Determining Fas Signaling Pathways by Quantifying Receptor Clustering

Abstract

Abstract The TNF-cytokine family receptor Fas (CD95) mediates T cell apoptosis by recruiting a death-inducing signaling complex (DISC) composed of the adaptor protein (FADD) and Caspase-8. Recently, it has been shown that Fas is also directs non-death signaling and enhances effector cell differentiation in T cells. Mutations which reduce the ability of Fas to localize to lipid raft microdomains and cluster upon activation abrogate the death-inducing potential of Fas while preserving non-apoptotic signaling. Caspase-8, a key mediator of both apoptotic and non-apoptotic signal, has been shown to form filamentous structures in vitro and upon overexpression in mammalian cells, but it is unknown if this process occurs in vivo to facilitate signaling. To investigate these supramolecular phenomena, we developed Fas and Caspase-8 fused to photoactivatable fluorescent proteins to be expressed in both primary T cells and T cell lines for super resolution imaging. Additionally, we have produced supported planar lipid bilayers presenting Fas ligand (FasL) and T cell stimulatory ligands to mimic a physiologic signaling environment. Photoactivatable localization microscopy (PALM) is used to observe and quantify clustering Fas molecules and formation of caspase-8 containing signaling complexes at nanometer scale resolution. Structure illuminated microscopy (SIM) and PALM will be used to observe the behavior of Fas relative to cellular-scale actin remodeling and TCR clustering. Our findings will reveal the behavior of supramolecular complexes mediating apoptosis and non-apoptotic signaling by Fas in the plasma membrane of living cells.