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Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation Background: 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. Results: 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. Conclusion: 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. Background believed that this reorganization is a prerequisite for the The formation of muscle during embryonic development elongation and fusion of myoblasts, and for the subse- involves the differentiation of myoblasts into long, quent alignment and organization of sarcomeres [3-7]. fibrous cells. In this differentiation process, myoblasts Microtubule reorganization is paralleled by reorganiza- withdraw from the cell cycle and fuse into multinucleate, tion of centrosomal proteins: myoblasts possess a mor- syncytial myotubes [1]. The microtubule cytoskeleton is phologically recognizable centrosome with characteristic reorganized from a radial network into a parallel array of marker proteins concentrated in the pericentriolar mate- filaments aligned along the long axis of the cells [2]. It is rial, whereas myotubes show perinuclear localization of a Page 1 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 multitude of centrosome proteins [8-10]. Consequently, nuclear surface next to the centrosome due to minus-end polymerization of microtubules is initiated in part from directed transport along microtubules [15], since the the surface of the nucleus [8,10,11]. It has been reported microtubule-organizing centre in these myoblasts is that reorganization of microtubules and relocalization of strongly focused at the centrosome and would thus favour centrosome proteins to the nuclear surface occurs after deposit of pericentrin in the surrounding area (Fig. 2A, fusion of myoblasts into myotubes [8]. However, the pre- cell marked with 'u'). It is noteworthy, however, that cise kinetics of this reorganization are unknown. Morover, already at the single cell stage microtubules were reorgan- it is unknown how centrosome proteins are attached to ized into a sun-like array radiating from the nuclear the nuclear surface, and how they are organized at the periphery within those cells displaying perinuclear relo- ultrastructural level. calization of centrosome proteins (Fig. 2A, cell marked with 'd'). Results and Discussion In order to determine at what stage of the differentiation To verify that these cells had indeed entered the differen- process relocalization of centrosome proteins occurs, we tiation programme, we performed immunofluorescence performed cell culture of mouse C2C12 myoblasts and trig- microscopy with differentiation markers. We noticed that gered differentiation by serum withdrawal. Alternatively, all cells in which the centrosome protein PCM-1 had relo- we used H-2K -tsA58 mouse myoblasts, carrying a thermo- calized to the nuclear surface showed cytoplasmic expres- labile T-antigen and allowing differentiation upon temper- sion of embryonic myosin, an isoform of myosin that is ature shift from 33 to 37°C [11,12]. In the undifferentiated specifically expressed upon onset of muscle cell differenti- state, the centrosomal proteins of both cell lines were ation (Fig. 2B). Furthermore, these cells expressed the dif- found in a single focus within the pericentriolar material ferentiation marker myogenin, a transcription factor that adjacent to the nucleus, while the protein PCM-1 ("pericen- localizes to the nucleus of differentiating cells (Fig. 2C). triolar material protein 1") localized to multiple 'centriolar This marker was absent from cells showing pericentriolar satellites', as described by [13] and [14]. One day after dif- PCM-1 staining. Conversely, these cells with pericentri- ferentiation of myoblasts was triggered, we observed cen- olar PCM-1 were the only ones that stained positively for trosome proteins at the nuclear periphery already at the the proliferation marker Ki-67 in the nucleus, whereas single cell stage, prior to fusion into myotubes (Fig. 1). nuclei in cells with relocalized PCM-1 did not contain Ki- 67 (Fig. 2D). Once relocalization of PCM-1 occurred, it We investigated the pattern of various proteins and found persisted until the final stages of differentiation: We that significant amounts of pericentrin and PCM-1 accu- detected PCM-1 around the nuclear surface after fusion of mulated at the nuclear periphery, whereas the centrosome multiple C2C12 cells into myotubes (Fig. 3A). Moreover, protein cdk5rap2 showed partial relocalization. Only nuclei in muscle from adult mice showed comparable minor amounts of gamma-tubulin were found at the staining of PCM-1 at their surface (Fig. 3B). nuclear periphery, consistent with data from [10], and the protein ninein was not found to relocalize to the nuclear We next sought to determine whether centrosome pro- periphery in our experiments. teins that had assembled around the nucleus were bound at the inside or at the outside of the nuclear envelope. For A closer look at differentiating C2C12 cells revealed that this purpose, we transfected mouse myoblasts with GFP- at the single cell stage, proteins such as pericentrin and tagged lamin A, a protein of the inner nuclear envelope. PCM-1 started to accumulate at the nuclear periphery in Deconvolution microscopy revealed that the centrosome the proximity of the centrosome, whereas areas of the protein PCM-1 localized in a rim slightly outside of lamin nuclear envelope distal from the centrosome showed less A (Fig. 4A), suggesting that PCM-1 associates with the centrosome protein enrichment (Fig. 1, first two rows). At outer surface of the nuclear envelope. Because the PCM-1 the same time, remnant pericentrin and cdk5rap2 were staining at the nuclear surface was visible in small clusters, still visible at the centrosome, and PCM-1 was still partly we tested whether these sites co-localized with nuclear localized in pericentriolar satellites (Fig. 1). The early relo- pores. However, double immunofluorescence with an calization of these proteins from the centrosome to antibody against a family of nuclear pore complex pro- nearby sites of the nuclear surface raises the question as to teins indicated that most of the PCM-1 clusters were dis- whether direct transfer from the centrosome is involved, tinct from nuclear pores (Fig. 4B). To reveal further or whether the proteins are recruited to the nuclear surface structural details, we performed immuno-electron micro- from a soluble cytoplasmic pool. Both scenarios seem scopy of ultra-thin cryosections of differentiated C2C12 possible: direct transfer from the centrosome to the cells. PCM-1 was detected with antibodies and gold-cou- nucleus might occur by diffusion. Alternatively, at the pled protein A. This technique preserved cellular mem- onset of differentiation, proteins such as pericentrin branes and showed that PCM-1 localized mainly outside might be recruited from a soluble pool and localize to the the nucleus, away from the outer nuclear membrane (Fig. Page 2 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Figure 1 Relocalization of centrosome proteins to the nuclear surface in differentiating myoblasts Relocalization of centrosome proteins to the nuclear surface in differentiating myoblasts. Cultures of C2C12 myoblasts were induced to differentiate by serum starvation for one day. At this stage, the culture contains undifferentiated myoblasts (u), as well as cells that started to differentiate (d). Immunofluorescence of these cultures was performed to visualize the centrosome proteins PCM-1 (red), as well as pericentrin, gamma-tubulin, cdk5rap2, and ninein (all green). DNA was stained in blue. Arrows indicate partial staining of the remnant centrosomes by pericentrin, gamma-tubulin, and cdk5rap2. Bar, 10 μm. Page 3 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Reorga Figure 2 nization of microtububules and centrosome protein in cells expressing myogenic differentiation markers Reorganization of microtububules and centrosome protein in cells expressing myogenic differentiation mark- ers. Culture of mouse myoblasts containing undifferentiated (u) cells, and cells that started to differentiate (d). The centro- some protein PCM-1 is stained in red, DNA is stained in blue. In green is marked (A) tubulin, (B) the differentiation marker 'embryonic myosin', (C) the differentiation marker myogenin, (D) the proliferation marker Ki-67. Bars, 10 μm. Identical magni- fications in A-C. Page 4 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Perinucle fusion of myoblasts in cle Figure 3ar localization o to myotubes, an f centrosome proteins persists a d in adult mouse mus- fter Perinuclear localization of centrosome proteins per- sists after fusion of myoblasts into myotubes, and in adult mouse muscle. (A) Myotube of fused C2C12 cells in culture. Immunofluorescence of PCM-1 (red); DNA is stained in blue. (B) Cryosection through leg muscle from a mouse. Left: immunofluorescence of PCM-1. Middle: nuclei, stained with the DNA marker DAPI. Right: phase contrast image, showing the striations of the muscle tissue. Bar, 10 μm. 4C). Consistent with our immunofluorescence data, immunogold labelling of PCM-1 was often seen in clus- ters (Fig. 4C, top right). The gold was mostly seen along grey electron-dense material surrounding the nuclear sur- face (Fig. 4C, arrows). This electron-dense material had a thickness between 30 and 40 nm, suggesting that was part of a tight matrix. To test for the biochemical behaviour of perinuclear PCM- 1-containing material, we purified nuclei from differenti- ated C2C12 cells that had fused into myotubes. These were separated from non-differentiated myoblasts by short treatment of cultures with trypsin, leading to selec- tive enrichment of the differentiated cells. Following frac- tionation of cells, we performed extraction of purified nuclei with buffer containing various concentrations of salt, detergents, or urea. We found that perinuclear PCM- The centrosome tures Figure 4 on the cytoplasm protein PCM- ic site of th 1 loe n caliz uclear env es to dens elope e struc- 1 localization was largely resistant to 1.5 M NaCl, to treat- The centrosome protein PCM-1 localizes to dense ment with 1% Triton X-100, or even to extraction with 6 structures on the cytoplasmic site of the nuclear M urea (Fig. 5A, B). However, PCM-1 was efficiently envelope. (A) Deconvolved image of a nucleus from a differ- removed from the nuclear surface in 8 M urea, or in buffer entiated H-2K -tsA58 cell, expressing GFP-lamin A (green), containing the denaturing detergent SDS (sodium and stained for PCM-1 (red) and DNA (blue). (B) Nucleus dodecyl sulfate) (Fig. 5A, B). Nuclei treated with SDS lost from a differentiated H-2K -tsA58 cell, stained for PCM-1 (red), and for nuclear pore complex proteins (NPC, green). their integrity even after short times of treatment, and Selected areas of the nuclei in (A) and (B) are shown could therefore not be quantified reproducibly. Alto- enlarged on the right. (C) Immuno-electron microscopy of gether, these results indicate that PCM-1 is part of an cryosections of differentiated C2C12 cells. Different views of insoluble matrix in differentiated muscle cells. Taking cross-sections of the nucleus are shown. PCM-1 is labelled into account that several centrosome proteins form a with antibody and protein A, coupled to 10 nm gold. White fibrous meshwork surrounding the centrioles in undiffer- arrows indicate the outline of a layer of electron-dense entiated cells [16], it seems plausible to assume that a sim- material at the outer nuclear surface. Bars in (B) and (C), 1 ilar fibrous meshwork forms around the nucleus during μm. differentiation. Proteins such as pericentrin that have large predicted