title: Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization.
abstract: BACKGROUND
  Cholesterol/sphingolipid-rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (muscle-specific kinase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear.
  
  
  RESULTS
  In this work, we attempted to identify the interactions between membrane raft components and cortical skeleton that regulate, upon signalling by agrin, the assembly and stabilization of synaptic proteins of the postsynaptic membrane domain at the NMJ. We provide evidence that in C2C12 myotubes, agrin triggers the association of a subset of membrane rafts enriched in AChR, the -MuSK and Cdc42 (cell division cycle 42) to the actin cytoskeleton. Disruption of the liquid-ordered phase by methyl-β-cyclodextrin abolished this association. We further show that actin and the actin-nucleation factors, N-WASP (neuronal Wiscott-Aldrich syndrome protein) and Arp2/3 (actin-related protein 2/3) are transiently associated with rafts on agrin engagement. Consistent with these observations, pharmacological inhibition of N-WASP activity perturbed agrin-elicited AChR clustering. Finally, immunoelectron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nanodomains at steady state that progressively coalesced on 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 rafts.
  
  
  CONCLUSIONS
  The present observations support the notion that membrane rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization through the recruitment of effectors of the agrin/MuSK signalling cascade. These mechanisms are believed to play an important role in vivo in the formation of the NMJ.
authors.affiliation: Cell Biology Program, Institut Jacques Monod, UMR 7592, CNRS/Université Paris 7-Denis Diderot, Bâtiment Buffon, 15 rue Hélène Brion, 75205 Paris Cedex 13, France.
authors.forename: Annie
authors.initials: A
authors.lastname: Cartaud
authors.forename: Françoise
authors.initials: F
authors.lastname: Stetzkowski-Marden
authors.forename: Agathe
authors.initials: A
authors.lastname: Maoui
authors.forename: Jean
authors.initials: J
authors.lastname: Cartaud
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000187:drug effects* / Q000378:metabolism; D018171:Agrin / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D009469:Neuromuscular Junction / Q000378:metabolism; D058105:Polymerization; D011412:Propanolamines / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000037:antagonists & inhibitors; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D018171:Agrin; D002227:Carbazoles; D011412:Propanolamines; D011950:Receptors, Cholinergic; C495772:Wasl protein, mouse; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D047392:beta-Cyclodextrins; C489942:wiskostatin; C511695:MuSK protein, mouse; D020794:Receptor Protein-Tyrosine Kinases