autophagic Lysosome Reformation in ... - Current Protocols

Visualizing Autophagic Lysosome Reformation in Cells Using In Vitro Reconstitution Systems

UNIT 11.24

Yang Chen,1,4 Qian Peter Su,2,4 Yujie Sun,3,5 and Li Yu1,5 1

State Key Laboratory of Membrane Biology, Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China 2 Institute for Biomedical Materials & Devices (IBMD), Faculty of Science, University of Technology Sydney, New South Wales, Australia 3 State Key Laboratory of Membrane Biology, Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, Peking University, Beijing, China 4 These authors contributed equally to this work 5 Corresponding author ([email protected]; [email protected])

Autophagy is a lysosome-based degradation pathway. Autophagic lysosome reformation (ALR) is a lysosomal membrane recycling process that marks the terminal step of autophagy. During ALR, LAMP1-positive tubules, named reformation tubules, are extruded from autolysosomes, and nascent lysosomes are generated from these tubules. By combining proteomic analysis of purified autolysosomes and RNA interference screening of identified candidates, we systematically elucidated the ALR pathway at the molecular level. Based on the key components clathrin, PtdIns(4,5)P2 , and the motor protein KIF5B, among others, we reconstituted this process in vitro. This unit describes a detailed method for visualizing ALR in cells during the autophagy process. This unit also present a protocol for reconstituting the ALR tubular protrusion and elongation process in vitro and three methods for preparing materials for in vitro reconstitution: (1) autolysosome purification from cultured cells, (2) liposome preparation, and (3) KIF5B purification and quality testing. C 2018 by John Wiley & Sons, Inc. Keywords: autolysosome purification r autophagic lysosome reformation (ALR) r autophagy r density-gradient centrifugation r liposome preparation r in vitro reconstitution

How to cite this article: Chen, Y., Su, Q. P., Sun, Y., & Yu, L. (2018). Visualizing autophagic lysosome reformation in cells using in vitro reconstitution systems. Current Protocols in Cell Biology, 78, 11.24.1–11.24.15. doi: 10.1002/cpcb.44

INTRODUCTION Autophagy is an evolutionarily conserved, lysosome-based degradation pathway that has significant physiological relevance. Dr. Yoshinori Ohsumi won the 2016 Nobel Prize in Physiology or Medicine for his research on autophagy in yeast. In mammalian cells, autophagy proceeds through formation of omegasomes on the endoplasmic reticulum, elongation of the autophagosomal membrane, closure of autophagosomes, formation of autolysosomes by autophagosome/lysosome fusion, cargo degradation, and finally autophagic lysosome reformation (ALR), which regenerates functional lysosomes. The process of ALR can be divided into the following steps: clathrin-mediated budding, tubule elongation mediated by the motor protein KIF5B, dynamin-mediated fission of In Vitro Reconstitution Current Protocols in Cell Biology 11.24.1–11.24.15, March 2018 Published online March 2018 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/cpcb.44 C 2018 John Wiley & Sons, Inc. Copyright

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Figure 11.24.1 Schematic illustration of the process of autophagic lysosome reformation including clathrin-mediated budding, KIF5B-mediated tubule elongation, and dynamin-mediated fission of tubules to form a proto-lysosome.

tubules to form a proto-lysosome, and maturation of the proto-lysosome into a functional lysosome (Fig. 11.24.1). In this unit, we provide protocols for visualizing the autolysosome tubulation process (Basic Protocol 1), for reconstituting the ALR tubulation and elongation process in vitro (Basic Protocol 2), and for preparing materials for in vitro reconstitution, including autolysosome purification from cultured cells (Support Protocol 1), liposome preparation (Support Protocol 2), and KIF5B purification and quality testing (Support Protocol 3). BASIC PROTOCOL 1

VISUALIZING THE ALR PROCESS IN CELLS Normal rat kidney (NRK) cells are used to visualize ALR in vivo. Other studies have used HeLa cells, NIH3T3 mouse fibroblasts, U2OS cells, macrophages, or hepatocytes to visualize ALR with different purposes (Chang, Lee, & Blackstone, 2014; Munson et al., 2015; Schulze et al., 2013; Sridhar et al., 2013; Zhang et al., 2016). The cells are first labeled with fluorescent lysosome and autophagosome markers and then subjected to long-term starvation in order to visualize ALR by confocal microscopy. When ALR occurs, tubules of various lengths protrude from autolysosomes. ALR-deficient cells show enlarged autolysosomes by confocal microscopy (Du et al., 2016; Rong et al., 2011, 2012). The starvation procedure can be replaced by other triggers of autophagy (Zhang et al., 2016). It is important to note that intact reformation tubules cannot be observed in cells fixed with 4% paraformaldehyde.