alpha-helical domains, and the potential Page 5 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Th Figure 5 e centrosome protein PCM-1 is part of a detergent-resistant perinuclear matrix in differentiated muscle cells The centrosome protein PCM-1 is part of a detergent-resistant perinuclear matrix in differentiated muscle cells. (A) Purified nuclei from differentiated C2C12 cells were incubated for 10 minutes in buffer, containing as indicated, 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. For details, see 'Methods'. Nuclei were spun onto coverslips and processed for immunofluorescence of PCM-1. DNA was stained with 4',6- diamidino-2-phenylindole. Bar, 10 μm. (B) Percentage of nuclei that stained positively for perinuclear PCM-1, following treat- ment as specified in (A). A minimum of three experiments were performed, counting more than 100 nuclei per experiment. to form coiled-coil interactions, might contribute to the cycle events such as S-phase and mitosis in vitro, and formation of such a meshwork. assemble mitotic spindles around various sources of DNA, including exogenously added nuclei (Fig. 6D). To further characterize the nature of the perinuclear mesh- work of centrosome proteins, we designed an in-vitro- We noticed that upon entry of these extracts into a pro- approach to determine whether its formation was reversi- metaphase-like state, C2C12 nuclei began to disassemble. ble in differentiated C2C12 cells upon re-entry into the DNA condensed into mitotic chromosomes, and the peri- cell cycle. Because muscle cells become postmitotic after nuclear 'matrix' of PCM-1 disintegrated into a system of differentiation, as shown by the lack of Ki-67 staining interwoven fibres, and finally disassembled into protein (Fig. 2D), cultures of differentiated C2C12 cells could not aggregates of varying sizes (Fig. 6B, C). We performed simply be driven into mitosis. To circumvent this prob- deconvolution microscopy on nuclei, stained with vari- lem, we isolated nuclei from differentiated cells and incu- ous markers, in S-phase and prometaphase. We found bated them in cycling egg extracts from Xenopus laevis that PCM-1, pericentrin, gamma-tubulin, as well as the (Fig. 6, A–C). These extracts are capable of mimicking cell nuclear envelope protein nesprin 1 localized to patches Page 6 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Figure 6 The perinuclear matrix of centrosome proteins disassembles in mitotic egg extract The perinuclear matrix of centrosome proteins disassembles in mitotic egg extract. Purified nuclei from differenti- ated C2C12 cells were incubated in Xenopus egg extract and driven through the cell cycle in vitro. (A) Nuclei in extract in S- phase. Left: PCM-1; right: DNA. Continued incubation of nuclei in extract entering prometaphase (B), and metaphase (C). Condensation of chromosomes (right) and disintegration of the perinuclear matrix of PCM-1 (left) are visible. (D) Incubation of Xenopus sperm in parallel extract reactions produced mitotic spindles. Left: Rhodamine-labelled tubulin; right: DNA. (E – H) Nuclei from differentiated C2C12 cells were incubated in Xenopus egg extract as in (B). Samples in S-phase and prometaphase were processed for immunofluorescence of PCM-1, pericentrin, gamma-tubulin, and nesprin 1, as indicated. Deconvolved sec- tions are shown. Left column: S-phase; second and third column: prometaphase; right column: merged signals of second and third columns. Bars, (D) 10 μm, (H) 5 μm. Page 7 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 on the nuclear envelope that were closely apposed, but ance), gamma-tubulin (monoclonal antibody GTU-88, without fully co-localizing with each other (Fig. 6, E–H, Sigma), cdk5rap2 (rabbit antibody 46024, raised against left column). In samples that had entered prometaphase, GST-tagged fusion protein, containing amino acids 1–247 we found that PCM-1, pericentrin, and nesprin 1 localized of cdk5rap2), ninein (rabbit antibody 1732, raised against to spots and interconnected fibres that co-localized partly, GST-tagged ninein fusion protein), alpha-tubulin (mono- whereas gamma-tubulin was found more diffusely distrib- clonal antibody DM1a, Sigma), Ki-67 (polyclonal anti- uted (Fig. 6, E–H). We therefore believe that PCM-1 and body M-19, Santa Cruz Biotechnology), embryonic pericentrin form distinct fibrillar structures at the outer myosin (monoclonal antibody, developed by Helen Blau, nuclear surface that are connected at various contact sites, Developmental Studies Hybridoma Bank, University of thus constituting a tight matrix. This matrix may be struc- Iowa), myogenin (monoclonal antibody F5D, Santa Cruz turally equivalent to the fibrous pericentriolar material in Biotechnology), nuclear pore complex proteins (mono- undifferentiated cells, and it can be disassembled or at clonal antibody Mab414, Covance), nesprin 1 (rabbit least loosened upon entry into mitosis. Consistently, antibody, Abcam). The rabbit antibodies against many centrosome proteins, including PCM-1 and pericen- cdk5rap2 and ninein were raised in our laboratory, and trin, are seen in undifferentiated cells during mitosis in a tested for specificity by immunoblotting of bacterial wide crescent-shaped area at the spindle poles or diffuse fusion protein and by immunofluorescence in cultured in the cytoplasm, whereas in interphase they are more cells. The specificity was further confirmed in experiments focused at the centrosome [14,17,18]. using siRNA against cdk5rap2 or ninein, leading to disap- pearance of the respective immunofluorescence signal (unpublished results). DNA was stained with 4',6-diamid- Conclusion In this manuscript, we demonstrate that relocalization of ino-2-phenylindole (DAPI). Histological sections of mus- centrosome proteins to the nuclear surface is an early event cle were prepared from pieces of mouse hind leg muscle, during the