C.O.1 Role of Amphiphysin-2 and N-WASP in nuclear positioning and T-tubule formation during myofiber differentiation

Abstract

Centronuclear myopathies (CNM) are congenital muscle disorders, characterized morphologically by small fibers with centrally-positioned nuclei and abnormal T-tubules. Although several mutations in Myotubularin, Dynamin-2 and Amphiphysin-2 and have been found in patients and studied in different cellular and mouse models, the molecular mechanism causing the pathology is unknown. In particular, the cytoskeletal anomalies leading to the mispositioning of nuclei within the fibers and T-tubules structure changes remain elusive. 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 have set up a 3D-like cellular model, in which agrin-stimulated primary myotubes, differentiate to form mature myofibers with peripheral nuclei and T-tubules. RNAi against Amphiphipysin-2 as well as microinjection of a mutated form associated with CNM prevented peripherally localization of nuclei and T-tubule structure was affected, thus replicating the hallmarks of CNM. Moreover, using the same model, we found an interaction between Amphiphysin 2 and N-WASP, an activator of the Arp2/3 complex, an actin nucleator. In addition, N-WASP silencing also lead to centrally located nuclei and inhibition of T-tubules formation. Furthermore, we observed, for the first time, that N-WASP co-localized with Amphiphysin 2 in T-tubules in healthy human and mouse muscles. Whether either Amphiphysin 2 or N-WASP are silenced in adult mouse muscle, by siRNA electroporation, T-tubules structure is strongly compromised, representing the earliest hallmark of centronuclear myopathies. This data sheds 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.