Résumé :
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Cholesterol/sphingolipid-rich membrane micro-domains or membrane rafts have been implicated in various aspects of receptor function such as receptor activation, trafficking and synaptic localization. More specifically in muscle, membrane rafts are involved in acetylcholine receptor (AChR) clustering by the neural factor agrin, a mechanism considered integral to neuromuscular junction (NMJ) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibers. Since membrane rafts are specialized platforms sustaining actin nucleation, we hypothesize that these micro-domains provide the suitable microenvironment favoring the agrin/MuSK signaling cascade eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signaling pathways operating through these micro-domains still remains unclear. In this work, we attempted to identify the interactions between membrane/raft components and cortical skeleton that regulate the assembly and stabilization of raft-based signaling domains at the NMJ. We provide evidence that in C2C12 myotubes agrin triggers the association of a subset of rafts enriched in AChR, MuSK and Cdc42 to the actin cytoskeleton. We further show that actin and the actin-nucleation factors N-WASP and Arp2/3 transiently associated with rafts upon agrin signaling. Consistent with these observations, pharmacological inhibition of N-WASP activity perturbed agrin-elicited AChR clustering. Finally, immuno-electron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nano-domains at steady state that progressively coalesced upon agrin activation. These rearrangements of membrane domains correlated with the reorganization of cortical actin cytoskeleton through concomitant and transient recruitment of the Arp2/3 complex to AChR-enriched raft micro-domains. The present observations support the notion that lipid rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization following agrin stimulation. These mechanisms are believed to play important role in vivo in the formation of the NMJ.
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