Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation
© Srsen et al; licensee BioMed Central Ltd. 2009
Received: 16 February 2009
Accepted: 21 April 2009
Published: 21 April 2009
Muscle fibres are formed by elongation and fusion of myoblasts into myotubes. During this differentiation process, the cytoskeleton is reorganized, and proteins of the centrosome re-localize to the surface of the nucleus. The exact timing of this event, and the underlying molecular mechanisms are still poorly understood.
We performed studies on mouse myoblast cell lines that were induced to differentiate in culture, to characterize the early events of centrosome protein re-localization. We demonstrate that this re-localization occurs already at the single cell stage, prior to fusion into myotubes. Centrosome proteins that accumulate at the nuclear surface form an insoluble matrix that can be reversibly disassembled if isolated nuclei are exposed to mitotic cytoplasm from Xenopus egg extract. Our microscopy data suggest that this perinuclear matrix of centrosome proteins consists of a system of interconnected fibrils.
Our data provide new insights into the reorganization of centrosome proteins during muscular differentiation, at the structural and biochemical level. Because we observe that centrosome protein re-localization occurs early during differentiation, we believe that it is of functional importance for the reorganization of the cytoskeleton in the differentiation process.
The formation of muscle during embryonic development involves the differentiation of myoblasts into long, fibrous cells. In this differentiation process, myoblasts withdraw from the cell cycle and fuse into multinucleate, syncytial myotubes . The microtubule cytoskeleton is reorganized from a radial network into a parallel array of filaments aligned along the long axis of the cells . It is believed that this reorganization is a prerequisite for the elongation and fusion of myoblasts, and for the subsequent alignment and organization of sarcomeres [3–7]. Microtubule reorganization is paralleled by reorganization of centrosomal proteins: myoblasts possess a morphologically recognizable centrosome with characteristic marker proteins concentrated in the pericentriolar material, whereas myotubes show perinuclear localization of a multitude of centrosome proteins [8–10]. Consequently, polymerization of microtubules is initiated in part from the surface of the nucleus [8, 10, 11]. It has been reported that reorganization of microtubules and relocalization of centrosome proteins to the nuclear surface occurs after fusion of myoblasts into myotubes . However, the precise kinetics of this reorganization are unknown. Morover, it is unknown how centrosome proteins are attached to the nuclear surface, and how they are organized at the ultrastructural level.
Results and Discussion
We investigated the pattern of various proteins and found that significant amounts of pericentrin and PCM-1 accumulated at the nuclear periphery, whereas the centrosome protein cdk5rap2 showed partial relocalization. Only minor amounts of gamma-tubulin were found at the nuclear periphery, consistent with data from , and the protein ninein was not found to relocalize to the nuclear periphery in our experiments.
We noticed that upon entry of these extracts into a prometaphase-like state, C2C12 nuclei began to disassemble. DNA condensed into mitotic chromosomes, and the perinuclear 'matrix' of PCM-1 disintegrated into a system of interwoven fibres, and finally disassembled into protein aggregates of varying sizes (Fig. 6B, C). We performed deconvolution microscopy on nuclei, stained with various markers, in S-phase and prometaphase. We found that PCM-1, pericentrin, gamma-tubulin, as well as the nuclear envelope protein nesprin 1 localized to patches on the nuclear envelope that were closely apposed, but without fully co-localizing with each other (Fig. 6, E–H, left column). In samples that had entered prometaphase, we found that PCM-1, pericentrin, and nesprin 1 localized to spots and interconnected fibres that co-localized partly, whereas gamma-tubulin was found more diffusely distributed (Fig. 6, E–H). We therefore believe that PCM-1 and pericentrin form distinct fibrillar structures at the outer nuclear surface that are connected at various contact sites, thus constituting a tight matrix. This matrix may be structurally equivalent to the fibrous pericentriolar material in undifferentiated cells, and it can be disassembled or at least loosened upon entry into mitosis. Consistently, many centrosome proteins, including PCM-1 and pericentrin, are seen in undifferentiated cells during mitosis in a wide crescent-shaped area at the spindle poles or diffuse in the cytoplasm, whereas in interphase they are more focused at the centrosome [14, 17, 18].
In this manuscript, we demonstrate that relocalization of centrosome proteins to the nuclear surface is an early event during the differentiation of myoblasts, which occurs prior to their fusion into myotubes. This may imply that the relocalization is important for subsequent differentiation events, for example by affecting microtubule organization. Further, we show that centrosome proteins form a filamentous matrix around the outer surface of the nuclear envelope. Our biochemical experiments demonstrate that this matrix is highly insoluble, but disassembles in mitotic cytoplasm. The assembly and disassembly characteristics of this matrix may provide general insights into the organization of the pericentriolar material of centrosome proteins. Future work will be needed to determine the molecular mechanisms that lead to re-localization of centrosome proteins to the nuclear surface upon muscular differentiation.
C2C12 cells were grown in Dulbecco's modified Eagle's medium, containing 0.5% chicken embryonic extract and 20% fetal calf serum. Differentiation was induced by serum starvation for one or more days, by replacing the regular growth medium with Dulbecco's modified Eagle's medium containing 5% horse serum. H-2Kb-tsA58 myoblasts were cultured and differentiated as described . Transfections of H-2Kb-tsA58 cells with GFP-tagged lamin A  were performed using Lipofectamine Plus transfection agent (Invitrogen).
