title: Laminin is instructive and calmodulin dependent kinase II is non-permissive for the formation of complex aggregates of acetylcholine receptors on myotubes in culture.
abstract: Previous work has shown that myotubes cultured on laminin-coated substrates form complex aggregates of synaptic proteins that are similar in shape and composition to neuromuscular junctions (NMJs). Here we show that laminin instructs the location of complex aggregates which form only on the lower surface when laminin is coated onto culture dishes but over the entire cell when laminin is added in solution. Silencing of myotubes by agents that block electrical activity (tetrodotoxin, verapamil) or by inhibitors of calmodulin dependent kinase (CaMKII) render the myotube permissive for the formation of complex aggregates. Treatment with laminin alone will facilitate the formation of complex aggregates hours later when myotubes are made permissive by inhibiting CaMKII. The AChR agonist carbachol disperses pre formed aggregates suggesting that non-permissiveness may involve active dispersal of AChRs. The permissive period requires ongoing protein synthesis. The latter may reflect a requirement for rapsyn, which turns over rapidly, and is necessary for aggregation. Consistent with this geldanamycin, an agent that increases rapsyn turnover disrupts complex aggregates. Agrin is well known to induce small clusters of AChRs but does not induce complex aggregates even though aggregate formation requires MuSK, a receptor tyrosine kinase activated by agrin. Dystroglycan (DG) is the major laminin receptor mediating complex aggregate formation with some contribution from β1 integrins. In addition, there is a pool of CaMKII associated with DG. We discuss how these permissive and instructive mechanisms bear on NMJ formation in vivo.
authors.affiliation: Centre for Research in Neuroscience, and Dept. of Neurology, McGill University Health Centre, 1650, Cedar Ave., Montreal, Quebec, H3G 1A4, Canada.
authors.forename: Raphael
authors.initials: R
authors.lastname: Vezina-Audette
authors.affiliation: Centre for Research in Neuroscience, and Dept. of Neurology, McGill University Health Centre, 1650, Cedar Ave., Montreal, Quebec, H3G 1A4, Canada.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Tremblay
authors.affiliation: Centre for Research in Neuroscience, and Dept. of Neurology, McGill University Health Centre, 1650, Cedar Ave., Montreal, Quebec, H3G 1A4, Canada. Electronic address: sal.carbonetto@mcgill.ca.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Carbonetto
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D018171:Agrin / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016227:Benzoquinones / Q000494:pharmacology; D002038:Bungarotoxins / Q000737:chemistry / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D015870:Gene Expression; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D007797:Laminin / Q000494:pharmacology*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009124:Muscle Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D066329:Protein Aggregates / Q000187:drug effects*; D011950:Receptors, Cholinergic / Q000235:genetics / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D013779:Tetrodotoxin / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
chemical_list: D018171:Agrin; D016227:Benzoquinones; D002038:Bungarotoxins; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D047029:Lactams, Macrocyclic; D007797:Laminin; D009124:Muscle Proteins; D066329:Protein Aggregates; D011950:Receptors, Cholinergic; D026941:Sodium Channel Blockers; D015222:Sodium Channels; C051609:peripheral membrane protein 43K; D013779:Tetrodotoxin; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D002217:Carbachol; D014700:Verapamil; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; C001277:geldanamycin
keywords: CaMKII; Dystroglycan; Electrical activity; Integrin; Laminin; MuSK; Synapse formation