Association of the Death-inducing Signaling Complex with Microdomains after Triggering through CD95/Fas

Abstract

In this investigation we show that the death-inducing signaling complex (DISC) associates with glycosphingolipid-enriched microdomains (GEM) upon CD95/Fas engagement. We primarily analyzed the ganglioside pattern and composition of GEM after triggering through CD95/Fas and observed that GM3 is the main ganglioside constituent of GEM. Stimulation with anti-CD95/ Fas did not cause translocation of gangliosides within or from the GEM fraction. Scanning confocal microscopy showed that triggering through CD95/Fas induced a significant GM3-caspase-8 association, as revealed by nearly complete colocalization areas. Coimmunoprecipitation experiments demonstrated that GM3 and GM1 were immunoprecipitated by anti-caspase-8 only after triggering through CD95/Fas. This association was supported by the recruitment of caspase-8, as well as of CD95/Fas, to GEM upon CD95/Fas engagement, as revealed by the analysis of linear sucrose gradient fractions. It indicates that the DISC associates with GEM; no changes were observed in the distribution of caspase-9. The disruption of GEM by methyl-␤-cyclodextrin prevented DNA fragmentation, as well as CD95/Fas clustering on the cell surface, demonstrating a role for GEM in initiating of Fas signaling.These findings strongly suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in CD95/Fas receptor-mediated apoptotic pathway.

Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells

“…6 In particular, they may be involved in the control of apoptosis via positive interactions involving CD95/Fas or via negative interactions involving Bad. 31,32 Indeed, some apoptotic molecules, such as CD95/Fas, FADD, procaspase-8 and procaspase-10 are recruited into microdomains, 33 whereas relocalization of Bad from lipid rafts to mitochondria resulted in apoptosis.…”

“…It indicates that mitochondria from CEM cells show a less complex ganglioside pattern as compared to whole cells. 6 Densitometric analysis revealed a significant increase of GD3 content in mitochondria after triggering through CD95/Fas ( Figure 1C). Conversely, a decrease of GD3 content was observed in the supernatant withdrawn of mitochondria.…”

“…Both untreated and Fas-stimulated cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature, as previously reported. 6 Alternatively, cells were fixed with acetone-methanol (1 : 1, v/v) for 10 min at 41C and then soaked in balanced salt solution (BSS; Sigma Chem. Co.).…”

“…5 During apoptosis, the death-inducing signaling complex (DISC) associates with these domains upon CD95/Fas engagement. 6,7 In particular, a role for gangliosides as structural components of the multimolecular signaling complex involved in CD95/Fas receptor-mediated apoptotic pathway was suggested. 6 Although not all cell types show the raft-dependency of CD95/Fas clustering, 7,8 in lymphocytic cells, the GEM-dependency of the initiation of receptor clustering and signaling was demonstrated by using cholesterol depleting agents, such as methyl-b-cyclodextrin (MbCD).…”

“…6,7 In particular, a role for gangliosides as structural components of the multimolecular signaling complex involved in CD95/Fas receptor-mediated apoptotic pathway was suggested. 6 Although not all cell types show the raft-dependency of CD95/Fas clustering, 7,8 in lymphocytic cells, the GEM-dependency of the initiation of receptor clustering and signaling was demonstrated by using cholesterol depleting agents, such as methyl-b-cyclodextrin (MbCD). 9 In addition, it was hypothesized 10,11 that a portion of intracellular GD3 can play a role in apoptotic machinery.…”