Microtubule motors involved in nuclear movement during skeletal muscle differentiation

Nuclear positioning is a determining event in several cellular processes, such as fertilization, cell migration, and cell differentiation. The structure and function of muscle cells, which contain hundreds of nuclei, have been shown to rely in part on proper nuclear positioning. Remarkably, in the course of muscle differentiation, nuclear movements along the myotube axis might represent the event required for the even positioning of nuclei in the mature myofiber. Here we analyze nuclear behavior, time in motion, speed, and alignment during myotube differentiation and temporal interference of cytoskeletal microtubule-related motors. Using specific inhibitors, we find that nuclear movement and alignment are microtubule dependent, with 19 microtubule motor proteins implicated in at least one nuclear behavior. We further focus on Kif1c, Kif5b, kif9, kif21b, and Kif1a, which affect nuclear alignment. These results emphasize the different roles of molecular motors in particular mechanisms. INTRODUCTION The skeletal muscle fiber is a syncytium resulting from the fusion of hundreds of myoblasts (Abmayr and Pavlath, 2012). Muscle cells represent the main vertebrate cell type and are specialized to contract, and thus are responsible for voluntary movements. During muscle development or regeneration, nuclei are first found in the center of myotubes and then migrate to the periphery of mature skeletal myofibers (Bruusgaard et al., 2003). Proper nuclear positioning is required for muscle function (Metzger et al., 2012), and nuclear mispositioning is a hallmark of different muscle disorders, such as centronuclear myopathies (Jungbluth et al., 2008). Multiple nuclear movement events occur during myofiber development to localize precisely each individual nucleus (Cadot et al., 2012; Metzger et al., 2012; Wilson and Holzbaur, 2012, 2015; Falcone et al., 2014). The first nuclear migration event in muscle development takes place just after fusion, when the nucleus from a fusing myoblast rapidly migrates toward the center of the myotube. This process is called centration (Cadot et al., 2012). Second, nuclei spread to become evenly spaced along the length of developing myotubes (Metzger et al., 2012), a step called spreading. These two events were proposed to rely on the microtubule (MT) network and on some motor proteins. Finally, as myotubes mature into myofibers, nuclei migrate from the center to the periphery of the fiber by a mechanism dependent on NWasp, an actin regulator, and amphiphysin-2, a BAR protein mutated in centronuclear myopathies. This nuclear migration is termed dispersion (Falcone et al., 2014). Nuclear positioning in cells appears to be a key mechanism required for multiple developmental processes in order to maintain functionally efficient muscle or brain (Dauer and Worman, 2009). These processes are described as being driven by MTs or actin (Gundersen and Worman, 2013). Even though recent advances in understanding nuclei position in developing muscle cells reveal an important role of MTs and some MT motors (kinesins and dyneins; Cadot et al., 2012; Metzger et al., 2012; Wilson and Holzbaur, 2012), the contribution of other MT motors to nuclei position during myofiber formation remains unexplored. Monitoring Editor Laurent Blanchoin CEA Grenoble Received: Jun 14, 2016 Revised: Feb 1, 2017 Accepted: Feb 1, 2017 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E16-06-0405) on February 8, 2017. V.G. , E.R. G. , and B.C. conceived and designed the experiments; V.G. and B.C. performed the experiments; V.G. and B.C. analyzed the data; V.G. , E.R. G. , and B.C. drafted the article; B.C. prepared the digital images. †Present address: Institut NeuroMyogène, CNRS UMR 5310, INSERM U1217, Université de Lyon, Ecole Normale Supérieure de Lyon, 69364 Lyon Cedex 07, France. *Address correspondence to: E. R. Gomes (edgargomes@medicina.ulisboa.pt), B. Cadot (bruno.cadot@upmc.fr). © 2017 Gache et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: GFP, green fluorescent protein; MT, microtubules; siRNA, small interfering RNA. V. Gachea,†, E. R. Gomesa,b,*, and B. Cadota,* aCenter of Research in Myology, INSERM UPMC UMR974, Centre National de la Recherche Scientifique, FRE3617, 75013 Paris, France; bInstituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisbon, Portugal MBoC | BRIEF REPORT