Spatio-temporal control of CD95-activation mode.
CD95 is a transmembrane receptor whose engagement by its ligand (CD95L) can induce either apoptosis or cell survival (1). Apoptosis is initiated by activation of caspases in a death-inducing signaling complex through the interactions at the intracellular death domain of CD95. Phosphorylation of a tyrosine within this domain recruits another complex with kinase activity, which outcompetes the death-inducing complex (2, 3). SH2- domain containing proteins bind to the phosphorylated tyrosine, ultimately leading to increased PI3K or ERK activity, depending on the cell type (1). Thus, the cell’s fate is governed by a molecular switch controlling the recruitment of either a death- or a kinase-inducing complex. At the organismal level, CD95 is involved in cell clearing in development and degenerative diseases, and CD95-mediated kinase activity may induce inflam- mation or cancer progression. Despite its involvement in such fundamental processes, the molecular mecha- nism behind CD95’s switch remains unclear. In the current funding period, we could show that cell-cell contacts mediate a global increase of tyrosine kinase activity, tipping the CD95-mediated cellular outcome from death to survival. One important feature of cell-cell contact is the selective compartmentalization of membrane do- mains favoring a distinct cooperativity of signaling modules (4). However, the contribution of membrane com- partmentalization to CD95 activation has not been explicitly investigated. We hypothesize that changes in the local membrane microenvironment of CD95 serve as a switch between the protein’s different activation modes. In the current funding period, beyond demonstrating the impact of cell-cell contacts on CD95’s activation mode (Gülcüler Balta et al., in press, 2019), we have initiated the analysis of the local lipid microenvironment. First, we have run molecular dynamic simulations to predict the lipid species with the highest probability of being in contact with CD95. Second, we have analysed the lipid composition of CD95 wild type (wt) and CD95 knock- out (CD95KO) cancer cells. Analysis of the global lipidomics and glycosphingolipidomics profiles allowed us to identify CD95-dependent changes in specific lipid species. Notably, identified lipid species were also pre- dicted to be in contact with CD95 via molecular dynamics simulations of CD95-lipid interactions. Further, we established superresolution microscopy techniques for the quantitative assessment of CD95 receptor clusters at the plasma membrane. In the coming funding period, we plan to further characterize CD95’s lipid microen- vironment and the molecular mechanism by which CD95 shapes this microenvironment. In addition, we will examine how these changes to the plasma membrane modulate the local viscosity, curvature and submem- brane cortical cytoskeleton. Together, this investigation will shed light on how the local lipid-protein membrane composition impacts the cellular response to receptor activation. Our long-term plan is to modulate these spe- cialized membrane domains, such as lipid rafts, by rational targeting and disruption of their interactions to further understand CD95’s role in dictating a cell’s fate.