Imaging of Mobile Stable Nanoplatforms in the Live Cell Plasma Membrane

Abstract

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of “lipid rafts” 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 direct imaging of nanoscopic stable platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. A special photobleaching protocol was used 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 distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analogue Bodipy-GM1 which preferentially partitions into liquid ordered phases. For both markers we found cholesterol-dependent homo-association in the plasma membrane of living CHO and Jurkat T cells in the resting state, thereby demonstrating the existence of small, mobile, stable platforms containing these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures the mGFP-GPI homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.