Materials NRK cells Cell culture medium (see recipe) 0.02% to 0.25% trypsin-EDTA Amaxa Cell Line Nucleofector kit (e.g., Lonza, cat. no. VCA-1002) with transfection buffer Plasmids: LAMP1-cherry or LAMP1-YFP GFP-LC3 or CFP-LC3 Phosphate-buffered saline (PBS; see recipe) Dulbecco’s Modified Eagle Medium (DMEM), serum- and glutamate-free (e.g., Gibco, cat. no. 11960)

Visualizing Autophagic Lysosome Reformation

10-cm cell culture dish Transfection cuvettes (e.g., Bio-Rad, cat. no. 1652086) Amaxa Biosystems Nucleofector Device 3.5-cm glass-bottom dish (e.g., In Vitro Scientific, cat. no. D35-10-1.5-N) 37°C, 5% CO2 incubator Confocal microscope with live-cell imaging system (e.g., Olympus F1000)


Current Protocols in Cell Biology

Figure 11.24.2 Live-cell image of the tubular autophagic lysosome reformation structures. NRK cells over-expressing CFP-LC3 and LAMP1-cherry were starved for 8 hr. Scale bar: 5 μm.

Cell preparation 1. Grow NRK cells using cell culture medium in a 10-cm dish. Once the cells are 80% confluent, trypsinize cells by incubating in 0.02% to 0.25% trypsin-EDTA for 2 min at 37°C, and collect 1 × 106 cells (approximately one-sixth of the cells from a 10-cm dish) in 100 μl transfection buffer. Then mix the cells with 2 μg LAMP1-cherry and 2 µg GFP-LC3. The plasmids encode autophagosome and lysosome markers fused with fluorescent proteins. The combination could either be LAMP1-cherry/GFP-LC3 or LAMP1-YFP/CFPLC3. Both combinations work well. Plasmids are available upon request to the authors.

2. Transfer the mixture to the transfection cuvette. Perform transfection using the Amaxa Cell Line Nucleofector kit. The program used is X-001 for NRK cells.

3. Plate one-third of the cells in a 3.5-cm glass-bottom dish. Wait at least 14 hr for cells to settle down and express the proteins from the exogenous plasmids. Use of glass-bottom dish is recommended since it is compatible with confocal microscopy.

Cell starvation 4. Wash cells twice with 2 ml PBS prewarmed to 37°C. 5. Add 2 ml DMEM prewarmed to 37°C. Serum- and glutamate-free DMEM is used as starvation medium.

6. Incubate cells in a 37°C incubator with 5% CO2 for 6 to 8 hr for observation of ALR (see step 7). Usually, ALR occurs at 8 hr of starvation in NRK cells. However, the time varies between 6 and 12 hr of starvation depending on the cell conditions. Thus, we recommend starting the visualization at 6 hr poststarvation and continuing until 12 hr poststarvation.

Visualization 7. Use a live-cell imaging system on a confocal microscope to visualize the ALR phenomenon at 6 to 10 hr poststarvation. Adjust the pinhole of the confocal microscope to 80 to 120 μm (Fig. 11.24.2). IN VITRO RECONSTITUTION ASSAY OF ALR When it comes to pinpointing the exact molecules involved in ALR and understanding the physical principles involved in the ALR process, in vitro reconstitution systems are second to none. In the presence of microtubules coated on a glass chamber, the motor protein KIF5B can mediate the tubulation process from purified autolysosomes

BASIC PROTOCOL 2 In Vitro Reconstitution

11.24.3 Current Protocols in Cell Biology

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or man-made liposomes. Tubule protrusion and elongation can be reconstituted in vitro. Based on this basic reconstitution system, many more adjustments can be made in order to understand the extended molecular mechanisms underlying ALR. Similar systems can be applied to study lysosome motility and the dynamic tubulation of mitochondria (Su et al., 2016; Wang et al., 2015). Using this in vitro system, we found that most autolysosomes (500 to 1000 nm) form tubular structures, while lysosomes, which are smaller in size (100 to 200 nm), are mainly transported along the microtubule tracks. Using purified mitochondria, we also achieved in vitro reconstitution of KIF5B-mediated tubulation of mitochondria on a glass chamber.

