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 plectin's association with NMJs led to the accumulation and anchorage of IF networks at the synapses, we performed costaining of AChRs and desmin IFs. Consistent with our observations made with ex vivo–differentiated myotubes (Figure 2A), en face confocal imaging revealed dense desmin IF networks juxtaposed to the NMJ in wt muscle, whereas in Pax7-Cre/cKO muscle, desmin IFs were detached from the synapse and showed collapse around synaptic nuclei (Figure 9A). Three-dimensional reconstructions of confocal Z-stack images of muscle fibers costained for desmin, AChRs, and synaptophysin provided clear evidence for the retraction of desmin IFs from NMJs in plectin-deficient muscle (Figure 9B and Supplemental Videos S7 and S8). Similarly, cytokeratins, a second, although in muscle fibers less abundantly expressed IF type (Ursitti et al., 2004), showed accumulation around NMJs in wt but not Plec−/− muscle fibers (Figure 9C). Similarly, using antibodies to nestin, an IF protein that is specifically expressed at NMJs and myotendinous junctions of adult skeletal muscle fibers (Carlsson et al., 1999), showed that nestin-positive filaments were separated from the postsynaptic apparatus in plectin-negative fibers (Figure 9D). With the use of vimentin-specific antibodies, in neither wt nor Plec−/− soleus muscle fibers were positive filaments observed (Figure 9E).
Affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria.