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 study the molecular mechanism behind plectin's contribution to NMJ integrity, we first investigated cluster formation of AChRs in myotubes that had been differentiated ex vivo from immortalized plectin-positive (Plec+/+) and plectin-deficient (Plec−/−) myoblast cell lines (Winter et al., 2014). Plectin-deficient myotubes, like their normal counterparts, are multinucleated and show signs of full differentiation, such as spontaneous contraction (Winter et al., 2014). In addition, Plec−/− myotubes closely mimic the pathology of EBS-MD myofibers, including the development of desmin-positive protein aggregates (Winter et al., 2014). To induce AChR clustering, myotubes were exposed to recombinant neural agrin, and AChRs were visualized by Alexa 488–α-bungarotoxin (BTX) labeling (Figure 1A). A morphometric analysis revealed that in plectin-deficient myotubes, the formation of large clusters (≥5 μm2 in size) was strikingly reduced compared to wild-type (wt) cells (Figure 1C). Moreover, such clusters showed reduced fluorescence intensity compared with corresponding ones in plectin-positive myotubes (Figure 1B). On the other hand, in plectin-deficient myotubes, we observed a significantly increased number of (micro) clusters of sizes <5 μm2 (Figure 1D). To test the responsiveness of Plec−/− myotubes to agrin-induced AChR-clustering, we quantified large clusters at different time points after initiating the treatment. In contrast to Plec+/+ myotubes, where the number of AChR clusters increased consistently with time (Figure 1E), no increase was observed in Plec−/− myotubes. In control experiments in which AChR expression levels in myoblasts and myotubes were assessed by immunoblotting, no differences were observed between Plec+/+ and Plec−/− cells, regardless of whether or not they had been treated with agrin (Supplemental Figure S1).
Affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria.