Plasmamembrane Organization in Signaling

Abstract

The composition of the plasma membrane has long been modeled as a fluid mosaic. (Singer and Nicolson, 1972) Studies in the last few years have identified microdomains like lipid rafts and caveolae that constrain membrane proteins within a small region of the cellular plasma membrane. These domains facilitate anchoring of different signaling proteins, like H-Ras, that has been shown to co-localize with nano domains upon activation (Lommerse et al. 2005, Rotblat et al. 2004). It is believed that these nanodomains function as important platforms for a multitude of signaling cascades that are initiated at the plasma membrane. Given that many of the transmembrane signals will need a coordinated domain organization, it is of importance to investigate properties like size, shape, stability and their mutual interaction.Here we transfected 3T3-cells to express the membrane anchor of H-Ras linked to Dendra2 or mEos2. Photo Activated Localization Microscopy (PALM) and Stimulated Emission Depletion Microscopy (STED) were used to make high resolution images of H-Ras distribution on the membrane as a probe for inner membrane domains. The GPI-anchored protein CD59 is used as a probe for the outer membrane domains to investigate colocalization of internal and external membrane domains. Using a SNAP-tag we covalently link Alexa647n to CD59 and image with direct stochastic optical microscopy (dSTORM).