Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn-plectin 1f complexes.
Bottom Line: Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells.In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span.Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion.
Affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria.Show MeSH
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Mentions: To assess whether AChR cluster formation and stabilization in cells were directly linked to plectin and especially to P1f, the isoform specifically associated with NMJs (Supplemental Figure S2B), we performed rescue experiments in which Plec−/− myoblasts were transiently transfected with cDNA expression plasmids encoding fusion proteins of GFP and various intact and mutated isoforms of plectin. We found that after transfection of P1f-GFP expression plasmids into myoblasts and their differentiation to myotubes, not only was P1f-GFP primarily localized at the sarcolemma of the cells, showing a specific enrichment at AChR clusters (Figure 4A), but, remarkably, it also reestablished desmin IF network association with the receptors (Figure 4A). A quantitative analysis revealed that the number of AChR clusters was significantly increased compared with nontransfected Plec−/− myotubes, approaching the level detected in plectin-positive myotubes (Figure 4B). In contrast, when nuclear/ER membrane–associated isoform P1 (P1-GFP) and Z-disk–associated isoform P1d (P1d-GFP) were expressed, neither of them colocalized with the receptor complex, nor did they reestablish desmin IF network accumulation in defined sarcolemmal areas, indicating that they failed to promote AChR cluster formation (Figure 4, A and B). Similarly, when the experiments were performed with plasmids encoding IF-binding–incompetent P1f (P1f-Ins16), neither reanchoring of IF networks at AChR clusters nor improved receptor clustering was observed in myotubes (Figure 4, A and B). On the other hand, a significant increase in the number of AChR clusters, paralleled by desmin reanchorage, was observed upon forced expression of P1f-rodless, a P1f version lacking the rod but containing the other domains of the plectin molecule, including its IFBD (Figure 4, A and B). Codistribution of P1f with AChR clusters was observed also upon its overexpression in wt myotubes (Figure 4A). When the compactness of receptor clusters in P1f-reconstituted versus unreconstituted myotubes was evaluated by quantifying the fluorescence intensities of Cy5–α-BTX–labeled clusters, only the expression constructs that encoded full-length or rodless P1f were found to effect the formation of more compact receptor clusters, whereas those encoding isoforms P1 or P1d or P1f-Ins16 were not (Figure 4C).
Affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria.