Imaging of Mobile Stable Lipid Rafts in the Live Cell Plasma Membrane and their Involvement in Cellular Signaling During Heat Shock

Abstract

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, also termed lipid or membrane rafts. Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. We present here a method which allows for the first time the demonstration that single rafts diffuse as stable platforms in the live cell plasma membrane. Our method senses rafts by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. The special photobleaching protocol TOCCSL (Thinning Out Clusters while Conserving Stoichiometry of Labeling1) was used to reduce the surface density of labeled mobile rafts down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per raft was determined by single molecule brightness analysis. For demonstration, we used the consensus markers Bodipy-GM1, a fluorescent lipid analogue, and glycosylphosphatidylinositol-anchored monomeric GFP. For both markers we found cholesterol-dependent association in the plasma membrane of living CHO and Jurkat T cells in the resting state, indicating the presence of small, mobile, stable rafts hosting these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures mGFP-GPI homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.1. Moertelmaier, M., Brameshuber, M., Linimeier, M., Schutz, G. J. & Stockinger, H. Thinning out clusters while conserving stoichiometry of labeling. Appl Phys Lett 87, 263903 (2005).