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Nuclear Alignment in Myotubes Requires Centrosome Proteins Recruited by Nesprin-1

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Nuclear Alignment in Myotubes Requires Centrosome Proteins Recruited by Nesprin-1 © 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 4227-4237 doi:10.1242/jcs.191767 RESEARCH ARTICLE Nuclear alignment in myotubes requires centrosome proteins recruited by nesprin-1 Aude Espigat-Georger, Vyacheslav Dyachuk*, Cécile Chemin, Laurent Emorine and Andreas Merdes‡,§ ABSTRACT through N-terminal calponin homology or plectin-binding domains Myotubes are syncytial cells generated by fusion of myoblasts. and spectrin repeats. At least four nesprin proteins exist in humans Among the numerous nuclei in myotubes of skeletal muscle fibres, and mice (nesprins 1, 2, 3 and 4), of which nesprin-4 is specifically the majority are equidistantly positioned at the periphery, except for expressed only in secretory epithelia (Zhang et al., 2001; clusters of multiple nuclei underneath the motor endplate. The correct Wilhelmsen et al., 2005; Roux et al., 2009). positioning of nuclei is thought to be important for muscle function and In muscle cells, nesprin-1 has been implicated in nuclear requires nesprin-1 (also known as SYNE1), a protein of the nuclear positioning in the syncytial cytoplasm and in clustering a subset envelope. Consistent with this, mice lacking functional nesprin-1 of synaptic nuclei at the neuromuscular junction (Grady et al., show defective nuclear positioning and present aspects of 2005; Zhang et al., 2007b, 2010; Lei et al., 2009). Consistent with Emery–Dreifuss muscular dystrophy. In this study, we perform this, nesprin-1 is particularly enriched on nuclei beneath the small interfering RNA (siRNA) experiments in C2C12 myoblasts neuromuscular junction (Apel et al., 2000), as well as localizing less undergoing differentiation, demonstrating that the positioning of strongly to non-synaptic nuclei. Interference with the function or the nuclei requires PCM-1, a protein of the centrosome that relocalizes localization of nesprin-1 abolishes correct myonuclear anchorage to the nuclear envelope at the onset of differentiation in a manner that and leads to respiratory failure and death in mice (Zhang et al., is dependent on the presence of nesprin-1. PCM-1 itself is required 2007b, 2010; Puckelwartz et al., 2009). However, it remains unclear for recruiting proteins of the dynein–dynactin complex and of kinesin by what mechanism nesprin-1 mediates nuclear anchorage. In motor complexes. This suggests that microtubule motors that are various studies on non-muscular cells, nesprins have been implied attached to the nuclear envelope support the movement of nuclei to interact with the actin cytoskeleton (Starr and Han, 2002; Zhen along microtubules, to ensure their correct positioning in the myotube. et al., 2002; Padmakumar et al., 2004). In addition, nesprins and related proteins have been shown to interact with components of the KEY WORDS: Centrosome protein, Myotube, Nesprin, Nuclear microtubule network, such as kinesin, dynein and dynactin, and positioning with the centrosome (Malone et al., 2003; Roux et al., 2009; Zhang et al., 2009; Zhou et al., 2009; Fridolfsson et al., 2010; Yu et al., INTRODUCTION 2011; Wilson and Holzbaur, 2012, 2015). Several groups have Muscle dysfunctions in several diseases have been correlated with reported that proteins of the centrosome, such as PCM-1, pericentrin mutations in nuclear envelope genes, such as emerin, lamin A, and and γ-tubulin, are relocated from the pericentriolar material to the nesprins 1 and 2 (nesprins 1–4 are also known as SYNE1–SYNE4, nuclear envelope upon onset of myoblast differentiation, and that a respectively). In particular, mutations in these proteins have been substantial amount of microtubules grow from the nuclear surface reported in human patients and in mouse models with Emery– following this reorganization (Tassin et al., 1985a; Bugnard et al., Dreifuss muscular dystrophy, as well as ataxias and 2005; Srsen et al., 2009; Fant et al., 2009). In this study, we show cardiomyopathies (Ellis, 2006; Gros-Louis et al., 2007; Wheeler that nesprin-1 is essential for the relocalization of centrosome et al., 2007; Zhang et al., 2007a; Attali et al., 2009; Puckelwartz proteins and components of microtubule motor complexes to the et al., 2009, 2010). It has been speculated that these mutations might nuclear envelope in differentiating mouse myoblasts, and that affect signalling pathways in muscle cells, but the exact role of nesprin-1 and the centrosome protein PCM-1 are needed for regular nuclear envelope components in myopathies remains unclear. positioning of nuclei in these cells. Whereas emerin and lamins are proteins that associate with the inner nuclear membrane, the nesprins are transmembrane proteins that