Techniques for Direct Imaging of Nanoplatforms in the Live Cell Plasma Membrane

Abstract

We recently developed a method termed TOCCSL (‘Thinning out Clusters while Conserving Stoichiometry of Labeling') 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/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.To further validate our method we extended TOCCSL by utilizing two-color co-localization and photo-activation. While two-color TOCCSL allows for direct imaging of mobile nanoplatforms containing different probe molecules and thus supporting the lipid raft concept, photo-activation based TOCCSL addresses an additional population of observed probes. Since TOCCSL is suitable for characterizing the mobile fraction of marker-proteins/lipids, the information about slowly diffusing or immobile nanoplatforms is not accessible. This can be circumvented by substituting the fluorescent marker by a photo-activatable protein linked to the molecule of interest. By irreversibly switching a small fraction of markers from a dark into the fluorescent state single molecule brightness and diffusion analysis after activation will add to the characterization of nanodomains.Key Publication: Brameshuber et al., JBC 2010, 285(53): 411765-71