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

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

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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 that contained both nesprin- after differentiation, followed by quantitative PCR (qPCR). Using 1-positive and -negative nuclei. Nuclei that were depleted of PCR primers complementary to the spectrin-repeat-containing nesprin-1 showed only 37±15% (mean±s.d., n=20) of the

Fig. 1. Localization of nesprins during C2C12 differentiation. (A) Immunolocalization of nesprin-1 (rabbit anti-nesprin-1), nesprin-2, nesprin-3 (green) and pericentrin (red) in proliferating myoblasts and in differentiated myotubes. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). The dashed white lines indicate the edge of the myotube. Scale bar: 10 µm. (B) Immunoblots of cell lysates from C2C12 myoblasts (myobl.) and myotubes, stained with rabbit anti-nesprin-1, mouse anti-nesprin-1 (monoclonal ‘MANNES1A’) and mouse anti-nesprin-3 antibodies. As a loading control, α-tubulin was probed. The position of protein molecular mass markers (in kDa) for both nesprins 1 and 3 is indicated on the left. (C) qPCR of nesprin-1 and nesprin-2 cDNA, obtained by reverse transcription of total RNA from C2C12 myoblasts and myotubes. Levels of nesprin-1 and nesprin-2 (mean±s.d., n=2) are displayed relative to those obtained by qPCR of housekeeping cDNAs (ribosomal protein S16 and hypoxanthine guanine phosphoribosyl transferase), calculated by Biorad CFX Manager software.

Nesprin-1 levels obtained from myotubes were set to 1 for normalization. Journal of Cell Science

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Fig. 2. Nesprin-1 is essential for the recruitment of centrosome proteins to the nuclear envelope in differentiated C2C12 cells. (A,B) Immunoblots of cell lysates from C2C12 myotubes, treated with control RNA, or with siRNA against nesprin-1 or nesprin-3. Blots were probed with antibodies against nesprin-1, nesprin-3, PCM-1 and α-tubulin as indicated. (C,D) Pericentrin localization (red) in control C2C12 myotubes (top row), and in (C) nesprin-1-depleted or (D) nesprin-3-depleted myotubes. Green, nesprin-1 or nesprin-3; blue, DNA. The asterisks in the bottom rows of C and D indicate nuclei of non-depleted myoblasts that have fused into myotubes with nesprin-1-depleted or nesprin-3- depleted myoblasts, respectively. (E,F) PCM-1 localization (red) in control or nesprin-1-depleted (E) or nesprin-3- depleted (F) myotubes. Green, nesprin-1 or nesprin-3, respectively; red, PCM-1; blue, DNA. (G) Control C2C12 myotubes, (top row) or myotubes treated with 5 µg/ml Brefeldin A for 90 min (BFA, bottom row). Immunofluorescence of Golgi protein GM130 (green), and pericentrin (red). Blue, DNA. (H) Localization of pericentrin (red) in myotubes treated with control RNA or siRNA against calnexin (green). Blue, DNA. Scale bar: 10 µm.

pericentrin intensity of control nuclei. The effects of nesprin-1 Pericentrin levels on nesprin-3-depleted nuclear envelopes were siRNA could be reproduced with double-stranded RNAs (dsRNAs) 89±20% (n=21) of the level of non-depleted nuclear envelopes. As a against two different targeting sequences (see Materials and control, silencing of nesprin-2 was attempted (see Materials and Methods), thus reducing the risk of unspecific off-target effects. Methods), but was difficult to monitor due to the absence of In contrast to nesprin-1 depletion, the removal of nesprin-3 did not nesprin-2 immunofluorescence in C2C12 myotubes and the substantially alter the amount of nuclear-envelope-bound absence of specific nesprin-2 signal on immunoblots. Treatment pericentrin (Fig. 2D). Quantification of the pericentrin signal was with siRNA against nesprin-2 was without effect on the localization performed in myotubes containing nesprin-3-depleted and of centrosome proteins (Fig. S1B). When investigating the fate of undepleted nuclei (Fig. 2D, asterisk) in the same cytoplasm. centrosome proteins other than pericentrin, we found that depletion Journal of Cell Science

