Human Subcutaneous Adipose Tissue Adipocytes Demonstrate Two ...

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Sunday, February 12, 2017

suitable for dissecting collective activities by the separation of autonomous and non-autonomous molecular processesin vivo ranging from subcellular to organism level. Since a series of drugs are available for molecular tattoo, the technology can be implemented by a wide range of fields in the life sciences. Supported by the Hungarian Research and Innovation Fund (VKSZ_14-1-2015-0052). 733-Pos Board B498 Human Subcutaneous Adipose Tissue Adipocytes Demonstrate Two Physiological States: Insulin Responsive or Insulin Refractory Chad D. McCormick, Hang Waters, Ludmila Bezrukov, Brad Busse, Andrew Demidowich, Paul S. Blank, Jack A. Yanovski, Joshua J. Zimmerberg. NICHD, NIH, Bethesda, MD, USA. Insulin resistance is a precursor to Type II Diabetes. Peripheral insulin resistance, as exemplified by the response of tissues like adipose to an insulin challenge, is a tissue average traditionally monitored by glucose uptake assays. We hypothesize that these assays may not reflect cellular level heterogeneous behavior and instead report a weighted response of insulin responsive and refractory adipocytes. We developed new assays to monitor the adipocyte insulin response in the context of ex vivo tissue samples. Within one hour of biopsy from subjects recruited to the NIH Clinical Center, we tested if AKT phosphorylation, one of the major signaling nodes of the canonical insulin signaling pathway, is sufficient to monitor the number of insulin responsive or refractory cells in fixed human tissue. Immunostaining revealed two adipocyte populations: low pAKT cells, primarily seen in the absence of insulin stimulation, and high pAKT cells, primarily seen in tissue from healthy subjects after insulin stimulation. The fraction of tissue with a large pAKT response to insulin correlates well with the fraction of tissue with GLUT4, the insulin-stimulated glucose transporter, localized in the adipocyte plasma membrane. The pAKT fraction also supports a two-component model: insulin responsive versus insulin refractory adipocytes, rather than a graded continuum of insulin responses, when the pAKT fraction is matched with each subject’s insulin sensitivity index (SI), calculated using the insulin-modified frequently sampled intravenous glucose tolerance test (FSIVGTT). These results agree with previous work from our lab using isolated cells in which the healthier SI value a subject has, the greater fraction of their subcutaneous adipocytes respond to insulin. 734-Pos Board B499 Single Cell Examination of Membrane Fluidity and Cellular Respiration Krishna Ojha, John Ertle, Michael C. Konopka. Chemistry, The University of Akron, Akron, OH, USA. Bulk measurements of oxygen consumption rates by bacteria have previously been shown to be related to the cells’ average membrane fluidity (as measured by the diffusion of fluorescently-labeled molecules). Within a bacterial population, there is also significant cell-to-cell variation in both the diffusion coefficient of membrane probes and oxygen consumption rates of individual cells. One possibility is the diffusion of ubiquinone, an electron carrier in the electron transport chain (ETC), is being limited by the fluidity of the membrane which is causing the heterogeneity. We describe Fluorescence Recovery After Photobleaching (FRAP) measurements to monitor cell-to-cell variation in membrane diffusion coefficient. These results are compared to two approaches to look at what influence it might have on respiration at the single cell level. The first uses a fluorescent indicator of the activity of the ETC. The second directly measures the consumption of oxygen by individual cells using a phosphorescent Pt-porphyrin dye. 735-Pos Board B500 An Improved Single Molecule Imaging Vivo Method for In Vivo Stoichiometric and Functional Analysis of Protein Complexes Warren R. Zipfel1, Avtar Singh2, Maria Sirenko3, Alexander Song4, Paul Kammermeier5. 1 Biomedical Engineering, Cornell University, Ithaca, NY, USA, 2 Applied Physics, Cornell University, Ithaca, NY, USA, 3Cornell University, Ithaca, NY, USA, 4Princeton University, Princeton, NJ, USA, 5University of Rochester Medical Center, Rochester, NY, USA. Elucidating the composition and stoichiometry of membrane bound and cytosolic protein complexes is critical to understand their biological function and the underlying molecular mechanisms involved. Existing techniques used to determine subunit stoichiometry in single molecule experiments may significantly bias experimental results due to the need for either extremely low expression levels required to obtain concentrations suitable for single molecule imaging, or in the case of protein complex isolation for in vitro characterization, perturbation of the normal stoichiometric relationships by the isolation method used. Here we present an alternative approach in which protein complexes are assembled at physiological concentrations and subsequently diluted by conditionally controlled cell fusion to obtain protein levels suitable for