RESULTS span a wide distance from the perinuclear space to the cytoplasm. In Nesprin-1 expression correlates with the recruitment of the perinuclear space, nesprins are anchored to proteins of the SUN pericentriolar proteins at the nuclear envelope family by their conserved C-terminal KASH domain, whereas in the To identify factors that recruit centrosome proteins to the surface of cytoplasm, they are believed to interact with the cytoskeleton the nuclear envelope during myoblast differentiation, we investigated the potential role of proteins of the outer nuclear membrane. To date, Centre de Biologie du Développement, UniversitéPaul Sabatier/CNRS, Toulouse the only known proteins specifically localized at the outer nuclear 31062, France. membrane are members of the KASH family; in mouse muscle cells, *Present address: Department of Neuroscience, Karolinska Institutet, Stockholm these include nesprins 1, 2 and 3 (Hetzer, 2010). 17177, Sweden. ‡Present address: National Scientific Center of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences, Vladivostok 690041, Russia. We reinvestigated the localization of nesprins in C2C12 cells, given that conflicting reports on the localization of nesprin-1 and -2 § Author for correspondence ([email protected]) in skeletal muscle cells have been published (Apel et al., 2000; A.E., 0000-0001-8801-2951; A.M., 0000-0002-3739-2728 Zhang et al., 2001, 2005; Mislow et al., 2002). Because nesprins exist as alternative splice variants, we performed immunofluorescence and Received 3 May 2016; Accepted 26 September 2016 western blot experiments with antibodies against the C-terminal Journal of Cell Science 4227 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4227-4237 doi:10.1242/jcs.191767 KASH domain that is present in nuclear-envelope-bound isoforms. sequences found in a large variety of possible splice variants of In undifferentiated C2C12 myoblasts, nesprin-2 and nesprin-3 were nesprin-1 or nesprin-2, we obtained strong signals for nesprin-1 only expressed at substantial levels and localized to the nuclear envelope, upon differentiation of C2C12 into myotubes, in contrast to nesprin- whereas the expression of nesprin-1 was very weak (Fig. 1A,B). 2, for which levels dropped in differentiating cells (Fig. 1C). Upon differentiation into myotubes, the nesprin-1 signal increased and became prominent at the nuclear envelope, while at the same Nesprin-1 but not nesprin-3 is needed for the recruitment of time the centrosome protein pericentrin relocalized to the nuclear pericentriolar proteins to the nuclear envelope surface. The accumulation of nesprin-1 at the nuclear envelope Because nesprin-2 was found to be absent from the nuclear envelope correlated with the appearance of a 115-kDa band on western blots, in myotubes, we tested whether nesprins 1 or 3 are involved in the corresponding to the nesprin-1α isoform (Fig. 1B), which is the recruitment of centrosome proteins to the nuclear envelope in prominent isoform expressed in human muscle cells (Randles et al., differentiating myoblasts. We depleted up to 80% of nesprin-1α or 2010). Concomitantly, the nesprin-2 signal disappeared from the nesprin-3 protein by siRNA treatment of C2C12 cells (Fig. 2A,B). nuclear envelope, whereas nesprin-3 remained (Fig. 1A). This As a consequence, the recruitment of pericentrin to the nuclear pattern of nesprin localization at the nuclear envelope was confirmed envelope was inhibited in nesprin-1-depleted cells that had fused using two different sets of antibodies against nesprins 1 and 2 into myotubes (Fig. 2C). Interestingly, numerous myotubes had (Fig. S1A,B). Depending on the antibody used, nesprin-2 nesprin-1-positive as well as nesprin-1-negative nuclei in the same immunofluorescence in C2C12 myotubes was seen in intranuclear cytoplasm. This was likely due to the fact that nesprin-1 depletion structures as previously described (Zhang et al., 2005) or was diffuse had already started prior to fusion of C2C12 cells, enabling non- in the cytoplasm (Fig. 1A, Fig. S1B). The nature of the intranuclear depleted cells to fuse with depleted cells. Thus, in heterogeneous structures is unknown, but RNA interference experiments suggest myotubes, only nuclear envelopes with a high level of nesprin-1 that these might represent unspecific targets of the antibody also displayed enriched amounts of pericentrin (Fig. 2C, asterisk). (Fig. S1B). Given that immunoblotting against nesprin-2 failed Given that the pericentrin levels at the nuclear envelope varied to provide specific bands, we examined expression levels of between myotubes, we quantified the immunofluorescence nesprin-2 in comparison to those of nesprin-1 by performing intensity within a perinuclear rim of ∼0.5 µm thickness. For these reverse transcription from C2C12 RNA obtained prior to and measurements, we analysed myotubes
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