4229 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4227-4237 doi:10.1242/jcs.191767 of nesprin-1 also led to the reduction of PCM-1 at the nuclear seen in non-transfected neighbouring cells. In control transfections, envelope (Fig. 2E), γ-tubulin (see Fig. 4A) and CDK5RAP2 (data we expressed mCherry-tagged tubulin-α or mEmerald-tagged not shown). None of these reductions were seen after depletion of nesprin-3 (Fig. 3H,I). Tagged α-tubulin incorporated into the nesprin-3 (Fig. 2F). We verified by immunoblotting that the microtubule network, but failed to relocalize PCM-1 to the nuclear depletion of nesprin-1 or nesprin-3 did not affect the overall protein envelope (only 2±1% of a total of 1224 cells with nuclear-envelope- levels of centrosome proteins, such as PCM-1 (Fig. 2A,B). To test bound PCM-1, in three different experiments). Likewise, tagged whether the anchoring of centrosome proteins to the nuclear nesprin-3 localized to the nuclear envelope in addition to structures envelope involved proteins of the Golgi or of the endoplasmic resembling the Golgi, but induced relocalization of PCM-1 only in reticulum (ER), we tested the effects of the Golgi-disrupting drug 9±3% of the transfected cells (n=124; Fig. 3H). Moreover, Brefeldin A and siRNA against the ER-resident chaperone calnexin comparison of the ratio of fluorescence intensity of PCM-1 (Fig. 2G,H). Although it is known that Golgi membranes between the nuclear envelope (within a rim of 0.5 µm thickness) accumulate around the nuclear surface upon differentiation of and the neighbouring cytoplasm revealed that nesprin-1 expression muscle cells (Tassin et al., 1985b), and that ER membranes are increased PCM-1 immunofluorescence by a factor of 1.7±0.3 (n=5), continuous with the outer nuclear membrane, neither of these whereas nesprin-3 expression led to an insignificant increase of seemed to be essential for the anchorage of centrosome proteins, 1.1±0.1. Taken together, this suggests that PCM-1 relocalization to because neither Brefeldin A nor calnexin siRNA reduced the the nuclear envelope upon nesprin-1α expression is specific. To accumulation of pericentrin at the nuclear envelope (Fig. 2G,H). exclude that those C2C12 cells displaying PCM-1 at the nuclear Because silencing of nesprin-1 expression prevented the envelope had undergone premature differentiation in culture, we accumulation of centrosome proteins at the nuclear envelope, we performed immunofluorescence for the differentiation marker tested whether displacement of nesprins from the nuclear membrane myosin II heavy chain (MYH1E) (Fig. 3J). We quantified that caused a similar effect. We found that a high overexpression of the only 1% of mCherry–nesprin-1α-expressing cells stained positively nesprin-1 KASH domain displaced endogenous nesprin-1 from the for myosin II heavy chain (in a total of 970 cells), demonstrating that nuclear envelope in myotubes and prevented the relocalization of relocalization of PCM-1 to the nuclear envelope could be induced centrosome proteins to the nuclear envelope (Fig. 3A,B). This was by nesprin-1α, independently of a full differentiation program. also achieved by depletion of the SUN proteins 1 and 2, which localize to the inner nuclear membrane (Fig. 3C,D), and which are Abnormal nuclear positioning in myotubes depleted of known to anchor nesprins to the nuclear envelope (Padmakumar et al., nesprin-1 or PCM-1 2005; Crisp et al., 2006). To test the association between nesprin-1 Because the localization of the microtubule nucleator γ-tubulin was and centrosome proteins biochemically, we performed reduced at nesprin-1-depleted nuclear envelopes in myotubes immunoprecipitation of nesprin-1 from extracts of nuclear envelopes (Fig. 4A, second column, right nucleus), we tested whether the prepared from C2C12 myotube nuclei (see Materials and Methods microtubule network in these cells was altered. Unlike control and Fig. S1C for details). The centrosome protein PCM-1 was found myotubes, nesprin-1-depleted myotubes contained a large number to co-immunoprecipitate under these conditions, whereas γ-tubulin of nuclei (∼70%) that failed to re-grow microtubules efficiently was absent from the precipitate (Fig. 3E). Consistent with this, after recovery from nocodazole-induced depolymerization immunoprecipitation of PCM-1 co-precipitated a portion of the (Fig. 4B), suggesting that the absence of centrosome proteins nesprin-1α pool in the reverse experiment (Fig. 3E). from the nuclear surface prevented microtubule nucleation. To ensure that the depletion of nesprin-1 did not cause unspecific Surprisingly, in cell cultures that had not been subjected to interference with centrosome protein relocalization because of a microtubule depolymerization, both control myotubes and global disorganization of the nuclear envelope, we stained for nesprin-1-depleted myotubes displayed a similar microtubule nuclear pore complexes using monoclonal antibody 414, which network organization (Fig. 4C). To test whether the number of recognizes a subset of nucleoporins (Davis and Blobel, 1986), and microtubules in the vicinity of nuclei were reduced upon nesprin-1 for nesprin-3. Both were still correctly localized in nesprin-1- depletion, we tracked individual microtubules within a distance of depleted myotubes, indicating that the structure of the nuclear ∼1 µm of the nuclear surface, in image stacks of myotubes. Optical envelope was not globally affected (Fig. 3F; Fig. S1D). sections of complete myotubes were grouped by performing sum Taken together, our data suggest that nesprin-1 is specifically projections of partial stacks, allowing the tracking of individual involved in anchoring centrosome proteins to the nuclear envelope microtubules over a large volume (Fig. 4D). We counted ∼600 during myoblast differentiation and myotube formation. To test microtubules close to each nucleus, for controls as well as for whether nesprin-1 is sufficient for the recruitment of centrosome nesprin-1-depleted nuclei (Fig. 4E). Long microtubules, oriented proteins to the nuclear envelope, we expressed exogenous mCherry- parallel to the axis of the myotube, were usually seen curving around tagged nesprin-1α in undifferentiated C2C12 myoblasts, that is, the surface of the nuclei in both conditions, with only 12±2% under conditions that show only minor amounts of endogenous (mean±s.d.; control) compared to 14±1% (nesprin-1 depletion) of nesprin-1 on the nuclear envelope. We observed that the tagged microtubules oriented approximately perpendicular to this axis exogenous nesprin-1α localized to the nuclear envelope even in (Fig. 4F). Analysis of deconvolved 3D data sets of microtubules undifferentiated C2C12 cells (Fig. 3G). Immunofluorescence of the suggested that end-on contacts of microtubules with the nuclear centrosome protein PCM-1 revealed that 47±1% (mean±s.d.) of envelope were rare, although the exact origins and ends of C2C12 cells that incorporated mCherry–nesprin-1α at the nuclear microtubules were difficult to trace. To find out whether there was envelope also showed relocalization of PCM-1 (data from four a substantial number of microtubules emanating from the nuclear different experiments counting a total of 7163 cells). In these cells, surface in myotubes at a steady state (with an established PCM-1 was either entirely recruited to the nuclear surface (top row equilibrium of polymerized versus non-polymerized tubulin), we of Fig. 3G, left), or a portion of the PCM-1 remained in the pool of quantified the immunofluorescence signal intensity of microtubules cytoplasmic pericentriolar satellites (second and third row of in myotube segments containing nuclei (segment 1 in scheme

Fig. 3G, left). PCM-1 accumulation at the nuclear envelope was not Fig. 4G), in tangential segments at the periphery of nuclear Journal of Cell Science

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Fig. 3. See next page for legend. Journal of Cell Science

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Fig. 3. SUN and KASH proteins are required for the recruitment of PCM-1, yielding 55% of cells with aligned nuclei (Fig. 5A, graph). centrosome protein PCM-1 to the nuclear envelope. (A) Localization of The depletion led to removal of 74% of PCM-1 in cultures overall endogenous nesprin-1 (red) in C2C12 myotubes overexpressing (top) control (Fig. 5B, quantification of immunoblot), but for the quantification EGFP or (bottom) the EGFP-tagged nesprin-1 KASH domain (green). (B) Localization of endogenous PCM-1 (red) in C2C12 myotubes, expressing of nuclear positioning, only myotubes lacking PCM-1 at the nuclear various levels of the EGFP-tagged nesprin-1 KASH domain (green). Nuclei were envelope were counted. We verified that depletion of PCM-1 did not stained with DAPI (blue). (C) Immunoblots of lysates of C2C12 myoblasts affect the overall protein levels of nesprin-1 (Fig. 5B). Moreover, we (myobl.) and myotubes, stained with antibodies against SUN1 and SUN2, as well verified by immunofluorescence that nuclear envelopes depleted of as immunoblots of lysates treated with control RNA or with siRNA against SUN1 PCM-1 still retained nesprin-1 (Fig. 5B, right). or SUN2, probed with the respective antibodies. Immunoblots of corresponding To understand the potential mechanisms that caused cell lysates, probed with antibody against α-tubulin (α-tub.), are shown as loading controls. The position of protein molecular mass markers (in kDa) for both misalignment of nuclei in myotubes depleted of nesprin-1 or immunoblots of SUN1 and SUN2 is indicated on the right of the second column. PCM-1, we investigated proteins that are known to mediate (D) Localization of pericentrin (red) in myotubes depleted simultaneously of microtubule-dependent transport. Treatment of differentiated SUN1 and SUN2 (immunofluorescence of SUN2 in green). Blue, DNA. C2C12 cultures with erythro-9-[3-(2-hydroxynonyl)]adenine (E) Immunoprecipitation (IP) of nesprin-1 (left column) or PCM-1 (right column) (EHNA), an ATPase inhibitor that has been shown to interfere from nuclear envelope preparations from C2C12 cells (see Materials and with dynein function (Bouchard et al., 1981; Penningroth et al., Methods and Fig. S1C). For comparison, total C2C12 lysates, as well as the 1982), led to inhibition of nuclear alignment (Fig. 5A, graph). The eluates from magnetic beads without antibody, or from magnetic beads coupled with control IgG, were loaded. Immunoprecipitations were performed from effect of the dynein inhibitor ciliobrevin A was also tested, but fractions containing octyl beta-D-glucopyranoside (OGP) and NaCl, as depicted ciliobrevin A was found to significantly prevent the fusion of in lane 4 of Fig. S1C. Immunoblots were probed with antibodies against PCM-1, myoblasts into myotubes (data not shown). Moreover, the protein nesprin-1 and γ-tubulin. The loading control shows a Ponceau-stained region of p150glued (also known as DCTN1, hereafter referred to as p150), a the western blots, depicting the IgG heavy chains used for immunoprecipitation. component of the dynactin complex that has been implicated in (F) Localization of nuclear pore complexes, detected with monoclonal antibody dynein activation, as well the kinesin light chain 1 (KLC1) were 414 (NPC, red) in myotubes treated with control RNA or siRNA against nesprin-1 (green). Blue, DNA. (G) Expression of mCherry–nesprin-1α (red) in C2C12 found to localize to the nuclear envelope in control myotubes myoblasts prior to differentiation. Arrowheads point to areas of the nuclear (Fig. 5C,D). Depletion of nesprin-1, and also of PCM-1, prevented envelope with massive PCM-1 accumulation (green in the merged image). Blue, the localization of both p150 and KLC1 to the nuclear envelope DNA. (H,I) Expression of mCherry–tubulin-α (red) (H) or mEmerald–nesprin-3 (Fig. 5C,D). By contrast, depolymerization of the microtubule (green) (I), as a controls. Immunofluorescence of PCM-1 is shown in green (H), network in myotubes did not remove p150 from the nuclear – α and in red (I). Blue, DNA. (J) Expression of mCherry nesprin-1 (red) and envelope (Cadot et al., 2012; Fig. 5E), suggesting that motor- immunofluorescence of the differentiation marker myosin II heavy chain (green). Blue, DNA. Scale bar: 10 µm. complex-associated proteins are bound to the nuclear surface in a manner that is dependent on nesprin-1 and PCM-1, but independent of microtubules. envelopes (segment 2) and in areas free of nuclei (segment 3). Both segments 1 and 2, at or near nuclei, showed a microtubule intensity DISCUSSION that was 10–20% higher compared to nuclei-free cytoplasmic In the present study, we employed a cell culture model of muscle segments, raising the possibility that there was a small contribution differentiation to show that centrosome proteins are anchored to the of myotube nuclei to microtubule nucleation or microtubule nuclear envelope by nesprin-1, and that this interaction is necessary anchoring. However, this percentage was not significantly altered for the recruitment of motor proteins that position nuclei in syncytial in myotubes with nesprin-1-depleted nuclei (Fig. 4G), indicating myotubes. Our findings are consistent with earlier studies, in which that the small number of microtubules at the nuclear envelope did nesprin-related KASH domain proteins have been implicated in not require substantial amounts of nuclear-envelope-bound nuclear migration and positioning (Starr, 2007), such as UNC-83 centrosome proteins under equilibrium conditions, unlike in C. elegans (Starr et al., 2001), and MSP-300 and Klarsicht in microtubule re-growth after depolymerization, which is favoured Drosophila photoreceptor cells and in striated muscle (Mosley- only if many nucleation sites are instantly available (Fig. 4A,B). Bishop et al., 1999; Patterson et al., 2004; Elhanany-Tamir et al., 2012). In mammals, the KASH domain of nesprins is required for Positioning of myotube nuclei, dependent on nesprin-1 and nuclear positioning in neurons and in muscle cells, as elimination of PCM-1 KASH-encoding exons or displacement of endogenous nesprins by The most notable change in myotubes depleted of nesprin-1 was an overexpressed KASH results in defective nuclear migration, abnormal positioning of nuclei within the syncytial cytoplasm localization and anchorage (Grady et al., 2005; Zhang et al., (Fig. 5A): whereas 76% of control myotubes showed nuclei aligned 2007b, 2009, 2010; Wilson and Holzbaur, 2012, 2015). Nuclear evenly along the long axis of the cell, only 21% of nesprin-1- movement has previously been suggested to be mediated by a depleted myotubes contained aligned nuclei (Fig. 5A, graph). physical interaction between nesprins or related proteins such as Moreover, the spacing between nuclei was reduced and multiple ZYG-12 and the centrosome, which is at the centre of a radial nuclei often concentrated into small packages (Fig. 5A). To microtubule network (Malone et al., 2003; Zhang et al., 2009). In determine whether centrosome proteins mediated any aspect of early differentiated myotubes, the centrosome no longer exists as a nesprin-1-dependent nuclear positioning, we depleted PCM-1, microtubule-organizing centre, but its constituent proteins are given that this has previously been characterized as a protein relocalized all around the surface of the nucleus (Tassin et al., necessary for the recruitment of many other centrosome 1985a; Bugnard et al., 2005; Srsen et al., 2009). We show that this components (Dammermann and Merdes, 2002), including depends on the differentiation-specific expression of the nesprin-1α binding of pericentrin to the nuclear envelope (Fig. S1E; in the isoform, and that other nesprins are not involved in this process. We reverse experiment, that is, depletion of pericentrin, PCM-1 was not did not find nesprin-2 at nuclear membranes of newly differentiated altered). As compared to nesprin-1 depletion, a similar although C2C12 cells, although splice variants of nesprin-2 have previously weaker effect on nuclear positioning was seen after depletion of been identified in skeletal muscle (Duong et al., 2014; Zhang et al., Journal of Cell Science