Materials Acetone (e.g., Sigma-Aldrich, cat. no. 270725) 1 M KOH Nitrogen gas Tubulin protein (e.g., Cytoskeleton, cat. no. HTS03-A) HiLyte 647–labeled tubulin (e.g., Cytoskeleton, cat. no. TL670M) Tubulin buffer (e.g., Cytoskeleton, cat. no. BST01-010) Tubulin glycerol buffer (e.g., Cytoskeleton, cat. no. BST05-001) GTP (e.g., Cytoskeleton, cat. no. BST06-001) Liquid nitrogen Paclitaxel (e.g., Cytoskeleton, cat. no. TXD01) Motility assay buffer (MAB-BRB80; see recipe) Anti-tubulin antibody (e.g., Abcam, cat. no. ab6160) Casein 1 mg/ml PE-rhodamine (e.g., Avanti, cat. no. 810150) 1 mg/ml PtdIns(4,5)P2 -TopFluor (e.g., Avanti, cat. no. 840046) Man-made liposomes or purified autolysosomes (see Support Protocols 1 and 2) Purified full-length KIF5B (see Support Protocol 3) PBS (see recipe) CellTracker CM-DiI Dye (e.g., Thermo Fisher Scientific, cat. no. C7001) ATP working solution (see recipe) Coverslips, 24 × 50 mm (e.g., Fisher Scientific, cat. no. 12-545-F) Staining jar Ultrasonic cleaner Slides, 24 × 60 mm (e.g., Shitai, cat. no. 10127101P) Double-sided tape, 200 MP 37°C water bath High-speed centrifuge Glass tubes for high-speed centrifuge Total internal reflection fluorescence (TIRF) microscope with camera for time-lapse imaging Preparation of flow chambers for in vitro assays 1. Clean coverslips for in vitro reconstitution assays with acetone followed by 1 M KOH in a staining jar in an ultrasonic cleaner. Store in distilled, deionized water until use to keep the surface hydrophilic. When coverslips are needed, dry using compressed nitrogen.


Since the experiment depends on the sensitive detection of single molecules, it is crucial to clean the coverslips as specified to eliminate background signals caused by autofluorescent particles or nonspecific binding of fluorescent molecules.

2. Assemble the flow chamber for the in vitro reconstitution assay.



Figure 11.24.3 Assembly of a flow chamber for the in vitro assay. Double-sided tape is placed on the coverslip. The slide is then placed on top of the double-sided tape to make the channels. Pipette tips are used to add samples from one side of the channels, and filter paper is used to absorb the liquid from the other side.

The flow chamber is made from a coverslip, a slide, and double-sided tape and contains three individual channels. One side of the tape is stuck to the coverslip (24 × 50 mm), and the other side of the tape is stuck to the slide (24 × 60 mm) on top to form the channels. The flow chamber needs to be made in a Mega-level ultra-clean hood to avoid autofluorescent particles. Refer to Figure 11.24.3 for assembling the flow chamber.

Preparation of microtubule filaments 3. Dissolve tubulin powder (either unlabeled tubulin or HyLite 647-labeled tubulin) to a final concentration of 4 mg/ml in a buffer which is made by mixing 5 vol. general tubulin buffer supplemented with 1 mM GTP and 1 vol. tubulin glycerol buffer. Aliquot the tubulin solution, and store at −80°C before flash freezing in liquid nitrogen. The tubulin powder is easy to dissolve; no vortexing is needed. The final buffer composition with dissolved tubulin is 1 mM GTP and 5% glycerol. The solution of tubulin is clear, and the solution of HiLyte 647–labeled tubulin is light blue. The tubulin solution should be protected from light.

4. Mix the unlabeled tubulin (47 μl) and HiLyte 647–labeled tubulin (2 μl) at a molar ratio of 25:1. Add GTP and paclitaxel to the tubulin solution to give a final volume 50 µl with a GTP concentration of 1 mM and a paclitaxel concentration of 20 μM. 5. Incubate and polymerize the tubulin mixture in a 37°C water bath for 30 min, and then centrifuge 30 min at 20,000 × g, 37°C, to remove the tubulin dimers or oligomers. Discard the supernatant, and save the pellet, which contains the polymerized tubulin. The supernatant contains tubulin dimers or oligomers, which need to be removed.