single-molecule observation while preserving the complexes in a near-native live cell environment. Methods such as stepwise photobleaching can then be used to study the subunit stoichiometry of membrane receptors, or for cytosolic complexes, this strategy greatly facilities fluorescence correlation spectroscopy based methodologies which can provide a quantitative assessment of cytoplasmic oligomerization state. We present examples that illustrate the advantages of the method for both membrane and intracellular cytosolic investigations. The method, called Single Protein Recovery After Dilution (SPReAD), is a simple and versatile means of extending the concentration range of single molecule measurements to what are often more normal cellular levels with minimal perturbation of protein complex stoichiometry. 736-Pos Board B501 Visualizing Heterogeneous Single-Molecule Dynamics of Molecular Assemblies in Live Cells Michael Lacy1, David Baddeley2, Julien Berro1. 1 Molecular Biophysics and Biochemistry, Yale University, West Haven, CT, USA, 2Cell Biology, Yale University, West Haven, CT, USA. Molecular assemblies can have highly heterogeneous dynamics within the cell. Classic fluorescence microscopy methods, such as FRAP (Fluorescence Recovery After Photobleaching), have been invaluable to characterize these dynamics at the micrometer scale. However, it has been particularly difficult to characterize molecular heterogeneities inside diffraction limited zones within multimolecular assemblies in live cells. We have developed a novel fluorescent labeling and imaging protocol, called Single Molecule Recovery After Photobleaching (SMRAP), which has allowed us to reveal the heterogeneous dynamics of the eisosome, a multi-protein structure on the cytoplasmic face of the plasma membrane in fungi. By fluorescently labeling only a small fraction of cellular Pil1p, the core eisosome protein in fission yeast, we were able to visualize whole eisosomes before photobleaching and, after photobleaching, the recovery of individual Pil1p molecules that bound to the structure with ~30 nm precision. Further analysis of these sparsely labeled, dynamic structures allowed us to show that Pil1p turnover is spatially heterogeneous. We observed that Pil1p molecules from the cytoplasm bind and unbind at the ends of eisosomes, but not along the interior, supporting a new model of the eisosome as a dynamic filament. We expect our new SMRAP method will be easily and broadly applicable to any molecular assembly in the cell, since it only requires sparse labelling of proteins of interest and a standard TIRF setup with single molecule detection capabilities.

Single-Molecule Spectroscopy I 737-Pos Board B502 Single-Molecule Counting Applied to Immunoassays Patrick J. Macdonald, Qiaoqiao Ruan, Kerry M. Swift, Sergey Y. Tetin. Abbott Laboratories, Libertyville, IL, USA. We investigated the sensitivity of single-molecule TIRF counting in diagnostic immunoassay applications. This work focused on using single-molecule techniques purely for detection, with the assay itself taking place on a separate platform with the detectable label being eluted for single-molecule measurement. Such an approach both limits the amount of background in the final measurement and sets up a universal detection procedure for a potentially wide variety of immunoassays. We took advantage of the low volume required for singlemolecule measurements and demonstrated a sample reloading approach to further concentrate the sample on the single-molecule surface. SM reloading—for a biotin-streptavidin surface capture reaction—is a remarkably robust procedure, independent of starting concentration, and so can be used for assaying unknown samples. We tested model assays with single-molecule detection to substantiate this approach and demonstrate the potential of single-molecule counting for diagnostic applications. 738-Pos Board B503 Comparing Antibody-Antigen Binding in Serum Versus Buffer with Fluorescence Correlation Spectroscopy David Ortiz, Isabel Yannatos, Abhinav Nath. Medicinal Chemistry, University of Washington, Seattle, WA, USA. Therapeutic proteins or ‘biologics’ such as monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs) or Fc-fusions comprise a growing percentage of drugs in the development pipeline. A majority of techniques used to characterize these drugs require dilution into standard buffers, conditions that do not reflect the complexity of crowded biological environments such as serum. Here, we use fluorescence correlation spectroscopy (FCS) to directly assess the effect of biological solutions on the binding affinity of mAbs to antigen. FCS is a single-molecule technique that measures the mean time a labeled particle takes to diffuse across a small (~1fL) confocal volume. Diffusion time is directly proportional to hydrodynamic radius, thus binding can be