CD95 Receptor Organization on the Plasma Membrane and its Influence on Apoptosis

Abstract

CD95 is a plasma membrane receptor that can mediate apoptosis after activation by its specific ligand CD95L. Upon stimulation the death receptor recruits various proteins that form the so-called death inducing complex (DISC). The DISC contains the protease procaspase-8 that becomes active by dimerization and self-processing. This process is thought to require high local concentration, that can be achieved by CD95 and DISC clustering. Several mechanisms have been proposed to achieve this clustering, including CD95 self-association, lipid raft localization and oligomerization of the DISC protein FADD.Our goal is to understand how DISC clusters correlate with CD95 spatial organization on the membrane and more generally how this organization can regulate cell sensitivity to CD95-induced apoptosis.We visualize the distribution of CD95 by immunofluorescence and GFP fusion, using confocal microscopy, stimulated emission depletion microscopy and total internal reflection microscopy. As this distribution appears as a dotty pattern (fig.), we determine the dot density in different cell types. We investigate the influence of system perturbations such as cholesterol depletion and actin depolymerization on the CD95 distribution and on apoptosis sensitivity. We also investigate the dependence of clustering on the receptor amount.View Large Image | View Hi-Res Image | Download PowerPoint Slide