6. Resuspend the pellet, which contains microtubule filaments, with 50 μl MABBRB80 containing 20 μM paclitaxel, and keep the tube in a 37°C water bath for at least 1 day (no more than 1 week) before use.

In vitro reconstitution of autolysosome tubulation 7. Add 15 μl of 2.5 μg/ml anti-tubulin antibody to each channel of the flow chamber, and incubate for 5 min to coat the chamber with the antibody. Replace the solution with 50 μl of 3 mg/ml casein in MAB-BRB80, and incubate for 5 min to block the channel in order to eliminate nonspecific binding of other proteins.

In Vitro Reconstitution


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Figure 11.24.4 tubules (MTs).

Image showing flow chamber channels coated with HiLyte 647–labeled micro-

8. To immobilize the microtubule filaments on the coverslip surface, wash the channel with 50 μl MAB-BRB80 containing 20 μM paclitaxel. Then add the 1 mg/ml microtubule filament solution from step 6, and incubate for 5 min. The liquid exchange in the channel is achieved by adding the solution from one side of the channel and absorbing the liquid passing through the channel with filter paper at the other side of the channel.

9. Check the density of the microtubule filaments under TIRF illumination with excitation at 640 nm (Fig. 11.24.4). 10. For different vesicles, perform labeling and binding as follows: a. For PtdIns(4,5)P2 –containing man-made liposomes: Mix the lipids with 5% PErhodamine or PtdIns(4,5)P2 -TopFluor during liposome preparation. Dilute the liposomes to 40 μM, and incubate 50 μl with 80 nM full-length KIF5B for 5 min on ice. The final concentration of the man-made liposomes is 1 mM.

b. For autolysosome tubulation: Resuspend the purified autolysosomes in about 500 μl PBS. Take 30 μl autolysosomes for one sample, and supplement with cell tracker CM-DiI dye. Incubate at 37°C for 5 min to fluorescently label the autolysosomes. Centrifuge the autolysosomes 20 min at 20,000 × g, 37°C. Wash the labeled autolysosomes once with PBS, and collect the pellet. Resuspend the labeled autolysosomes again in about 50 μl PBS. Incubate 80 nM full-length KIF5B with 0.3 mg/ml resuspended autolysosomes. 11. Introduce the motor-coated vesicles (50 μl), prepared as in step 10, into the microtubule-coated flow channels for 10 to 15 min, and then wash with 50 μl MAB-BRB80 containing paclitaxel. Then add 60 μl ATP working solution to the channels. The ATP working solution contains an ATP regeneration system (phosphocreatine and creatine kinase) and an oxygen scavenger system (glucose and GLOX).

12. Take a time-lapse movie of the tubulation process using an excitation wavelength of 561 nm with HiLo illumination (Fig. 11.24.5). Visualizing Autophagic Lysosome Reformation



Figure 11.24.5

In vitro reconstitution of autolysosome tubulation and speed calculation.

AUTOLYSOSOME PURIFICATION FROM CULTURED CELLS One of the membrane sources for the reconstitution system is purified autolysosomes from cells under starvation. The purity and quality of autolysosomes is important for tubule formation in vitro. The purification step is based on an OptiPrep density gradient.

SUPPORT PROTOCOL 1

Materials NRK cells Cell culture medium (see recipe) PBS (see recipe) DMEM, serum- and glutamate-free (e.g., Gibco, cat. no. 11960) 0.02% to 0.25% trypsin-EDTA Lysosome isolation kit (e.g., Sigma-Aldrich, cat. no. LYSISO1-1KT) containing: Extraction buffer OptiPrep Dilution buffer Trypan blue 15-cm culture dishes 37°C incubator 50-ml conical tube Microcentrifuge and ultracentrifuge Dounce homogenizer 1.5-ml microcentrifuge tube 5-ml ultracentrifuge tube Additional reagents and equipment for immunoblot and immunofluorescence (see Du et al., 2016) Cell preparation 1. Culture NRK cells in 15-cm dishes. When the density of cells reaches 80% to 90% confluence, wash twice with PBS, and incubate with DMEM for 4 hr. For one batch of purification, six dishes of cells are needed. Serum- and glutamate-free DMEM is used as starvation medium.