differentiation of myoblasts, which occurs prior embedded and frozen in Tissue-Tek (Sakura), using a to their fusion into myotubes. This may imply that the relo- Leica cryostat. Sections were subsequently fixed in metha- calization is important for subsequent differentiation events, nol at -20°C and processed for immunofluorescence. for example by affecting microtubule organization. Further, we show that centrosome proteins form a filamentous Immunoelectron microscopy was performed on differen- matrix around the outer surface of the nuclear envelope. Our tiated C2C12 cells that were fixed overnight in 4% para- biochemical experiments demonstrate that this matrix is formaldehyde in 0.2 M sodium phosphate buffer, pH 7.4. highly insoluble, but disassembles in mitotic cytoplasm. The Ultra-thin frozen sections were prepared as described assembly and disassembly characteristics of this matrix may [20], and labelled with rabbit antibody against PCM1, fol- provide general insights into the organization of the pericen- lowed by protein A conjugated with 10 nm gold. triolar material of centrosome proteins. Future work will be needed to determine the molecular mechanisms that lead to Biochemical methods re-localization of centrosome proteins to the nuclear surface C2C12 cells were differentiated for four days, to obtain a upon muscular differentiation. high yield of differentiated, fused myotubes. After removal of culture medium, cells were treated with 1× Methods Trypsin/EDTA (Gibco) for several seconds, leading to Cell culture selective detachment of fused myotubes. Detachment was C2C12 cells were grown in Dulbecco's modified Eagle's monitored by phase contrast microscopy. Detached cells medium, containing 0.5% chicken embryonic extract and were collected and trypsin was neutralized by addition of 20% fetal calf serum. Differentiation was induced by serum growth medium. Following centrifugation and two starvation for one or more days, by replacing the regular washes with PBS (phosphate-buffered saline), cells were growth medium with Dulbecco's modified Eagle's medium re-suspended in buffer containing 0.2 M KCl, 0.1 M PIPES containing 5% horse serum. H-2K -tsA58 myoblasts were cul- pH 7.4, 0.2 M MgCl , 10 μM cytochalasin B, 0.1 mM phe- tured and differentiated as described [11]. Transfections of H- nylmethyl sulfonylfluoride, and 10 μg/ml of leupeptin, 2K -tsA58 cells with GFP-tagged lamin A [19] were performed pepstatin, and chymostatin. After an additional centrifu- using Lipofectamine Plus transfection agent (Invitrogen). gation and wash in the same buffer, cells were homoge- nized in a douncer with tight-fitting pestle. The material Microscopy was layered onto a 30% sucrose cushion and centrifuged Cells were grown on glass coverslips and fixed in metha- at 850 × g for 10 minutes. The pellet, containing nuclei, nol at -20°C for 10 minutes. Immunofluorescence was was re-suspended in the same buffer as used for homoge- performed using standard procedures. Antibodies used in nization, and centrifuged for 450 × g for 5 minutes. The this study were against PCM-1 (rabbit and mouse anti- resulting nuclei were stored in 50% glycerol, 250 mM PCM-1, [14]), pericentrin (rabbit anti-pericentrin, Cov- sucrose, 80 mM KCl, 20 mM NaCl, 5 mM EGTA, 15 mM Page 8 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 3. Holtzer H, Croop J, Dienstman S, Ishikawa H, Somlyo A: Effects of PIPES pH 7.4, 1 mM dithiothreitol, 0.5 mM spermidin, cytochalasin-B and colcemide on myogenic cultures. Proc Natl 0.2 mM spermin, 0.1 mM phenylmethyl sulfonylfluoride, Acad Sci USA 1975, 72:513-517. and 10 μg/ml of leupeptin, pepstatin, and chymostatin. 4. Antin PB, Forry-Schaudies S, Friedmann TM, Tapscott SJ, Holtzer H: Taxol induces postmitotic myoblasts to assemble interdigi- To examine the solubility of perinuclear PCM-1, extrac- tating microtubule-myosin arrays that exclude actin fila- tion of nuclei was performed for 10 minutes in the same ments. J Cell Biol 1981, 90:300-308. 5. Saitoh O, Arai T, Obinata T: Distribution of microtubules and buffer without glycerol, containing 0.1% Triton X-100, or other cytoskeletal filaments during myotube elongation as 1% Triton X-100, or 1.5 M NaCl, or 6 M urea, or 8 M urea, revealed by fluorescence microscopy. Cell Tissue Res 1988, or 2% sodium dodecyl sulfate. Subsequently, immunoflu- 252:263-273. 6. Pizon V, Gerbal F, Diaz CC, Karsenti E: Microtubule-dependent orescence was performed, following centrifugation of transport and organization of sarcomeric myosin during extracted nuclei through a cushion of 30% glycerol onto skeletal muscle differentiation. EMBO J 2005, 24:3781-3792. glass coverslips. Cell cycle experiments were performed by 7. Straube A, Merdes A: EB3 regulates microtubule dynamics at the cell cortex and is required for myoblast elongation and incubation of purified nuclei in cytostatic factor-arrested fusion. Curr Biol 2007, 17:1318-1325. extracts from Xenopus laevis eggs that were prepared as 8. Tassin AM, Maro B, Bornens M: Fate of microtubule-organizing centers during myogenesis in vitro. J Cell Biol 1985, 100:35-46. described [21]. Extracts were stimulated to cycle through 9. Connolly JA, Kiosses BW, Kalnins VI: Centrioles are lost as and incubation for S-phase by addition of 0.4 mM CaCl embryonic myoblasts fuse into myotubes in vitro. Eur J Cell approximately 40 to 60 minutes. The passage through S- Biol 1986, 39:341-345. 10. Bugnard E, Zaal KJ, Ralston E: Reorganization of microtubule phase was verified in parallel experiments by monitoring nucleation during muscle differentiation. Cell Motil Cytoskeleton DNA synthesis, by incorporating bromodeoxyuridine into 2005, 60:1-13. 11. Musa H, Orton C, Morrison EE, Peckham M: Microtubule assem- nuclei, followed by immunofluorescence [22]. After 90 bly in cultured myoblasts and myotubes following nocoda- minutes, extracts had reached prophase, and a new zole induced microtubule depolymerisation. J Muscle Res Cell mitotic state was stabilized by addition of a fresh 50% vol- Motil 2003, 24:301-308. 