CD95 death‐inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells

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Abstract
We investigated the membrane localization of CD95 in type I and type II cells, which differ in their ability to recruit and activate caspase‐8. We found that CD95 was preferentially located inlipid rafts of type I cells, while it was present both in raft and non‐raft plasma membrane sub‐domains of type II cells. After stimulation, CD95 located in phospholipid‐rich plasma membrane was recruited to lipid rafts in both types of cells. Similarly, CD95 cross‐linking resulted in caspase‐independent translocation of FADD/MORT1 and caspase‐8 to the lipid rafts, which was prevented by a death domain‐defective receptor. CD95 internalization was then rapid in type I and delayed in type II cells and showed a substantial correlation with the kinetics of Fas‐associated death domain (FADD)and caspase‐8 recruitment to lipid rafts. Finally, electron microscopy analysis showed that after CD95 stimulation lipid rafts aggregated in large clusters that were internalized in endosomal vesicles, where caspase‐8 underwent massive processing. Taken together, our data demonstrate that CD95 death‐inducing signaling complex formation and internalization in type I and type II cells occur in lipidrafts, which are a major site of caspase‐8 activation.