Résumé :
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Centronuclear myopathies (CNMs) are congenital muscle disorders, described for the first time in 1966 by A. Spiro, characterized morphologically by small fibers with centrally-positioned nuclei. Although, at present, mutations in at least three genes (Myotubularin I, Amphiphysin II/Bin1 and Dynamin-2) have been found in patients little is known about the detailed mechanism causing the pathology. In particular, the cytoskeletal anomalies leading to the mis-positiong of nuclei within the fibers remain elusive, even though no muscle degeneration is associated with these diseases. The relevance of a physiologic model, where it would be possible to follow the nuclear positioning process and manipulate protein expression and function, is glaring. Bearing this in mind, we set up a 3D-like cellular model, in which agrin-stimulated primary myotubes differentiate to form mature myofibers with peripheral nuclei and patterned T-tubules. RNAi against Amphiphysin II (AmphII), MTM-1 or DNM2, as well as microinjection of CNM-associated mutated forms of AmphII caused nuclei become centrally located and disorganization of T-tubules, thus replicating the hallmarks of CNMs. Moreover, using the same model, we found an interaction between AmphII and N-WASP, an activator of the Arp2/3 complex that serves as an actin nucleator. Furthermore, we found that N-Wasp co-localizes with AmphII in T-tubules in muscle. In addition, siRNA against N-WASP also led to centrally located nuclei and inhibition of T-tubule formation. These data shed light on new candidate molecules implicated in the CNM disease and, more broadly, on the pathways leading to nuclear positioning and t-tubule formation during muscle maturation.
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