12. Morgan JE, Beauchamp JR, Pagel CN, Peckham M, Ataliotis P, Jat PS, ume-equivalent of cytostatic factor-arrested extract. Noble MD, Farmer K, Partridge TA: Myogenic cell lines derived Immunofluorescence of nuclei was performed following from transgenic mice carrying a thermolabile T antigen: a centrifugation onto glass coverslips as described above. model system for the derivation of tissue-specific and muta- tion-specific cell lines. Dev Biol 1994, 162:486-498. The cell cycle state of extracts was monitored in parallel 13. Kubo A, Sasaki H, Yuba-Kubo A, Tsukita S, Shiina N: Centriolar sat- samples incubated with Xenopus laevis sperm and rhod- ellites: molecular characterization, ATP-dependent move- ment toward centrioles and possible involvement in amine-labelled tubulin, to monitor chromosome conden- ciliogenesis. J Cell Biol 1999, 147:969-980. sation and spindle formation. 14. Dammermann A, Merdes A: Assembly of centrosomal proteins and microtubule organization depends on PCM-1. J Cell Biol 2002, 159:255-266. Authors' contributions 15. Young A, Dictenberg JB, Purohit A, Tuft R, Doxsey SJ: Cytoplasmic V.S. and X. F. contributed equally to this manuscript, by dynein-mediated assembly of pericentrin and gamma tubu- participating in the conception of this work, and by per- lin onto centrosomes. Mol Biol Cell 2000, 11:2047-2056. 16. Dictenberg JB, Zimmerman W, Sparks CA, Young A, Vidair C, Zheng forming microscopy and biochemical experiments. C.R. Y, Carrington W, Fay FS, Doxsey SJ: Pericentrin and gamma- performed electron microscopy experiments. R.H. and tubulin form a protein complex and are organized into a novel lattice at the centrosome. J Cell Biol 1998, 141:163-174. A.M. performed experiments on Xenopus egg extracts. 17. Doxsey SJ, Stein P, Evans L, Calarco PD, Kirschner M: Pericentrin, A.M. coordinated this study and drafted the manuscript. a highly conserved centrosome protein involved in microtu- All authors read and approved the final manuscript. bule organization. Cell 1994, 76:639-650. 18. Kubo A, Tsukita S: Non-membranous granular organelle con- sisting of PCM-1: subcellular distribution and cell-cycle- Acknowledgements dependent assembly/disassembly. J Cell Sci 2003, 116:919-928. We thank our colleagues for technical help and stimulating discussions. We 19. Ruchaud S, Korfali N, Villa P, Kottke TJ, Dingwall C, Kaufmann SH, Earnshaw WC: Caspase-6 gene disruption reveals a require- thank Dr Michelle Peckham (University of Leeds) for providing mouse H- ment for lamin A cleavage in apoptotic chromatin condensa- 2K -tsA58 myoblasts. Monoclonal antibody against embryonic myosin, tion. EMBO J 2002, 21:1967-1977. developed by Helen Blau, was obtained from the Developmental Studies 20. Slot JW, Geuze HJ: Cryosectioning and immunolabeling. Nat Hybridoma Bank developed under the auspices of the NICHD and main- Protoc 2007, 2:2480-2491. 21. Murray AW: Cell cycle extracts. Methods Cell Biol 1991, tained by the University of Iowa. We also thank Despina Xanthakis 36:581-605. (Department of Cell Biology, UMC Utrecht) for help with the immuno- 22. Merdes A, Cleveland DW: The role of NuMA in the interphase electron microscopy. The work was supported in part by a Wellcome nucleus. J Cell Sci 1998, 111:71-79. Trust Senior Research Fellowship to A.M., by a Wellcome Trust Prize Fel- lowship 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. References 1. Lassar AB, Skapek SX, Novitch B: Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr Opin Cell Biol 1994, 6:788-794. 2. Warren RH: Microtubular organization in elongating myo- genic cells. J Cell Biol 1974, 63:550-566. Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Cell Biology Springer Journals

Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

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Copyright © 2009 by Srsen et al; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Biological Microscopy; Life Sciences, general
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19383121
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Abstract

Background: 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. Results: 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. Conclusion: 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. Background believed that this reorganization is a prerequisite for the The formation of muscle during embryonic development elongation and fusion of myoblasts, and for the subse- involves the differentiation of myoblasts into long, quent alignment and organization of sarcomeres [3-7]. fibrous cells. In this differentiation process, myoblasts Microtubule reorganization is paralleled by reorganiza- withdraw from the cell cycle and fuse into multinucleate, tion of centrosomal proteins: myoblasts possess a mor- syncytial myotubes [1]. The microtubule cytoskeleton is phologically recognizable centrosome with characteristic reorganized from a radial network into a parallel array of marker proteins concentrated in the pericentriolar mate- filaments aligned along the long axis of the cells [2]. It is rial, whereas myotubes show perinuclear localization of a Page 1 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 multitude of centrosome proteins [8-10]. Consequently, nuclear surface next to the centrosome due to minus-end polymerization of microtubules is initiated in part from directed transport along microtubules [15], since the the surface of the nucleus [8,10,11]. It has been reported microtubule-organizing centre in these myoblasts is that reorganization of microtubules and relocalization of strongly focused at the centrosome and would thus favour centrosome proteins to the nuclear surface occurs after deposit of pericentrin in the surrounding area (Fig. 