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Fig. 4. Nesprin-1-depletion does not alter microtubule organization in myotubes. (A) Left column, localization of nesprin-1 (green) and γ-tubulin (red) in a control myotube. Right column, nesprin-1 (green) and γ-tubulin (red) in a myotube formed with a non-depleted (left) and nesprin-1-depleted nucleus (right). (B) Regrowth of microtubules (red) at 3 min after recovery from depolymerization in control or nesprin-1-depleted C2C12 cells. Green, nesprin-1. Graph, quantification of the microtubule repolymerization activity at the nuclear envelope in myotubes (nuclear envelopes with visible microtubule stubs ≥2 µm length). n=100. Errors bars correspond to 2×s.e.m. (confidence interval at 95%). Nuclei were stained with DAPI (blue). (C) Immunofluorescence of microtubules in control or nesprin-1-depleted C2C12 myotubes. Negative-contrast-images of single deconvolved optical sections of an image stack are shown, in addition to merged images, depicting microtubules in green, nesprin-1 in red and nuclei in blue. (D) Enlarged perinuclear region of a control myotube, showing sum projections of microtubules from three consecutive partial stacks of 1.2 µm thickness (each obtained from six sections at 0.2 µm distance; position of sections are indicated in each image). The track of a selected single microtubule is indicated by arrowheads. On the right, a merged image is shown in three colours (#14-19, blue; #20-25, green; #26-31, red, where # is the number of the section from top to bottom). (E) Individual microtubules were tracked manually in complete image stacksof myotubes (n=5 for controls or nesprin-1 siRNA), reconstructed from partial stacks as shown in D. The number of microtubules running within a distance of ∼1µm to the nuclear surface were counted. Results are mean±s.d. (F) Microtubules tracked in E were analysed for their orientation. The percentages of microtubules running parallel to the long axis of the myotubes are shown. Results are mean±s.d. (G) The immunofluorescence signal intensities of microtubules were determined in sum projections from entire image stacks of myotubes in myotube segments containing nuclei (segment 1 in scheme), in tangential segments at the periphery of nuclear envelopes (segment 2) and in areas free of nuclei (segment 3). n=5 for controls or nesprin-1 siRNA. Each value obtained for segment 1 or 2 was divided through the value of a neighbouring segment 3. The resulting values equalled percentages of segments 3 (defined as 100%). Results are mean±s.d. Scale bars: 10 µm.