Cells were grown on glass coverslips and fixed in methanol at -20°C for 10 minutes. Immunofluorescence was performed using standard procedures. Antibodies used in this study were against PCM-1 (rabbit and mouse anti-PCM-1, ), pericentrin (rabbit anti-pericentrin, Covance), gamma-tubulin (monoclonal antibody GTU-88, Sigma), cdk5rap2 (rabbit antibody 46024, raised against GST-tagged fusion protein, containing amino acids 1–247 of cdk5rap2), ninein (rabbit antibody 1732, raised against GST-tagged ninein fusion protein), alpha-tubulin (monoclonal antibody DM1a, Sigma), Ki-67 (polyclonal antibody M-19, Santa Cruz Biotechnology), embryonic myosin (monoclonal antibody, developed by Helen Blau, Developmental Studies Hybridoma Bank, University of Iowa), myogenin (monoclonal antibody F5D, Santa Cruz Biotechnology), nuclear pore complex proteins (monoclonal antibody Mab414, Covance), nesprin 1 (rabbit antibody, Abcam). The rabbit antibodies against cdk5rap2 and ninein were raised in our laboratory, and tested for specificity by immunoblotting of bacterial fusion protein and by immunofluorescence in cultured cells. The specificity was further confirmed in experiments using siRNA against cdk5rap2 or ninein, leading to disappearance of the respective immunofluorescence signal (unpublished results). DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). Histological sections of muscle were prepared from pieces of mouse hind leg muscle, embedded and frozen in Tissue-Tek (Sakura), using a Leica cryostat. Sections were subsequently fixed in methanol at -20°C and processed for immunofluorescence.
Immunoelectron microscopy was performed on differentiated C2C12 cells that were fixed overnight in 4% paraformaldehyde in 0.2 M sodium phosphate buffer, pH 7.4. Ultra-thin frozen sections were prepared as described , and labelled with rabbit antibody against PCM1, followed by protein A conjugated with 10 nm gold.
C2C12 cells were differentiated for four days, to obtain a high yield of differentiated, fused myotubes. After removal of culture medium, cells were treated with 1× Trypsin/EDTA (Gibco) for several seconds, leading to selective detachment of fused myotubes. Detachment was monitored by phase contrast microscopy. Detached cells were collected and trypsin was neutralized by addition of growth medium. Following centrifugation and two washes with PBS (phosphate-buffered saline), cells were re-suspended in buffer containing 0.2 M KCl, 0.1 M PIPES pH 7.4, 0.2 M MgCl2, 10 μM cytochalasin B, 0.1 mM phenylmethyl sulfonylfluoride, and 10 μg/ml of leupeptin, pepstatin, and chymostatin. After an additional centrifugation and wash in the same buffer, cells were homogenized in a douncer with tight-fitting pestle. The material was layered onto a 30% sucrose cushion and centrifuged at 850 × g for 10 minutes. The pellet, containing nuclei, was re-suspended in the same buffer as used for homogenization, and centrifuged for 450 × g for 5 minutes. The resulting nuclei were stored in 50% glycerol, 250 mM sucrose, 80 mM KCl, 20 mM NaCl, 5 mM EGTA, 15 mM PIPES pH 7.4, 1 mM dithiothreitol, 0.5 mM spermidin, 0.2 mM spermin, 0.1 mM phenylmethyl sulfonylfluoride, and 10 μg/ml of leupeptin, pepstatin, and chymostatin. To examine the solubility of perinuclear PCM-1, extraction of nuclei was performed for 10 minutes in the same buffer without glycerol, containing 0.1% Triton X-100, or 1% Triton X-100, or 1.5 M NaCl, or 6 M urea, or 8 M urea, or 2% sodium dodecyl sulfate. Subsequently, immunofluorescence was performed, following centrifugation of extracted nuclei through a cushion of 30% glycerol onto glass coverslips. Cell cycle experiments were performed by incubation of purified nuclei in cytostatic factor-arrested extracts from Xenopus laevis eggs that were prepared as described . Extracts were stimulated to cycle through S-phase by addition of 0.4 mM CaCl2 and incubation for approximately 40 to 60 minutes. The passage through S-phase was verified in parallel experiments by monitoring DNA synthesis, by incorporating bromodeoxyuridine into nuclei, followed by immunofluorescence . After 90 minutes, extracts had reached prophase, and a new mitotic state was stabilized by addition of a fresh 50% volume-equivalent of cytostatic factor-arrested extract. Immunofluorescence of nuclei was performed following centrifugation onto glass coverslips as described above. The cell cycle state of extracts was monitored in parallel samples incubated with Xenopus laevis sperm and rhodamine-labelled tubulin, to monitor chromosome condensation and spindle formation.
We thank our colleagues for technical help and stimulating discussions. We thank Dr Michelle Peckham (University of Leeds) for providing mouse H-2Kb-tsA58 myoblasts. Monoclonal antibody against embryonic myosin, developed by Helen Blau, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa. We also thank Despina Xanthakis (Department of Cell Biology, UMC Utrecht) for help with the immuno-electron microscopy. The work was supported in part by a Wellcome Trust Senior Research Fellowship to A.M., by a Wellcome Trust Prize Fellowship to X.F., by a grant from the France-Berkeley Fund to R.H. and A.M., and by grant 12471 from the 'Association Française contre les Myopathies', awarded to A.M.
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