2. Wash the cells once with PBS. Then, trypsinize cells with 2 ml of 0.02% to 0.25% trypsin-EDTA for 5 min at 37°C. Neutralize the trypsin with cell culture medium, and collect cells in 50-ml conical tube. 3. Centrifuge cells 5 min at 600 × g, 4°C. Wash the cell pellet once with 50 ml PBS, and then centrifuge 5 min at 600 × g, 4°C, to collect the pellet.



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Figure 11.24.6 somes.

Sample after density-gradient ultracentrifugation for separation of autolyso-

4. Resuspend the cell pellet with 500 μl ice-cold extraction buffer, and transfer to a clean homogenizer. Break the cells on ice with a Dounce homogenizer, avoiding the generation of bubbles. Cell homogenization should be done at 4°C.

5. Use trypan blue solution to monitor the extent of homogenization. Stop when 80% to 85% of cells are broken. The extent of cell homogenization should be carefully monitored. Too little homogenization will result in too low a yield of membranes. Too much homogenization will possibly disrupt the autolysosome membranes.

Density gradient All samples and buffers should be kept ice-cold, and the remaining steps should proceed at 4°C. 6. Transfer the cell fraction to a 1.5-ml microcentrifuge tube. Then rinse the homogenizer with 500 μl fresh extraction buffer, and combine this with the cell fraction to bring the volume to 1 ml. 7. Centrifuge 10 min at 1,000 × g, 4°C, to remove the unbroken cells and nuclei. 8. Transfer the supernatant to a 1.5-ml microcentrifuge tube, and centrifuge 20 min at 20,000 × g, 4°C, to collect the membranes in the pellet. 9. Resuspend the pellet in 400 μl ice-cold extraction buffer. 10. To prepare the sample solution, first mix 253 μl of 60% OptiPrep with 137 μl dilution buffer, and then add this to the 400 μl pellet suspension to make a final sample volume of 800 μl (19% final). Visualizing Autophagic Lysosome Reformation

11. Load the OptiPrep density gradient into a 5-ml ultracentrifuge tube. The volume and density from bottom to top is: 0.8 ml of 27%, 1 ml of 22.5%, 0.8 ml of 19% (sample), 1 ml of 16%, 0.9 ml of 12.5%, and 0.3 ml of 8%.



12. Centrifuge 4 hr at 150,000 × g, 4°C. The sample after centrifugation is shown in Figure 11.24.6.

Autolysosome recovery 13. Collect the top 2 fractions (0.5 ml each), separately, of the density-gradient sample, and add 1 ml PBS to each fraction to dilute the OptiPrep. Then centrifuge 20 min at 20,000 × g, 4°C, to recover the autolysosomes in the pellet. The top 1 ml of the gradient fraction contains the highest purity of autolysosomes as confirmed by immunoblotting and immunofluorescence analysis of autolysosome markers LC3 and LAMP1.

14. Test the purified autolysosomes for the presence of markers LC3 and LAMP1 either by immunoblotting or by immunostaining (see Du et al., 2016).

LIPOSOME PREPARATION In the in vitro reconstitution process, either purified autolysosomes or man-made liposomes can be used as the membrane source. The former is more physiologically relevant, and the latter makes it possible to precisely adjust the amount of each lipid type to study the function of different lipids in ALR. Also, liposomes provide a more homogeneous system for measuring different parameters involved in ALR. Thus, liposome preparation is a critical step in studying ALR in vitro.

SUPPORT PROTOCOL 2

Materials 1 mg/ml PtdIns(4,5)P2 (e.g., Avanti, cat. no. 840046P) 25 mg/ml PE ( e.g., Avanti, cat. no. 850725P) 25 mg/ml PS (e.g., Avanti, cat. no. 840034) 25 mg/ml PC (e.g., Avanti, cat. no. 850457) 25 mg/ml PE-rhodamine (e.g., Avanti, cat. no. 810150P) Nitrogen gas Liposome buffer (see recipe) Liquid nitrogen 2-ml glass vial Glass syringe and needle 37°C incubator with variable shaking DEPC-treated 1.5-ml microcentrifuge tubes Extruder (e.g., Avanti Mini Extruder) 1-µm filter Preparation of liposomes 1. Prepare the lipid mixture in glass vials as follows: 27.5 µl of 1 mg/ml PtdIns(4,5)P2 0.45 µl of 25 mg/ml PE 0.5 µl of 25 mg/ml PS 1.36 µl of 25 mg/ml PC

25% PtdIns(4,5)P2 15% PE 15% PS 45% PC

If the liposomes need to be fluorescently labeled, add 1% PE-rhodamine. The final liposome mixture contains 25% PIP2 (volume: 100 μl; molar concentration: 1 mM; size: 100 nm). Use only a glass syringe to take the lipids from the stock vial. All manipulations of lipids in organic reagents should be done in glass equipment (i.e., glass vials and glass syringes).