2005). It is possible that nuclear and non-nuclear forms of nesprin-2 differentiation stages, nesprin-3, has a cytoplasmic domain that is are expressed at more advanced stages of muscular differentiation, largely dissimilar from nesprin-1α (only 8% of sequence identity for example, upon sarcomere formation, that are not mimicked outside its KASH domain), and does not recruit centrosome proteins under our experimental conditions (Zhang et al., 2005). The only (Fig. 2D,F). The detection of premature PCM-1 relocalization to the other nesprin present at the nuclear envelope during the early nuclear envelope prior to differentiation upon mCherry–nesprin-1α Journal of Cell Science

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Fig. 5. Nuclear positioning in myotubes is dependent on nesprin-1 and PCM-1, with both involved in recruiting microtubule motor proteins to the nuclear envelope. (A) Alignment of nuclei along the axis of myotubes (dotted line) in control, nesprin-1- or PCM-1-depleted C2C12 cells. Immunofluorescence of nesprin-1 and PCM-1 is shown. Nuclei were stained with DAPI. Graph showing the percentage of myotubes with correctly aligned nuclei along the axis of the cell. From the left: control, nesprin-1-depleted, PCM-1-depleted and EHNA-treated C2C12 cells. n=300 cells for each condition, error bars correspond to 2×s.e.m. (confidence interval at 95%). (B) Left, PCM-1-depleted C2C12 myotubes (siRNA PCM-1), as well as control myotubes were analysed by immunoblotting, using antibodies against PCM-1, nesprin-1 or α-tubulin. Right, PCM-1-depleted myotubes were analysed by immunofluorescence using antibodies against PCM-1 (red) and nesprin-1 (green). Nuclei were stained with DAPI (blue). A myotube is shown with a PCM-1-depleted and an undepleted nucleus side by side. (C) Localization of p150glued in control, nesprin-1 and PCM-1-depleted C2C12 myotubes. Green, nesprin-1 or PCM-1; red, p150glued; blue, DNA. (D) Localization of KLC1in control, nesprin-1 and PCM-1-depleted myotubes. Owing to incompatibility of multiple rabbit antibodies for co-staining of KLC1, nesprin-1, and PCM-1, the depletion of nesprin-1 and PCM-1 were verified indirectly by performing immunofluorescence staining with mouse antibody against pericentrin (green), which is absent from the nuclear envelope under these conditions (see Fig. S1E). Red, KLC1; blue, DNA. (E) p150glued localization (red) in control cells, and in cells after depolymerization of microtubules (green) by nocodazole treatment. Blue, DNA. Scale bar: 10 µm. expression led us to conclude that nesprin-1α is a receptor for centrosome proteins, and that these modifications are missing in our centrosome proteins at the nuclear envelope. It is possible transfected cells at the myoblast stage. that additional proteins are involved for efficient anchoring, How do centrosome proteins affect nuclear positioning in because we only obtained partial recruitment of PCM-1. Another myotubes? Whereas in non-muscular cells, forces can be possibility would be that differentiation-specific post-translational transmitted to the nucleus by the radial centrosomally anchored modifications of nesprin-1α are needed for a strong interaction with microtubule network owing to the physical link between the Journal of Cell Science