2A, fusion of myoblasts into myotubes [8]. However, the pre- cell marked with 'u'). It is noteworthy, however, that cise kinetics of this reorganization are unknown. Morover, already at the single cell stage microtubules were reorgan- it is unknown how centrosome proteins are attached to ized into a sun-like array radiating from the nuclear the nuclear surface, and how they are organized at the periphery within those cells displaying perinuclear relo- ultrastructural level. calization of centrosome proteins (Fig. 2A, cell marked with 'd'). Results and Discussion In order to determine at what stage of the differentiation To verify that these cells had indeed entered the differen- process relocalization of centrosome proteins occurs, we tiation programme, we performed immunofluorescence performed cell culture of mouse C2C12 myoblasts and trig- microscopy with differentiation markers. We noticed that gered differentiation by serum withdrawal. Alternatively, all cells in which the centrosome protein PCM-1 had relo- we used H-2K -tsA58 mouse myoblasts, carrying a thermo- calized to the nuclear surface showed cytoplasmic expres- labile T-antigen and allowing differentiation upon temper- sion of embryonic myosin, an isoform of myosin that is ature shift from 33 to 37°C [11,12]. In the undifferentiated specifically expressed upon onset of muscle cell differenti- state, the centrosomal proteins of both cell lines were ation (Fig. 2B). Furthermore, these cells expressed the dif- found in a single focus within the pericentriolar material ferentiation marker myogenin, a transcription factor that adjacent to the nucleus, while the protein PCM-1 ("pericen- localizes to the nucleus of differentiating cells (Fig. 2C). triolar material protein 1") localized to multiple 'centriolar This marker was absent from cells showing pericentriolar satellites', as described by [13] and [14]. One day after dif- PCM-1 staining. Conversely, these cells with pericentri- ferentiation of myoblasts was triggered, we observed cen- olar PCM-1 were the only ones that stained positively for trosome proteins at the nuclear periphery already at the the proliferation marker Ki-67 in the nucleus, whereas single cell stage, prior to fusion into myotubes (Fig. 1). nuclei in cells with relocalized PCM-1 did not contain Ki- 67 (Fig. 2D). Once relocalization of PCM-1 occurred, it We investigated the pattern of various proteins and found persisted until the final stages of differentiation: We that significant amounts of pericentrin and PCM-1 accu- detected PCM-1 around the nuclear surface after fusion of mulated at the nuclear periphery, whereas the centrosome multiple C2C12 cells into myotubes (Fig. 3A). Moreover, protein cdk5rap2 showed partial relocalization. Only nuclei in muscle from adult mice showed comparable minor amounts of gamma-tubulin were found at the staining of PCM-1 at their surface (Fig. 3B). nuclear periphery, consistent with data from [10], and the protein ninein was not found to relocalize to the nuclear We next sought to determine whether centrosome pro- periphery in our experiments. teins that had assembled around the nucleus were bound at the inside or at the outside of the nuclear envelope. For A closer look at differentiating C2C12 cells revealed that this purpose, we transfected mouse myoblasts with GFP- at the single cell stage, proteins such as pericentrin and tagged lamin A, a protein of the inner nuclear envelope. PCM-1 started to accumulate at the nuclear periphery in Deconvolution microscopy revealed that the centrosome the proximity of the centrosome, whereas areas of the protein PCM-1 localized in a rim slightly outside of lamin nuclear envelope distal from the centrosome showed less A (Fig. 4A), suggesting that PCM-1 associates with the centrosome protein enrichment (Fig. 1, first two rows). At outer surface of the nuclear envelope. Because the PCM-1 the same time, remnant pericentrin and cdk5rap2 were staining at the nuclear surface was visible in small clusters, still visible at the centrosome, and PCM-1 was still partly we tested whether these sites co-localized with nuclear localized in pericentriolar satellites (Fig. 1). The early relo- pores. However, double immunofluorescence with an calization of these proteins from the centrosome to antibody against a family of nuclear pore complex pro- nearby sites of the nuclear surface raises the question as to teins indicated that most of the PCM-1 clusters were dis- whether direct transfer from the centrosome is involved, tinct from nuclear pores (Fig. 4B). To reveal further or whether the proteins are recruited to the nuclear surface structural details, we performed immuno-electron micro- from a soluble cytoplasmic pool. Both scenarios seem scopy of ultra-thin cryosections of differentiated C2C12 possible: direct transfer from the centrosome to the cells. PCM-1 was detected with antibodies and gold-cou- nucleus might occur by diffusion. Alternatively, at the pled protein A. This technique preserved cellular mem- onset of differentiation, proteins such as pericentrin branes and showed that PCM-1 localized mainly outside might be recruited from a soluble pool and localize to the the nucleus, away from the outer nuclear membrane (Fig. Page 2 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Figure 1 Relocalization of centrosome proteins to the nuclear surface in differentiating myoblasts Relocalization of centrosome proteins to the nuclear surface in differentiating myoblasts. Cultures of C2C12 myoblasts were induced to differentiate by serum starvation for one day. At this stage, the culture contains undifferentiated myoblasts (u), as well as cells that started to differentiate (d). Immunofluorescence of these cultures was performed to visualize the centrosome proteins PCM-1 (red), as well as pericentrin, gamma-tubulin, cdk5rap2, and ninein (all green). DNA was stained