2. Dry the solutions in nitrogen gas. Place the vial in a 37°C incubator for at least 30 min to remove residual chloroform. Add 1 ml liposome buffer into the vial, and shake at 37°C for 30 to 60 min.



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3. Transfer the mixture to a DEPC-treated 1.5-ml microcentrifuge tube. Freeze the tube in liquid nitrogen, and then thaw quickly in a 37°C water bath. Repeat the freeze-thaw cycle 15 to 20 times until the solution becomes clear. When transferring the mixture to the DEPC-treated tube, the color of the mixture will be milky.

Filter the lipid solution 4. Assemble the extruder, and wash it with deionized water. The procedure for extrusion is available at https://avantilipids.com/divisions/equipment/.

5. Wet the 1-μm filter and the two spacing pieces, and assemble the extruder with the filter and spacing pieces inserted. 6. Pass 1 ml liposome solution through the filter by pushing the syringe. The syringe should be plunged no less than 20 times.

7. Collect the liposomes in a DEPC-treated microcentrifuge tube, and store at 4°C for up to 1 week. SUPPORT PROTOCOL 3

KIF5B PURIFICATION AND QUALITY TESTING ALR is a membrane deformation process mediated by the motor protein KIF5B. Since the pulling force of KIF5B is critical in this process, protein with good motor activity and a high survival ratio is essential. We describe the purification of KIF5B and a recommended gliding assay to verify the motor activity of KIF5B, which will ensure successful in vitro reconstitution of ALR.

Materials Sf9 cells Insect-XPRESS protein-free cell medium with L-glutamine (e.g., Lonza, cat. no. 12-730Q) Virus containing full-length KIF5B construct Nickel-nitrilotriacetic acid (NTA) agarose (e.g., GE Healthcare, cat. no. 17-5318-06) Ni Sepharose 6 Fast Flow resin Elution buffer (see recipe) Storage buffer (see recipe) Casein MAB-BRB80 (see recipe) Paclitaxel Sheared microtubule filaments (see Basic Protocol 2) ATP working solution (see recipe) 50-ml conical tube Centrifuge Ultrafiltration tube Hydrophilic flow channels (see Basic Protocol 2) Confocal microscope TIRF microscope with camera for time-lapse imaging


Purification of full-length KIF5B 1. To express His-tagged KIF5B protein, use the Bac-to-Bac expression system (see Friedman & Vale, 1999). First, grow Sf9 cells in protein-free cell medium to a density of 2 × 106 cells/ml, and incubate with virus containing a full-length KIF5B construct.



Figure 11.24.7 Maximum intensity projection of the tracking trajectory (white lines) of the microtubule filaments.

2. After 60 hr, collect the cells in a 50-ml conical tube, and lyse them using freeze-thaw cycles. Centrifuge 40 min, 44,000 × g, 4°C. 3. Bind the soluble fraction in batches to NTA agarose, and then wash the resin. 4. Elute the His6-tagged KIF5B with elution buffer. 5. Concentrate the eluate using an ultrafiltration tube, and store in storage buffer at −80°C for up to 1 year.

KIF5B gliding assay 6. To confirm the activity of the motor protein, perform the gliding assay in hydrophilic flow channels, prepared as in Basic Protocol 2. Incubate the channel with 15 μl of 0.4 mg/ml full-length KIF5B for 5 min. Briefly wash the channel with 50 μl of 3 mg/ml casein in MAB-BRB80, and block the channel for 5 min. Wash the channel with 50 μl MAB-BRB80 containing 20 μM paclitaxel. 7. Prepare 50 μl sheared microtubule filaments in MAB-BRB80 with 20 μM paclitaxel, so that the final concentration of tubulin is about 0.04 mg/ml. Allow the microtubule solution to flow into the channel, and check the density of filaments under excitation at 640 nm. Usually, it takes about 2 to 3 min for the microtubule filaments to bind to the motor-coated surface.