4234 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4227-4237 doi:10.1242/jcs.191767 centrosome and the nucleus, a different mechanism is likely active KASH domain of nesprin-1 was deleted, and which mimicked in myotubes: we suggest that the relocalized centrosome proteins all aspects of Emery–Dreifuss muscular dystrophy (Zhang et al., 2007b, around the nuclear envelope anchor microtubule-dependent motors 2010; Puckelwartz et al., 2009). Interestingly, point mutations in the of the kinesin and dynein family, allowing nuclei to move laterally nesprin-1 gene have been found in a subgroup of patients suffering along microtubules and to position along the length of the myotube. from Emery–Dreifuss muscular dystrophy (Zhang et al., 2007a). The dependence of myonuclear distribution on microtubules was Given that defective nuclear positioning is reproduced in our cell originally inferred from pharmacological experiments and from culture model, and shown to involve PCM-1 in addition to nesprin-1, correlative studies of nuclear position and the microtubule network it will be important to test in the future whether the localization of (Englander and Rubin, 1987; Bruusgaard et al., 2006), and has more centrosome proteins and microtubule motors at the nuclear envelope recently been shown by Cadot et al. (2012) and Metzger et al. is affected in any way in Emery–Dreifuss muscular dystrophy, and (2012). Initial formation of microtubule filaments in myotubes whether this is involved in pathogenetic mechanisms. might involve microtubule-nucleating activity on the nuclear surface (Tassin et al., 1985a; Bugnard et al., 2005; Fant et al., MATERIALS AND METHODS 2009). However, microtubules in our early differentiated C2C12 Cell culture cultures were mostly seen in a parallel orientation along the length C2C12 mouse myoblasts (American Type Culture Collection) were cultured of the cell, with long microtubules running past nuclei instead of in Dulbecco’s modified Eagle’s Medium (DMEM)+Glutamax-I attaching end-on to their nuclear surface (Fig. 4C,E). Later in the (Invitrogen) supplemented with 20% fetal bovine serum (FBS) and 0.5% chicken embryonic extract. Cells were induced to differentiate by switching differentiation process, the microtubule network is further to differentiation medium (DMEM+Glutamax-I supplemented with 0.5% remodelled into an orthogonal-grid-like organization, with a large FBS, 5 µg/ml insulin and 5 µg/ml transferrin). fraction of microtubules growing off Golgi elements (Oddoux et al., 2013). It is possible that upon formation of a microtubule network in siRNA transfection myotubes, release of microtubule ends from their initial nucleation siRNAs were designed to target the following sequences: 5′-CCAGGGT- sites or generation of new distant nucleation sites occurs, as seen in GAAGAAGCTAAA-3′ and 5′-GCTCCTGCTGCTGCTTATT-3′ for many non-muscular cell systems undergoing differentiation nesprin-1, 5′-AGCCACAGAACTCCAAAAT-3′ for nesprin-2, 5′-GCTA- (Mogensen, 1999; Musa et al., 2003; Stiess et al., 2010). This CGTAGAATCATCACA-3′ for nesprin-3, 5′-TGAGCTTCGTGATTCTC- would explain loss of binding of microtubule ends at the nuclear AG-3′ for PCM-1 (Dammermann and Merdes, 2002), 5′-CGTCGGATG- surface, as seen in our study. Moreover, previously reported CTCTGGATTT-3′ and 5′-CAGGTGCCTTCGAAATATT-3′ for SUN-1, ′ ′ ′ microtubule nucleation from nuclear envelopes in myotubes might 5 -CCGCTGCTCTGAGACTTAT-3 and 5 -GCCCTTGCTGCAGACT- ′ ′ ′ have been experimentally enhanced by depolymerization of the TTT-3 for SUN-2, and 5 -AAGCATCATGCCATCTCTGCT-3 for calnexin. Control experiments were done using the scrambled siRNA microtubule network: under these conditions, the concentration of sequence 5′-UUCUCCGAACGUGUCACGU-3′ (Qiagen). C2C12 cells free tubulin is high, and re-nucleation can occur from sites that were seeded at 80,000 cells/cm2, and induced to differentiate 4 h later. might not specifically nucleate microtubules under physiological Transfection was performed after 24 h in differentiation medium, with conditions (Mitchison and Kirschner, 1985). Lipofectamine RNAiMAX (Invitrogen). The final concentrations of most The concept of nuclear positioning by gliding along microtubules siRNAs were 25–30 nM, except for PCM-1, for which a final concentration is supported by previous studies demonstrating an interaction of of 500 nM was necessary to achieve efficient depletion. For nesprin-1 kinesin and dynein–dynactin motor complexes with mammalian depletion, cells differentiated for 2–3 days were transfected twice with nesprins 1, 2 and 4, and with the related KASH-domain proteins HiPerfect reagent (Qiagen) and 150 nM siRNA at 48 h intervals. For ZYG-12 and UNC-83 in C. elegans (Malone et al., 2003; Roux western blot analysis, cells were recovered by scraping, washed in PBS, and ‘ ’ et al., 2009; Zhang et al., 2009; Fridolfsson et al., 2010; Yu et al., subsequently washed in wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 2 mM EDTA). Cells were resuspended in wash buffer containing 2011; Wilson and Holzbaur, 2012, 2015). In myotubes, we propose 0.5% Triton X-100, 1 mM DTT and a cocktail of protease inhibitors that this interaction is indirect, mediated by nuclear-envelope-bound (Complete/EDTA free, Roche). Cells were finally lysed by sonication. 70 µg centrosome proteins, because the depletion of the centrosome of extracts were subjected to SDS-PAGE. protein PCM-1 induces loss of dynactin and kinesin subunits from the nuclear surface. Given that PCM-1 is necessary for the targeting Immunoprecipitation experiments and assembly of a variety of proteins of the pericentriolar material Differentiated C2C12 cells, scraped from petri dishes, were centrifuged in (Dammermann and Merdes, 2002), anchoring of motor complexes 10 mM HEPES, pH 8.0, 1.5 mM MgCl2, 1 mM DTT, 1 mM PMSF, and nuclear alignment might occur through other centrosome followed by lysis with 20 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1% NP-40, proteins such as pericentrin and Cep135, both known to bind 10% glycerol, 5 mM EDTA, and a protease inhibitor cocktail (Complete/ directly to dynein and dynactin polypeptides (Purohit et al., 1999; EDTA free, Roche). After sonication and centrifugation at 16,100 g for Uetake et al., 2004). Consistent with this, the centrosome protein 30 min at 4°C, the resulting nuclear pellets were extracted with 20 mM Tris- β pericentrin was lost from the nuclear envelope when PCM-1 was HCl, pH 7.0, 400 mM NaCl, 1% octyl -D-glucopyranoside, 5 mM MgCl2, depleted (Fig. S1C). Besides contributing to nuclear positioning, 1 mM DTT and protease inhibitors. Protein extracts were diluted to a final concentration of 1 mg/ml in buffer containing 200 mM NaCl and 0.5% octyl the accumulation of centrosome proteins at the nuclear envelope β-D-glucopyranoside, and samples of 1 ml were incubated with rabbit anti- might serve additional roles in muscular cell types, given that a nesprin-1 or rabbit anti-PCM-1 antibody (see later for details) for several similar accumulation has been found in mono-nucleate mouse hours at 4°C. Immunoprecipitation was performed with 50 µl magnetic cardiomyocytes (Zebrowski et al., 2015). It has been speculated that protein A beads (Biorad) per sample, followed by rinsing four times in the in mammalian cardiomyocytes, that relocalization of centrosome same buffer, and elution in gel-loading buffer containing 2.3% SDS. proteins helps to maintain the post-mitotic cell cycle state (Zebrowski et al., 2015). Pharmacological treatments In skeletal muscle, the ordered positioning of nuclei would be Microtubules were depolymerized by treatment with 20 µM nocodazole for expected to be important for correct muscle function, given that 40 min at 37°C, followed by 40 min on ice. Cells were rinsed once with PBS abnormal positioning has been found in mouse models in which the and twice with growth medium, before re-polymerization was induced in Journal of Cell Science

