Direct Imaging of Mobile Nanodomains in the Live Cell Plasma Membrane by using a Two-Color Photobleaching Approach

Abstract

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 (Brameshuber,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 platforms 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 distribution 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/μm². 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 disappeared after cholesterol-oxidase treatment - thus supporting the lipid raft concept.