(PUFAs) affects MSC differentiation, promoting the osteogenic over the adipo- genic phenotype. Extending these studies to other cell functions, we have discovered a variety of exogenous mediators of the PM phenotype (in addition to u-3 PUFAs) and find that any deviation from optimal raft stability has a sup- pressive effect on antigen-mediated mast cell activation. These observations provide a quantitative framework to measure the PM phenotype, identify exog- enous fatty acids as potential mediators of this phenotype, and demonstrate the effect of perturbations of PM physical properties on cell function. 2587-Pos Board B279 Size and Acylation Influence the Lateral Mobility of Plasma Membrane Proteins in Live Cells Elin Edwald, Sarah L. Veatch. Biophysics/Chemical Biology, University of Michigan, Ann Arbor, MI, USA. Lipid-mediated membrane heterogeneity is proposed to be an important orga- nizing principle in mammalian cells. Using single fluorescent particle tracking, we quantify diffusion parameters of a large panel of fluorescent fusion mem- brane proteins ranging in size, mode of membrane anchoring, and putative phase-association. These include palmitoylated or non-palmitoylated versions of three transmembrane proteins (truncated linker of activated T-cell, trun- cated haemagglutinin, and b 2 adrenergic receptor) as well as three proteins anchored with lipid moieties (GPI, palmitoyl and myristoyl, or geranylger- anyl). We present an analysis that utilizes Brownian simulations to aid in in- terpreting heterogeneity. With the exception of two of the palmitoylated transmembrane proteins, diffusion of all constructs is unconfined and consis- tent with Brownian motion at 37  C at the time-scales investigated (20 ms-1 sec). We explore the contributions of lipid mixing to confinement by modu- lating cholesterol levels and through the use of biochemical perturbations that affect the temperature of the immiscibility phase transition in isolated plasma membrane vesicles. Among our findings is that diffusion and confine- ment are highly temperature sensitive. Furthermore, our results indicate a complicated size-dependence of diffusion suggesting that diffusion of small probes is particularly sensitive to dimerization when it occurs in either a bio- logical context or due to labeling techniques. Overall, we explore the contri- bution of phase-mediated membrane heterogeneity to protein mobility while highlighting several factors that can complicate the interpretation of lateral diffusion data. 2588-Pos Board B280 Induction of Endoplasmic Reticulum-Plasma Membrane Contacts is a Non-Conducting Function of the Kv2.1 Voltage-Gated Potassium Channel Philip D. Fox 1 , Diego Krapf 2 , Michael M. Tamkun 1 . 1 Biomedical Sciences, Colorado State University, Fort Collins, CO, USA, 2 Electrical and Computer Engineering, Colorado State University, Fort Collins, CO, USA. The voltage-gated potassium channel Kv2.1 localizes to micron diameter clusters on the soma of hippocampal pyramidal neurons and transfected HEK 293 cells. Channels localized to clusters are largely unable to flux K þ suggesting Kv2.1 possesses an auxiliary, non-conducting function. We present evidence that one non-conducting function of Kv2.1 is to induce the formation of endoplasmic reticulum (ER)-plasma membrane (PM) con- tacts. In HEK 293 cells, the cortical ER (cER) as observed by TIRF micro- scopy typically consists of a meshwork of tubules. Expression of Kv2.1 induced the enlargement of cER tubules into micron diameter sheets, mirror- ing the clustered localization of the channel. An ultrastructural analysis using thin-section electron microscopy and immuno-gold labeling indicated that cER is brought into close apposition to the PM (<30nm) opposite dense immuno-gold labeling for Kv2.1. The close apposition of cER observed opposite Kv2.1 immuno-gold was accompanied by thinning of the cER lumen. ER-PM contacts are typically demonstrated to be anchored by pro- teins which reside in the ER membrane and extend into the cytoplasm to bind anionic lipid species which reside in the inner leaflet of the PM. These data suggest that the integral PM protein Kv2.1 functions to direct the forma- tion of ER-PM contacts through interactions with unknown lipids or proteins in the ER. 2589-Pos Board B281 Direct Imaging of Mobile Nanodomains in the Live Cell Plasma Membrane by using a Two-Color Photobleaching Approach Mario Brameshuber, Christina Manner, Martin Fuerst, Eva Sevcsik, Gerhard J. Schuetz. Institute of Applied Physics - Biophysics, Vienna University of Technology, Vienna, Austria. We recently developed a method termed TOCCSL (Moertelmaier,APL-2005) (‘Thinning out Clusters while Conserving Stoichiometry of Labeling’) which allowed for the first time the direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane (Brameshu- ber,JBC-2010). Our method