4235 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 4227-4237 doi:10.1242/jcs.191767 growth medium at 37°C. Dynein ATPase activity was inhibited by using 432C, Covance), anti-KLC1 H75 (1:100, cat. no SC-25735, Santa Cruz erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA) at 0.1 mM for 24–48 h. Biotechnology), rabbit mAb anti-GM130 (1:100, cat. no ab52649, Abcam), The Golgi was disassembled by treatment with 5 µg/ml Brefeldin A for rabbit and mouse anti-PCM-1 (1:500, Dammermann and Merdes, 2002), 90 min. rabbit anti-SUN-1 and SUN-2 [raised against peptides NH2- CHKLEPVFDSPRMSR-CONH2 and NH2-CLGRFTYDQEGDSLQ- Molecular cloning and transfection CONH2 for SUN-1 (used at 1:50), and NH2-CVFKDSPLRTLKRKS- The mouse nesprin-1 cDNA IRAKp961K22116Q was obtained from CONH2 and NH2-CGTFAYDQDGEPIQT-CONH2 for SUN-2 (used at ‘Deutsches Ressourcenzentrum für Genomforschung’. A plasmid encoding 1:100), respectively; Covalab)]. We used Alexa-Fluor-488- and -568- mEmerald–nesprin-3 was obtained from Addgene (#54203), originally conjugated secondary antibodies (Molecular Probes) for provided by Dr Michael Davidson, Florida State University, Tallahassee, immunofluorescence, and horseradish-peroxidase-conjugated antibodies FL. Nesprin-1α and the nesprin-1 KASH domain were amplified by PCR (Jackson ImmunoResearch Laboratories) for immunoblotting, followed using the forward primers 5′-AAAAAAGAATTCATGGTGGTGGCAG- by ECL. AGGACTTG-3′ or 5′-AAAAAAGAATTCTCCCGTTCTGACCCCAGG- CC-3′, respectively, in combination with the reverse primer 5′-TTTTTT- Acknowledgements GGTACCTCAGAGTGGAGGAGGACCGTT-3′. The resulting DNA was We thank our colleagues at the Centre de Biologie du Développement (University digested with EcoRI and KpnI, and cloned into the respective sites of Toulouse III, France) for their help and for stimulating discussions. We thank Glenn pmCherry-C2 or pEGFP-C2 (Clontech). Transfection of myoblasts was E. Morris (Keele University, Staffordshire, UK), Rener Xu (Fudan University, performed using an Amaxa Nucleofector system according to the Shanghai, China) and Arnoud Sonnenberg (Netherlands Cancer Institute) for kindly providing antibody samples against nesprins 1, 2 and 3, respectively. manufacturer’s protocol (KitV, Lonzabio). Cells were induced to differentiate at 4 h to 6 h after transfection. For quantitative PCR (qPCR) Competing interests experiments, total RNA was isolated from C2C12 myoblasts and myotubes The authors declare no competing or financial interests. using an RNeasy Mini Plus kit (Qiagen). Equal amounts of RNA (1 µg) were added to a reverse transcriptase reaction mix (Superscript II Reverse Author contributions Transcriptase, Invitrogen) with random primers. The resulting cDNA was A.E.G. and V.D. designed and carried out experiments, and co-wrote the subjected to quantitative PCR using a Biorad C1000 thermal cycler, coupled manuscript; C.C. carried out experiments and analysed data; L.E. analysed data and to the CFX96 real-time system, using the SSoFast Evagreen Supermix co-wrote the manuscript; A.M. supervised the project, designed and carried out (Biorad) for 40 cycles. Primers were used for nesprin-1 (forward, 5′-CT- experiments, and co-wrote the manuscript. TCCTGTTCCGGATCCTC-3′; reverse, 5′-AGGTGAGTCCAATAAGC- AGCA-3′), nesprin-2 (forward, 5′-ATGTCACCAGCCCAGAGG-3′; Funding reverse, 5′-GACGGGCTACCAACTCCTTT-3′), as well as for two The project was supported in part by the Association Française contre les housekeeping genes, hypoxanthine guanine phosphoribosyl transferase Myopathies (12471, 14810 and 1481). (HGPRT1, forward, 5′-TCCTCCTCAGACCGCTTTT-3′; reverse, 5′- CCTGGTTCATCATCGCTAATC-3′), and ribosomal protein S16 (Rps16, Supplementary information Supplementary information available online at forward, 5′-AGGAGCGATTTGCTGGTGTGG-3′; reverse, 