8. Wash the channel with 50 μl MAB-BRB80 containing 20 μM paclitaxel to remove the unbound microtubule filaments. Then add 60 μl ATP working solution. The ATP working solution contains an ATP regeneration system (phosphocreatine and creatine kinase) and an oxygen scavenger system (glucose and GLOX).

9. Take a time-lapse movie with 500-msec exposure time using TIRF illumination. The speed of movement of the microtubule filaments is calculated using home-written Matlab codes. The tracking trajectories are represented by the maximum intensity projection in Figure 11.24.7.



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REAGENTS AND SOLUTIONS Use deionized or distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A.

ATP working solution 48 µl 80 mM MAB-BRB80 (see recipe) 2.0 µl 600 µM ATP (20 µM final) 0.6 µl 1 M phosphocreatine (10 mM final) 0.6 µl 30 mg/ml creatine kinase (300 µg/ml final) 0.6 µl 2 mM paclitaxel (20 µM final) 0.6 µl 1 M DTT (10 mM final) 2.0 µl 3 mg/ml casein (0.1 mg/ml final) 5.0 µl 30% (w/v) glucose (2.5% final) 0.6 µl 100× GLOX (1× final; see recipe) Prepare freshly immediately before use Add GLOX freshly before flowing into the channel.

Cell culture medium High-glucose DMEM (e.g., HyClone, cat. no. SH30022.01) supplemented with: 10% fetal bovine serum 100 U/ml penicillin 100 U/ml streptomycin 2 mM GlutaMAX-I Store at 4°C for up to 6 months GlutaMAX-I is usually purchased as a 100× solution.

Elution buffer 20 mM Tris·Cl (see APPENDIX 2A) 0.1 M NaCl 250 mM imidazole Adjust pH to 8.0 Store at 4°C for up to 6 months GLOX PBS (see recipe) supplemented with: 60 mg/ml glucose oxidase 6 mg/ml catalase Store at room temperature for up to 6 months Liposome buffer 50 mM Tris·Cl, pH 8.0 (see APPENDIX 2A) 150 mM NaCl Store at 4°C for up to 6 months Motility assay buffer (MAB-BRB80) 80 mM PIPES 1 mM MgCl2 1 mM EGTA Adjust pH to 6.8 with KOH Store at 4°C for up to 6 months Visualizing Autophagic Lysosome Reformation



Phosphate-buffered saline (PBS) 135 mM NaCl 4.7 mM KCl 10 mM Na2 HPO4 2 mM NaH2 PO4 Adjust pH to 7.4 with HCL or NaOH Store at 4°C for up to 6 months Storage buffer 50 mM HEPES-KOH, pH 7.4 300 mM NaCl 1 mM MgCl2 10% (w/v) sucrose 50 mM ATP Store buffer without ATP at 4°C for up to 6 months Add ATP immediately before use.

COMMENTARY Background Information The method described in this unit to visualize autophagic lysosome reformation (ALR) in cells is restricted to serum and glutamine starvation–induced autophagy. It is useful for dissecting the molecular mechanisms of ALR, but it does not fully replicate in vivo physiological conditions and therefore cannot fully explain the physiological role of ALR. Results from extended studies in both cells and mouse models have shown in vivo evidence for a role of ALR in hereditary spastic paraplegia (Chang et al., 2014; Varga et al., 2015). Because the in vitro system described in this unit is less sophisticated than in vivo conditions, it can be used to pinpoint the essential parameters and mechanisms of ALR. However, it cannot precisely simulate the buffer environment of cells or the viscosity of the microenvironment in which membrane deformation occurs. Also, the in vitro system can only reveal the roles of a few essential proteins and regulatory proteins. Different lipid kinases that have been shown to play important regulatory roles are not included (Munson et al., 2015; Schulze et al., 2013; Sridhar et al., 2013). A more sophisticated in vitro system is needed to unveil the detailed regulatory mechanisms of ALR. In the in vitro system described in this unit, two membrane sources are recommended. They both have advantages and limitations. Purified autolysosomes more closely mimic in vivo conditions, since ALR involves the protrusion of tubules from autolysosomes. However, the purification of autolysosomes is based on density-gradient centrifugation,

and therefore the autolysosome fraction actually consists of enriched autolysosomes together with contamination from other membranes and molecules that happen to have the same density. This weakens the precision of the in vitro system. Purity is not a concern if man-made liposomes are chosen as the membrane source since they are made from synthetic lipids. The concentration of lipids can be adjusted to study the function of a single lipid in ALR. Man-made liposomes can be formulated to closely mimic the lipid composition of autolysosomes, but the lipid composition of autolysosomes is not known with certainty. At the same time, the proteins associated with autolysosomes are all missing from man-made liposomes. This explains why the tubulation efficiency of man-made liposomes is much lower than liposomes supplemented with cytosol.

