Transient Effect of Calcium Influx on PIP2 Clusters and Cholesterol-Stabilized Nano-Domains in the Inner Plasma Membrane Leaflet of Intact Cells

400-Pos Board B180 Plasma Membrane Nanoplatforms are Dissolved by Oxidized Phospholipids Mario Brameshuber1, Eva Sevcsik1, Christina Manner1, Benedikt Rossboth1, Albin Hermetter2, Gerhard J. Schuetz1. Institute of Applied Physics Biophysics, Vienna University of Technology, Vienna, Austria, Department of Biochemistry, Graz University of Technology, Graz, Austria. We developed a method termed TOCCSL (‘Thinning out Clusters while Conserving Stoichiometry of Labeling’) which allows 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 is used to reduce the surface density of labeled mobile platforms down to the level of well-isolated diffractionlimited spots, without altering the single spot brightness. The statistical distribution of probe molecules per platform is determined by single molecule brightness analysis. As consensus raft marker we chose monomeric GFP linked via a GPI (glycosyl-phosphatidylinositol) anchor to the cell membrane. We found cholesterol-dependent homo-association of mGFP-GPI in the plasma membrane of living CHO cells, thereby demonstrating the existence of small, mobile, stable platforms hosting these probes. This strong homo-association could be released by addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), two oxidized lipids typically present in oxidatively modified low density lipoprotein. We found a clear dose-response relationship of mGPF-GPI nanodomain disintegration upon addition of POVP, correlating with the signal of an apoptosis marker. Applying similar concentrations of lyso-lipid did not dissolve nanodomains. To understand the mechanism behind nanodomain disintegration we inhibited the activation of acid sphingomyelinase (aSMase), which activates several apoptotic signaling pathways via ceramide generation. Inhibition of aSMase by NB-19 before addition of POVPC completely abolished the nanodomain-disintegration effect of oxidized phospholipids, thereby proofing a rather indirect effect of oxidized phospholipids on lipid nanodomains. Additionally we addressed the role of aSMase activity in the release of mGPF-GPI homo-association upon cholesterol extraction no correlation was found. In conclusion we could determine the mechanism how oxidized lipid species disrupt mGFP-GPI nanodomains in the live plasma membrane.