Skeletal muscle cells are multinucleate, and improper positioning of the nuclei contributes to muscle dysfunction. Skeletal-muscle cells contain large numbers of nuclei, positioned at regular intervals along the muscle fibre. Improperly positioned nuclei are a hallmark of certain muscle diseases. In this study, Mary Baylies and colleagues identify the microtubule-associated protein Ens/MAP7 and Khc/Kif5b as essential, evolutionarily conserved regulators of myonuclear positioning in Drosophila and cultured mammalian myotubes. Drosophila ens mutant larvae display decreased locomotion and incorrect myonuclear positioning, and these phenotypes are rescued by muscle-specific expression of Ens. This work confirms that correct nuclear positioning is important for muscle function. It also provides a system with which to identify further nuclear-positioning genes and assess their function, and to screen for drugs designed to alleviate muscle weakness in disease. The basic unit of skeletal muscle in all metazoans is the multinucleate myofibre, within which individual nuclei are regularly positioned1. The molecular machinery responsible for myonuclear positioning is not known. Improperly positioned nuclei are a hallmark of numerous diseases of muscle2, including centronuclear myopathies3, but it is unclear whether correct nuclear positioning is necessary for muscle function. Here we identify the microtubule-associated protein ensconsin (Ens)/microtubule-associated protein 7 (MAP7) and kinesin heavy chain (Khc)/Kif5b as essential, evolutionarily conserved regulators of myonuclear positioning in Drosophila and cultured mammalian myotubes. We find that these proteins interact physically and that expression of the Kif5b motor domain fused to the MAP7 microtubule-binding domain rescues nuclear positioning defects in MAP7-depleted cells. This suggests that MAP7 links Kif5b to the microtubule cytoskeleton to promote nuclear positioning. Finally, we show that myonuclear positioning is physiologically important. Drosophila ens mutant larvae have decreased locomotion and incorrect myonuclear positioning, and these phenotypes are rescued by muscle-specific expression of Ens. We conclude that improper nuclear positioning contributes to muscle dysfunction in a cell-autonomous fashion.

The activation of the cyclin-depdndent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates β-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of β-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-β3-tubulin^S172D/Emutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to β-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.

Nuclear movement and positioning within cells has become an area of great interest in the past few years due to the identification of different molecular mechanisms and functions in distinct organisms and contexts. One extreme example occurs during skeletal muscle development and regeneration. Skeletal muscles are composed of individual multinucleated myofibers with nuclei positioned at their periphery. Myofibers are formed by fusion of mononucleated myoblasts and during their development, successive nuclear movements and positioning events have been described. The position of the nuclei in myofibers is important for muscle function. Interestingly, during muscle regeneration and in some muscular diseases, nuclei are positioned in the center of the myofiber. In this review, we discuss the multiple mechanisms of nuclear positioning that occur during myofiber formation and regeneration. We also discuss the role of nuclear positioning for skeletal muscle function.

Mutations in amphiphysin‐2/BIN1, dynamin 2, and myotubularin are associated with centronuclear myopathy (CNM), a muscle disorder characterized by myofibers with atypical central nuclear positioning and abnormal triads. Mis‐splicing of amphiphysin‐2/BIN1 is also associated with myotonic dystrophy that shares histopathological hallmarks with CNM. How amphiphysin‐2 orchestrates nuclear positioning and triad organization and how CNM‐associated mutations lead to muscle dysfunction remains elusive. We find that N‐WASP interacts with amphiphysin‐2 in myofibers and that this interaction and N‐WASP distribution are disrupted by amphiphysin‐2 CNM mutations. We establish that N‐WASP functions downstream of amphiphysin‐2 to drive peripheral nuclear positioning and triad organization during myofiber formation. Peripheral nuclear positioning requires microtubule/Map7/Kif5b‐dependent distribution of nuclei along the myofiber and is driven by actin and nesprins. In adult myofibers, N‐WASP and amphiphysin‐2 are only involved in the maintenance of triad organization but not in the maintenance of peripheral nuclear positioning. Importantly, we confirmed that N‐WASP distribution is disrupted in CNM and myotonic dystrophy patients. Our results support a role for N‐WASP in amphiphysin‐2‐dependent nuclear positioning and triad organization and in CNM and myotonic dystrophy pathophysiology.