Résumé :
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Communication n° 354 Cholesterol/sphingolipid-enriched microdomains or lipid rafts are involved in intracellular trafficking of selected proteins and lipids, in the formation of signaling complexes and actin cytoskeleton organization. In neurons, rafts are associated with several postsynaptic proteins including AMPA and alpha-7 nicotinic acetylcholine receptors and are beleived to participate in the formation and maintenance of synapses. Here, we investigate whether lipid rafts are involved in the differentiation of the neuromuscular junction. To determine whether lipid rafts are involved in AChR clustering in myogenic cells, we employed two standard tools for specifying whether a protein is involved in raft processes: perturbation of lipid rafts by drugs that deplete membrane cholesterol, and detergent resistance of lipid-ordered domains followed by flotation in sucrose gradient centrifugation. Characteristic lipids of rafts, namely cholesterol and glycosphingolipids, were observed in situ at the vertebrate NMJ and depletion in cholesterol dispersed agrin-elicited AChR clusters in C2C12 myotubes. AChRs, as well as several postsynaptic signaling and/or scaffolding components which are known to be critical to the differentiation of the postsynaptic membrane at theNMJ, i.e. the muscle-specific kinase MuSK and syntrophin, are also recovered in light/raft fractions as characterized by caveolin-3 and flotillin-2 after fractionation by sucrose gradient centrifugation. Yet, immunofluorescence analysis of agrin-treated C2C12 cells revealed that these two bona fide rafts markers did not colocalize with AChR clusters, suggesting that AChR partition in a specialized subset of rafts. Collectively, these data indicate that the accumulation of AChR at the NMJ occurs through a raft-dependent clustering mechanism likely triggered by an agrin stimulated MuSK signal. Supported by the CNRS, the Universités Paris 6 and 7 and AFM.
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