in blue. Arrows indicate partial staining of the remnant centrosomes by pericentrin, gamma-tubulin, and cdk5rap2. Bar, 10 μm. Page 3 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Reorga Figure 2 nization of microtububules and centrosome protein in cells expressing myogenic differentiation markers Reorganization of microtububules and centrosome protein in cells expressing myogenic differentiation mark- ers. Culture of mouse myoblasts containing undifferentiated (u) cells, and cells that started to differentiate (d). The centro- some protein PCM-1 is stained in red, DNA is stained in blue. In green is marked (A) tubulin, (B) the differentiation marker 'embryonic myosin', (C) the differentiation marker myogenin, (D) the proliferation marker Ki-67. Bars, 10 μm. Identical magni- fications in A-C. Page 4 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Perinucle fusion of myoblasts in cle Figure 3ar localization o to myotubes, an f centrosome proteins persists a d in adult mouse mus- fter Perinuclear localization of centrosome proteins per- sists after fusion of myoblasts into myotubes, and in adult mouse muscle. (A) Myotube of fused C2C12 cells in culture. Immunofluorescence of PCM-1 (red); DNA is stained in blue. (B) Cryosection through leg muscle from a mouse. Left: immunofluorescence of PCM-1. Middle: nuclei, stained with the DNA marker DAPI. Right: phase contrast image, showing the striations of the muscle tissue. Bar, 10 μm. 4C). Consistent with our immunofluorescence data, immunogold labelling of PCM-1 was often seen in clus- ters (Fig. 4C, top right). The gold was mostly seen along grey electron-dense material surrounding the nuclear sur- face (Fig. 4C, arrows). This electron-dense material had a thickness between 30 and 40 nm, suggesting that was part of a tight matrix. To test for the biochemical behaviour of perinuclear PCM- 1-containing material, we purified nuclei from differenti- ated C2C12 cells that had fused into myotubes. These were separated from non-differentiated myoblasts by short treatment of cultures with trypsin, leading to selec- tive enrichment of the differentiated cells. Following frac- tionation of cells, we performed extraction of purified nuclei with buffer containing various concentrations of salt, detergents, or urea. We found that perinuclear PCM- The centrosome tures Figure 4 on the cytoplasm protein PCM- ic site of th 1 loe n caliz uclear env es to dens elope e struc- 1 localization was largely resistant to 1.5 M NaCl, to treat- The centrosome protein PCM-1 localizes to dense ment with 1% Triton X-100, or even to extraction with 6 structures on the cytoplasmic site of the nuclear M urea (Fig. 5A, B). However, PCM-1 was efficiently envelope. (A) Deconvolved image of a nucleus from a differ- removed from the nuclear surface in 8 M urea, or in buffer entiated H-2K -tsA58 cell, expressing GFP-lamin A (green), containing the denaturing detergent SDS (sodium and stained for PCM-1 (red) and DNA (blue). (B) Nucleus dodecyl sulfate) (Fig. 5A, B). Nuclei treated with SDS lost from a differentiated H-2K -tsA58 cell, stained for PCM-1 (red), and for nuclear pore complex proteins (NPC, green). their integrity even after short times of treatment, and Selected areas of the nuclei in (A) and (B) are shown could therefore not be quantified reproducibly. Alto- enlarged on the right. (C) Immuno-electron microscopy of gether, these results indicate that PCM-1 is part of an cryosections of differentiated C2C12 cells. Different views of insoluble matrix in differentiated muscle cells. Taking cross-sections of the nucleus are shown. PCM-1 is labelled into account that several centrosome proteins form a with antibody and protein A, coupled to 10 nm gold. White fibrous meshwork surrounding the centrioles in undiffer- arrows indicate the outline of a layer of electron-dense entiated cells [16], it seems plausible to assume that a sim- material at the outer nuclear surface. Bars in (B) and (C), 1 ilar fibrous meshwork forms around the nucleus during μm. differentiation. Proteins such as pericentrin that have large predicted alpha-helical domains, and the potential Page 5 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Th Figure 5 e centrosome protein PCM-1 is part of a detergent-resistant perinuclear matrix in differentiated muscle cells The centrosome protein PCM-1 is part of a detergent-resistant perinuclear matrix in differentiated muscle cells. (A) Purified nuclei from differentiated C2C12 cells were incubated for 10 minutes in buffer, containing as indicated, 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. For details, see 'Methods'. Nuclei were spun onto coverslips and processed for immunofluorescence of PCM-1. DNA was stained with 4',6- diamidino-2-phenylindole. Bar, 10 μm. (B) Percentage of nuclei that stained positively for perinuclear PCM-1, following treat- ment as specified in (A). A minimum of three experiments were performed, counting more than 100 nuclei per experiment. to form coiled-coil interactions, might contribute to the cycle events such as S-phase and mitosis in vitro, and formation of such a meshwork. assemble mitotic spindles around various sources of DNA, including exogenously added nuclei (Fig. 6D). To further characterize the nature of the perinuclear mesh- work of centrosome proteins, we designed an in-vitro- We noticed that upon entry of these extracts into a pro- approach to determine whether its formation was reversi- metaphase-like state, C2C12 nuclei began to disassemble. ble in differentiated C2C12 cells upon re-entry into the DNA condensed into mitotic chromosomes, and the peri- cell cycle. Because muscle cells become postmitotic after nuclear 'matrix' of PCM-1 disintegrated into a system of differentiation, as shown by the lack of Ki-67 staining interwoven fibres, and finally disassembled into protein (Fig. 2D), cultures of differentiated