Résumé :
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Liquid-ordered membrane microdomains or lipid rafts are involved in the clustering of nicotinic acetylcholine receptors at the neuromuscular junction (Stetzkowski-Marden et al., J. Lipid Res., 2006). Upon activation of the Muscle-specific tyrosine kinase receptor MuSK by the neurotropic factor agrin, a complex cascade of signalling events involving protein tyrosine phosphorylation and Rho family GTPases reorganize the actin-based cytoskeleton, eventually leading to AChR clustering. Among strategies developed by cells to initiate and sustain response to extracellular signals, membrane-raft dynamics controlled by proteins that are linked to the actin cytoskeleton has recently been recognized. In this work, we have explored the possibility that AChR clustering results from an actin-based coalescence of rafts containing critical postsynaptic components. We show that upon agrin treatment of C2C12 myotubes, actin and several actin assembly factors Cdc42, N-WASP, ARP 2/3 and cortactin were transiently recruited to raft fractions after Ca++/Mg++ isotonic buffer extraction.The recruitment of these proteins followed a complex mechanism: in the first few minutes upon agrin engagement, a dissociation of these assembly factors from rafts occured whereas, their robust and transient recruitment to the raft fractions was later observed. Our data point out a model of raft-actin cytoskeleton dynamics in which uncoupling of rafts from the skeleton allows raft coalescence to proceed through actin repolymerization. New strategies to investigate in situ raft-actin dynamics during agrin signalling are currently developed. This work was supported by the CNRS, Universités Paris 6 &7 and by grants from ANR and AFM. C.P. was a recipient of a Post-doctoral ANR fellowship.
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