Critical Parameters and Troubleshooting Cells react to starvation differently depending on cell type, cell confluency, and cell status. In normal rat kidney (NRK) cells, ALR usually peaks at 8 hr with about 60% to 80% of cells showing reformation tubules. ALR persists for a few hours but is no longer visible after 12 hr starvation. If no ALR tubules are observed, check the cell status and density before starvation. If no ALR tubules are observed in the in vitro system, the activity of the motor protein KIF5B should be checked first. The activity test should be done after protein purification, and the purified protein needs to be



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stored properly in aliquots to avoid multiple freeze-thaw cycles. Other than that, all other components should be freshly made or stored within acceptable times. The morphology of the purified autolysosomes or man-made liposomes can be checked by negative staining and transmission electron microscopy, since membrane ruptures or cracks sometimes occur. Last but not least, the procedures for cleaning the coverslips and assembling the flow chamber for single-molecule detection are very important. Since the detection method is singe-molecule level, all the procedures should be carried out in a Mega-level ultraclean hood to avoid contaminations of autofluorescent particles. All reagents should be of molecular grade, and all solutions must be filtered through a 2-μm syringe filter to remove impurities which could influence the singlemolecule fluorescent signal.

Anticipated Results About 60% to 80% of cells usually show reformation tubules. ALR peaks around 8 hr after the onset of starvation, then persists for a few hours before ceasing by 12 hr of starvation. The time points may vary between different cell types or with different triggers. Autolysosomes are positive for both LC3 and LAMP1, while protrusion tubules are only positive for LAMP1. The length of tubules varies, with some tubules extending across the whole cell and others only several multiples of the diameter of an autolysosome (Yu et al., 2010). In ALR-deficient mutant cells, such as KIF5B knockout cells, no tubules protrude from autolysosomes, and only enlarged autolysosomes can be observed (Du et al., 2016). Using the in vitro reconstitution system and its variants has enabled us to carry out in-depth mechanistic studies of autolysosome tubulation. The tubulation process happens in the presence of ATP, with a tubulation speed of 100 nm/sec, and 30% of the autolysosomes are tubulated. Tubulation was not observed when the KIF5B mutant T92N was used in the system (Du et al., 2016). Autolysosomes that were tubulated in vitro contained the lysosome membrane marker LAMP2 and could be visualized by immunofluorescence (Du et al., 2016).

Time Considerations Visualizing Autophagic Lysosome Reformation

To visualize ALR in cells, the cells should be transfected with the required fluorescent markers the day before, then allowed to settle down for more than 14 hr before starvation for 8 hr. ALR can then be visualized until 10 to

12 hr after the onset of starvation. The ALR tubules cannot be fixed for immunostaining or stored. The most time-consuming parts of the in vitro reconstitution protocol are the preparation of liposomes, purification of the motor protein, and isolation of autolysosomes. It takes about 2 weeks to purify the motor protein KIF5B. Thus, the protein should be prepared beforehand and stored at –80°C. It takes about 1 day to prepare the liposomes, and they can be used for up to 1 week if stored at 4°C. Preparation of microtubules takes 1 day, and they can also be kept for 1 week. Thus, liposomes and microtubules should be prepared a few days ahead of the experiment. Purification of autolysosomes from NRK cells requires 1 day, and the sample needs to be fresh for the reconstitution experiment. This step should therefore be done 1 day before the in vitro experiment. Determining the purity and characterization of autolysosomes takes 2 to 3 days because these steps require SDS-PAGE separation and immunoblotting of several marker proteins. This could be done after the reconstitution experiment. It takes about 1 hr to carry out one in vitro reconstitution assay in the flow channel, and data collection and processing requires 4 hr.

Acknowledgements Research in Li Yu’s laboratory was supported by the Ministry of Science and Technology of the People’s Republic of China (2016YFA0500202 and 2017YFA0503404) and the National Natural Science Foundation of China (31430053, 31621063, and 31671395). Research in Yujie Sun’s laboratory was supported by grants from the National Key R&D Program of China (no. 2017YFA0505300) and the National Science Foundation of China (21390412 and 31327901).

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