myotubes where Z-bodies, actin thin filaments and non-muscle myosin II (NMIIB) proteins form premyofibrils, which mature into myofibrils with the myosin isoform changed into muscle myosin II in the shaft of myotubes

INTRODUCTION Although significant progress has been made in understanding the structure of skeletal muscle, the molecular mechanisms by which morphological rearrangements occur during myogenesis are less clear. Microtubules and actin filaments have been shown to be active in generating the spatial organization in differentiating muscle cells (Antin et al., 1981; Holtzer et al., 1975; Siebrands et al., 2004; Toyama et al., 1982). A panel of kinesin-related motor proteins has been suggested to be involved, including Kif1b/c (Dorner et al., 1998; Faire et al., 1998), Kif3a/b/c (Ginkel and Wordeman, 2000) and Kif5b (Rahkila et al., 1997). Among them, Kif5b, the ubiquitously expressed isoform of kinesin-1 heavy chain, is abundant in intestine, heart and skeletal muscle (Hollenbeck, 1989). A study based on physical training showed that variation in the KIF5B sequence could affect the ability of the heart to adjust to regular exercise in humans, indicating a potential role of kinesin-1 in cardiac muscle capability (Argyropoulos et al., 2009). One of the possible reasons for the kinesin-1-associated phenotype in muscle is thought to be related to the mitochondria, as Kif5b is responsible for anterograde mitochondrial movement by interacting with the mitochondrial protein Miro through the linker protein Milton (Wang and Schwarz, 2009). Another possible reason might be the Kif5b-dependent localization of nuclei, which is important for muscle activity. As demonstrated in a recent study, Kif5b directly interacts with Map7 to regulate the positioning of nuclei in differentiating myotubes (Metzger et al., 2012). Kinesin1 has also been shown to interact with nesprin 4 through kinesin light chain (KLC) to regulate the localization of nuclei in several cell types (Roux et al., 2009), but whether this occurs in myogenesis needs further investigation. Immunostaining against kinesin heavy chain (KHC) showed that it might also function in ER-Golgi trafficking in skeletal muscles (Rahkila et al., 1997). However, the function of Kif5b in myofibrillogenesis and myotendinous junction (MTJ) formation has not been investigated. During myofibrillogenesis, the myofibril components are transported to their appropriate sites for myofibril assembly. As suggested by Sanger et al., assembly begins at the edges of myotubes where Z-bodies, actin thin filaments and non-muscle myosin II (NMIIB) proteins form premyofibrils, which mature into myofibrils with the myosin isoform changed into muscle myosin II in the shaft of myotubes (reviewed by Sanger et al., 2005). However, the spatial control of the myofibrillogenesis has not been investigated. During myogenesis, the intermediate filament (IF) proteins desmin and nestin are also transported to the growing tips of the myotubes (Towler et al., 2004), where they represent the sites of the myotendinous junctions after maturation. In parallel to the MTJ complex, which includes integrins, talin and vinculin, IF proteins form physical links between the sarcomeric units and the sarcolemma (Carlsson et al., 1999; Tidball, 1992). Conventional kinesin has been suggested to play a role in transportation of vimentin in fibroblasts (Kreitzer et al., 1999; Prahlad et al., 1998), and neurofilament subunits in neural cells (Xia et al., 2003; Yabe et al., 1999). However, whether kinesin-1 is involved in the transportation of desmin and nestin during myogenesis has never been investigated. To understand the role of Kif5b in muscle development and function, we generated mice with Kif5b conditionally knocked out in skeletal muscles. The mutant mice showed severe muscle dystrophy, exemplified by impaired myofibril assembly and their linkage to the myotendinous junctions, with myofibril components and IF proteins aggregated in the cell body. In vitro-differentiated myoblasts with Kif5b deficiency reproduced the in vivo phenotypes, and further demonstrated a role of Kif5b in anterograde transport of -sarcomeric actin and NMIIB, together with the IF proteins desmin and nestin during myogenesis. Detailed mapping revealed that a 64-amino acid -helix domain of Kif5b 1Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong. 2Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing, China. 3Beijing Institute of Geriatrics, Beijing Hospital, Ministry of Health, Beijing, China. 4Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong. 5HKU-Pasteur Research Centre, The University of Hong Kong, Hong Kong.