5′-GCTACC- http://jcs.biologists.org/lookup/doi/10.1242/jcs.191767.supplemental AGGGCCTTTGAGATGG-3′). Calibration curves were performed on each myoblast and myotube References cDNA, to measure the efficiency of the primer pairs used. Corresponding Apel, E. D., Lewis, R. M., Grady, R. M. and Sanes, J. R. (2000). Syne-1, a reaction mix containing RNA without reverse transcriptase was used as a dystrophin- and Klarsicht-related protein associated with synaptic nuclei at the negative control for the qPCR of each sample. Levels of nesprin-1 and neuromuscular junction. J. Biol. Chem. 275, 31986-31995. nesprin-2 cDNA were expressed relative to those of Rps16 and HGPRT1, Attali, R., Warwar, N., Israel, A., Gurt, I., McNally, E., Puckelwartz, M., Glick, B., using the Biorad CFX Manager software. Nevo, Y., Ben-Neriah, Z. and Melki, J. (2009). Mutation of SYNE-1, encoding an essential component of the nuclear lamina, is responsible for autosomal recessive arthrogryposis. Hum. Mol. Genet. 18, 3462-3469. Immunofluorescence microscopy Bouchard, P., Penningroth, S. M., Cheung, A., Gagnon, C. and Bardin, C. W. C2C12 cells grown on coverslips were fixed in methanol at −20°C and (1981). Erythro-9-[3-(2-Hydroxynonyl)]adenine is an inhibitor of sperm motility processed for immunofluorescence following standard protocols. For that blocks dynein ATPase and protein carboxylmethylase activities. Proc. Natl. staining of γ-tubulin, microtubules and dynactin complexes, cells were Acad. Sci. USA 78, 1033-1036. pre-permeabilized in PEM buffer (80 mM PIPES, 5 mM EGTA, 2 mM Bruusgaard, J. C., Liestøl, K. and Gundersen, K. (2006). Distribution of myonuclei and microtubules in live muscle fibers of young, middle-aged, and old mice. MgCl2, pH 6.8) containing 0.5% Triton X-100 (for 30 s to 5 min) or 0.05% – J. Appl. Physiol. 100, 2024-2030. saponine (5 15 min). The coverslips were rinsed with PEM containing 4% Bugnard, E., Zaal, K. J. M. and Ralston, E. (2005). Reorganization of microtubule polyethylene glycol and fixed with 4% paraformaldehyde in PEM for an nucleation during muscle differentiation. Cell Motil. Cytoskeleton 60, 1-13. initial period of 5 min, followed by a second fixation step with Cadot, B., Gache, V., Vasyutina, E., Falcone, S., Birchmeier, C. and Gomes, paraformaldehyde in carbonate buffer (50 mM, pH 10) for 10 min. E. R. (2012). 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(1:100, Zhang et al., 2007b), mouse mAb NSP-3 anti-nesprin-3 (1:100, Duong, N. T., Morris, G. E., Lam, L. T., Zhang, Q., Sewry, C. A., Shanahan, C. M. Ketema et al., 2007), mouse anti-α-tubulin clone B-5-2-1 (1:1000, cat. no and Holt, I. (2014). Nesprins: tissue-specific expression of epsilon and other short T5168, Sigma), rabbit anti-calnexin (1:200, cat. no SPA-860, Stressgen isoforms. PLoS ONE 9, e94380. Bioreagents), mouse anti-γ-tubulin TU-30 (1:1000, cat. no 11-465-C025, Elhanany-Tamir, H., Yu, Y. V., Shnayder, M., Jain, A., Welte, M. and Volk, T. Exbio), mouse anti-sarcomeric myosin MF20 (1:100, cat. no AB_2147781, (2012). Organelle positioning in muscles requires cooperation between two KASH DSHB), mouse anti-nuclear pore complex proteins, clone 414 (1:500, cat. no proteins and microtubules. J. Cell Biol. 198, 833-846. Ellis, J. A. (2006). Emery-Dreifuss muscular dystrophy at the nuclear envelope: 10 ab50008, Abcam), mouse anti-p150glued (1:100, cat. no 612708, BD years on. Cell. Mol. Life Sci. 63, 2702-2709. Transduction Laboratories), mouse anti-pericentrin (1:100, cat. no 611814, Englander, L. L. and Rubin, L. L. (1987). Acetylcholine receptor clustering and

BD Transduction Laboratories), rabbit anti-pericentrin (1:1000, cat. no PRB- nuclear movement in muscle fibers in culture. J. Cell Biol. 104, 87-95. Journal of Cell Science

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