sensed these platforms by their property to assemble the putative raft markers glycosylphosphatidylinositol-anchored monomeric GFP (mGFP-GPI) and GM1-Bodipy on a time-scale of seconds on the cell membrane of living CHO and Jurkat T-cells. In order to resolve plat- forms we used a special photobleaching protocol to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffraction- limited spots, without altering the single spot brightness. The statistical distri- bution of probe molecules per platform was determined by single-molecule brightness analysis. To further validate our method we extended TOCCSL by utilizing two-color co-localization in combination with stoichiometric photobleaching (Ruprecht,SoftMatter-2010). Glycosylphosphatidylinositol- anchored SNAP-tag (GPI-SNAP) was stably expressed by CHO cells and labeled with SNAP-AF488 and SNAP-AF647 at an equimolar ratio. For both colors we were able to observe a homogeneous surface staining with a density of 300-1000 molecules/mm 2 . To reduce the surface density we applied TOCCSL parallel to both color channels. An appropriate recovery time yielded a stoichiometrically reduced surface density enabling the localization of single fluorescent GPI-SNAPs in both color channels with a resolution down to a few ten nanometers. After correction of chromatic aberration we used an automatic algorithm to detect pairs of co-localized GPI-SNAPs within a diffraction limited distance. The number of co-localized GPI-SNAPs and their stability was found to be comparable to our previous measured mGFP-GPI data: around 30% of GPI-SNAPs homo-associated with a lifetime of seconds and disap- peared after cholesterol-oxidase treatment - thus supporting the lipid raft concept. 2590-Pos Board B282 Long-Term Live Observation of Membrane Protein Interaction with Lipid Nanodomains Show Dependence on Cell Cycle and Time After Transfection Muhammed F. Simsek, Arnd Pralle. Physics, University at Buffalo, Buffalo, NY, USA. The study of cholesterol-stabilized nanodomains in the cell membrane, aka lipid rafts, is complicated by differences in techniques, cell type, physiolog- ical parameters and possible changes of cell and membrane composition dur- ing the cell cycle and after transient transfection. We recently developed a non-destructive and non-perturbing technique combining fluorescence corre- lation spectroscopy (FCS) with spatially-resolved camera TIRF imaging, bimFCS. This method provides a quantification of the strength of the mem- brane protein - lipid nanodomain interaction, the membrane protein con- centration and its diffusion coefficient. As an optical method it can be combined with other imaging methods such as we record changes in cytoskel- eton structure simultaneously. bimFCS data was acquired with hourly inter- vals for periods longer than one day while keeping the cells growing at physiological temperature on the microscope stage. Transiently transfecting PtK2 or CHO cells with GPI-anchored membrane proteins associated with cholesterol-stabilized nanodomains, we observed a higher association with such domains in earlier stages of protein expression and slower diffusion compared to later times. Using synchronized cell cultures, we also quantify the membrane protein interaction with lipid nanodomains and record the im- ages of membrane cytoskeletal actin bundles as function of the cell cycle. To discriminate between possible membrane ultrastructure models, we combine the experimental results with diffusion simulations of molecules interacting with such domains in the presence and absence of linear actin meshwork acting as pinpoints for the domains. 2591-Pos Board B283 Characterizing the Cell Surface Structure and Antibody Recognition Forces on Intact Microbial Cells using Scanning Probe Microscopy Yoo Jin Oh 1 , Gerhard Sekot 2 , Memed Duman 1 , Lilia Chtcheglova 3 , Paul Messner 2 , Herwig Peterlik 4 , Christina Scha ¨ffer 2 , Peter Hinterdorfer 1 . 1 Institute for Biophysics, Johannes Kepler University Linz, Linz, Austria, 2 Department of NanoBiotechnology, NanoGlycobiology Unit, Universita ¨t fu ¨r Bodenkultur Wien, Vienna, Austria, 3 Center for Advanced bioanalysis (CBL), Linz, Austria, 4 Faculty of Physics, Universita ¨t Wien, vienna, Austria. Tannerella forsythia is among the most potent triggers of periodontal dis- eases and approaches to understand underlying mechanisms are currently intensively pursued. A ~22-nm thick, 2D crystallinesurface (S-) layer that completely covers T. forsythia cells is crucially involved in the bacterium- host cross-talk. The S-layer is composed of two intercalating glycoproteins (TfsA-GP, TfsB-GP) that are aligned into a periodic lattice. To characterize this unique S-layer structure at the nanometer-scale directly on intact T. forsythia cells, atomic force microscopy (AFM) based topography imaging Tuesday, February 18, 2014 511a