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 investigate whether the observed weaker association of AChR complexes with the underlying cytoskeleton in plectin-deficient myotubes was due to the detachment of desmin IFs and/or actin from the complexes, we performed costainings of Plec−/− and Plec+/+ myotubes, using Alexa 488–α-BTX for visualization of AChRs and antibodies to either desmin or actin. Confocal imaging revealed dense desmin IF networks juxtaposed to the AChRs in Plec+/+ myotubes, whereas in plectin-deficient myotubes, desmin IFs were detached from the AChR clusters and collapsed into massive aggregates (Figure 2A). In contrast, plectin-positive and -negative myotubes showed no differences in AChR association with the actin network (Figure 2A). To assess in a more direct way whether the association of IF network with AChRs had an effect on the formation and/or stability of clusters, we monitored agrin-induced clustering after disruption of desmin IF networks in immortalized Plec+/+ myotubes using okadaic acid (OA). OA, a potent phosphatase inhibitor, was previously shown to cause IF collapse in a number of cell types, including fibroblasts (Gregor et al., 2014) and keratinocytes (Strnad et al., 2002; Osmanagic-Myers et al., 2006). As shown in Figure 2, B and C, OA treatment of Plec+/+ myotubes led to a dramatic reduction in the number and density of agrin-induced clusters to levels even below those of plectin-deficient myotubes. To establish that OA-effected abnormal clustering was directly linked to IF retraction from the receptors and did not represent just a secondary effect of OA-induced hyperphosphorylation, we investigated receptor clustering upon treatment with withaferin A (WFA), a small molecule that covalently modifies the highly conserved α-helical coiled-coil 2B domain of type III IFs, leading to their aggregation in vitro and in vivo (Bargagna-Mohan et al., 2007). As shown in Figure 2, B and C, WFA affected AChR clustering in a way very similar to OA. Furthermore, to directly interfere with plectin's IF-anchoring function, we transfected into Plec+/+ myoblasts a cDNA expression plasmid encoding a defective plectin (plectin isoform 1f [P1f]–16-base pair insertion mutation [Ins16]) that carried a mutation near its IF-binding domain (IFBD); this mutation had previously been found to cause a human plectinopathy characterized by a severely disorganized desmin filament network (Schröder et al., 2002). The analysis of myotubes derived from such cells clearly revealed a retraction of the desmin IF network from the periphery of the cell and its collapse in interior regions (unpublished data). A quantitative analysis revealed a numerical reduction (Figure 2B), as well as a diminished density, of AChR clusters (Figure 2C). Overall the effects of all these treatments (OA, WFA, and forced expression of mutant plectin) on AChR clustering were quantitatively very similar, strongly suggesting that IF network collapse was indeed responsible for the diminished frequency and density of the receptor clusters (Figure 2, B and C).
Affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria.