Mechanisms of non-centrosomal MTOC formation at the nucleus in muscle cells

The accurate position of the nucleus within a cell is important for many biological processes such as cell migration or differentiation and relies on the connections between the nuclear envelope (NE) and the cytoskeleton. Nuclear positioning is particularly controlled during skeletal muscle formation, when post-mitotic myoblasts fuse to form multinucleated myotubes and when myotubes further differentiate into mature myofibers. Defects in nuclear positioning were shown to affect muscle function and are linked to many muscle diseases, such as Centronuclear Myopathies (CNM) and Emery- Dreifuss Muscular Dystrophy (EDMD). Nuclear movement during myogenic differentiation is highly dependent on microtubules (MTs) which adopt a unique organization during the muscle differentiation process. In comparison to proliferating myoblasts, where MTs are radially nucleated from the centrosome, MTs are nucleated from the NE in dense bundles parallel to the cell axis in differentiated myotubes. This dramatic reorganization of the MT network during muscle development is accompanied by a redistribution of the Golgi complex and proteins from the centrosome (e.g. Pericentrin, Pcm1, Akap450) to the NE. Thus, the nucleus takes over the function as a non-centrosomal microtubule- organizing center (MTOC) during myogenic differentiation. However, the molecular mechanisms underlying centrosomal protein recruitment and anchoring to the NE as well as their capacity to organize MTs are still unknown. Here, we performed a siRNA screen against 299 genes encoding NE-transmembrane or NE- associated proteins and identified Nesprin-1, a KASH (Klarsicht/ANC-1/SYNE homology) domain protein located at the outer nuclear membrane (ONM), involved in the recruitment of Pericentrin to the NE during myogenic differentiation. In C2C12 myotubes depleted for Nesprin-1 or in myotubes from a congenital muscular dystrophy patient carrying a nonsense mutation within the Nesprin-1/SYNE1 (23560 G&gt;T) gene, several centrosomal proteins, including Pericentrin, Akap450, Pcm1, and Cdk5Rap2, are mislocalized in the cytoplasm in close proximity to mislocalized Golgi fragments. During skeletal muscle formation, a muscle-specific isoform of Nesprin-1, namely Nesprin-1alpha, is increasingly expressed. Using the proximity-dependent biotin identification method, BioID, we could show that Nesprin-1alpha is highly associated with Akap450, Pericentrin and Pcm1 in C2C12 myotubes. Moreover, re-expression of Nesprin-1alpha in Nesprin-1-depleted cells is sufficient to rescue the observed recruitment defects, thus confirming Nesprin-1alpha as the major receptor at the NE to anchor centrosomal proteins. However, Nesprin-1alpha together with Sun1/Sun2 are not only important for recruiting centrosomal proteins but also for MTOC activity. MTs are nucleated from the NE in differentiated muscle cells, whereas MTs grow from cytoplasmic foci containing mislocalized Golgi fragments and centrosomal proteins in Nesprin-1- or Sun1/Sun2-depleted cells. Among the recruited centrosomal proteins, solely Akap450 seems to be required for MT nucleation from the NE. Using computer simulations and knockdown experiments, we demonstrate that Akap450-Nesprin- 1alpha-mediated MT nucleation from the nucleus has a biological relevance for nuclear positioning during myotube formation. Taken together, our data suggest a model where Nesprin-1alpha, when upregulated during myogenic differentiation, forms a complex with Sun1/Sun2 and thereby anchors centrosomal proteins at the nucleus. In turn, Akap450 plays a key role in inducing MT nucleation, thereby assuring proper nuclear positioning during skeletal muscle formation. These findings strengthen our understanding on how non-centrosomal MTOCs form in muscle and how defects within the underlying mechanism can potentially contribute to nuclear positioning defects in muscular dystrophies.

Topic: Nuclear Structure and Function
Subfield: Molecular Biology
Field: Biochemistry, Genetics and Molecular Biology
Domain: Life Sciences