Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment

Abstract

Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas–Fas and Fas–FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.

The benefits of clustering in TNF receptor superfamily signaling

“…Hexagonally arranged ligands generated a high amplitude signal in contrast to the low amplitude, broad signal generated by ligands with the wrong geometry ( 26 ). Super-resolution imaging has confirmed the importance of clustering in vivo in a Fas/FasL model but there is disagreement of the state of the ligand-free receptors ( 27 ). The Fas receptors appear largely monomeric and dimeric in the ligand-free state as observed by fluorescence energy transfer studies of C-terminal labeled Fas-fluorescence protein (Fas-FP) receptors.…”

RNA-Sequencing Characterization of lncRNA and mRNA Functions in Septic Pig Liver Injury

“…TNF protein superfamily members, including TNF-a, Fas cell surface death receptor ligand (FASL), and TNF-associated apoptosis-inducing ligand (TRAIL), are the main inducers of hepatocyte death. The TNF-a/tumor necrosis factor receptor (TNFR1), FAS/FASL, and TRAIL/TRAILR pathways can activate CASP8 and then initiate a proteolytic cascade that involves CASP3, CASP6, and CASP7, which ultimately leads to hepatocyte apoptosis [ 24 , 25 , 26 ]. We showed that TRAILR, FAS, CASP8, and CASP3 were upregulated in the LPS-stimulated piglet livers, and that the predicted lncRNA target genes were also enriched in the apoptosis pathway.…”

TNF-Related Apoptosis-Inducing Ligand: Non-Apoptotic Signalling

“…Likewise, it has been proposed that, upon cognate ligand binding, DR4, DR5 and Fas form, first of all, trimer complexes whose multimerization or crosslinking with neighbouring trimers occurs via the dimerization between receptor interfaces, either located opposite the ligand-binding interfaces, resulting in a hexameric honeycomb-like structure [ 141 ]. A dimerization motif found in the transmembrane helix domain of the receptors is also suspected, in addition, to play an important role for the assembly of the DISC, its stability and potency [ 138 , 141 , 142 ]. Moreover, as suggested for Fas, DISC stability may also be regulated at the level of the cytoplasmic domain, of some agonist receptors, by the adaptor protein FADD [ 143 , 144 , 145 ].…”