C2C12 cells could not aggregates of varying sizes (Fig. 6B, C). We performed simply be driven into mitosis. To circumvent this prob- deconvolution microscopy on nuclei, stained with vari- lem, we isolated nuclei from differentiated cells and incu- ous markers, in S-phase and prometaphase. We found bated them in cycling egg extracts from Xenopus laevis that PCM-1, pericentrin, gamma-tubulin, as well as the (Fig. 6, A–C). These extracts are capable of mimicking cell nuclear envelope protein nesprin 1 localized to patches Page 6 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 Figure 6 The perinuclear matrix of centrosome proteins disassembles in mitotic egg extract The perinuclear matrix of centrosome proteins disassembles in mitotic egg extract. Purified nuclei from differenti- ated C2C12 cells were incubated in Xenopus egg extract and driven through the cell cycle in vitro. (A) Nuclei in extract in S- phase. Left: PCM-1; right: DNA. Continued incubation of nuclei in extract entering prometaphase (B), and metaphase (C). Condensation of chromosomes (right) and disintegration of the perinuclear matrix of PCM-1 (left) are visible. (D) Incubation of Xenopus sperm in parallel extract reactions produced mitotic spindles. Left: Rhodamine-labelled tubulin; right: DNA. (E – H) Nuclei from differentiated C2C12 cells were incubated in Xenopus egg extract as in (B). Samples in S-phase and prometaphase were processed for immunofluorescence of PCM-1, pericentrin, gamma-tubulin, and nesprin 1, as indicated. Deconvolved sec- tions are shown. Left column: S-phase; second and third column: prometaphase; right column: merged signals of second and third columns. Bars, (D) 10 μm, (H) 5 μm. Page 7 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 on the nuclear envelope that were closely apposed, but ance), gamma-tubulin (monoclonal antibody GTU-88, without fully co-localizing with each other (Fig. 6, E–H, Sigma), cdk5rap2 (rabbit antibody 46024, raised against left column). In samples that had entered prometaphase, GST-tagged fusion protein, containing amino acids 1–247 we found that PCM-1, pericentrin, and nesprin 1 localized of cdk5rap2), ninein (rabbit antibody 1732, raised against to spots and interconnected fibres that co-localized partly, GST-tagged ninein fusion protein), alpha-tubulin (mono- whereas gamma-tubulin was found more diffusely distrib- clonal antibody DM1a, Sigma), Ki-67 (polyclonal anti- uted (Fig. 6, E–H). We therefore believe that PCM-1 and body M-19, Santa Cruz Biotechnology), embryonic pericentrin form distinct fibrillar structures at the outer myosin (monoclonal antibody, developed by Helen Blau, nuclear surface that are connected at various contact sites, Developmental Studies Hybridoma Bank, University of thus constituting a tight matrix. This matrix may be struc- Iowa), myogenin (monoclonal antibody F5D, Santa Cruz turally equivalent to the fibrous pericentriolar material in Biotechnology), nuclear pore complex proteins (mono- undifferentiated cells, and it can be disassembled or at clonal antibody Mab414, Covance), nesprin 1 (rabbit least loosened upon entry into mitosis. Consistently, antibody, Abcam). The rabbit antibodies against many centrosome proteins, including PCM-1 and pericen- cdk5rap2 and ninein were raised in our laboratory, and trin, are seen in undifferentiated cells during mitosis in a tested for specificity by immunoblotting of bacterial wide crescent-shaped area at the spindle poles or diffuse fusion protein and by immunofluorescence in cultured in the cytoplasm, whereas in interphase they are more cells. The specificity was further confirmed in experiments focused at the centrosome [14,17,18]. using siRNA against cdk5rap2 or ninein, leading to disap- pearance of the respective immunofluorescence signal (unpublished results). DNA was stained with 4',6-diamid- Conclusion In this manuscript, we demonstrate that relocalization of ino-2-phenylindole (DAPI). Histological sections of mus- centrosome proteins to the nuclear surface is an early event cle were prepared from pieces of mouse hind leg muscle, during the differentiation of myoblasts, which occurs prior embedded and frozen in Tissue-Tek (Sakura), using a to their fusion into myotubes. This may imply that the relo- Leica cryostat. Sections were subsequently fixed in metha- calization is important for subsequent differentiation events, nol at -20°C and processed for immunofluorescence. for example by affecting microtubule organization. Further, we show that centrosome proteins form a filamentous Immunoelectron microscopy was performed on differen- matrix around the outer surface of the nuclear envelope. Our tiated C2C12 cells that were fixed overnight in 4% para- biochemical experiments demonstrate that this matrix is formaldehyde in 0.2 M sodium phosphate buffer, pH 7.4. highly insoluble, but disassembles in mitotic cytoplasm. The Ultra-thin frozen sections were prepared as described assembly and disassembly characteristics of this matrix may [20], and labelled with rabbit antibody against PCM1, fol- provide general insights into the organization of the pericen- lowed by protein A conjugated with 10 nm gold. triolar material of centrosome proteins. Future work will be needed to determine the molecular mechanisms that lead to Biochemical methods re-localization of centrosome proteins to the nuclear surface C2C12 cells were differentiated for four days, to obtain a upon muscular differentiation. high yield of differentiated, fused myotubes. After removal of culture medium, cells were treated with 1× Methods Trypsin/EDTA (Gibco) for several seconds, leading to Cell culture selective detachment of fused myotubes. Detachment was C2C12 cells were grown in Dulbecco's modified Eagle's monitored by phase contrast microscopy. Detached cells medium, containing 0.5% chicken embryonic extract and were collected and trypsin was neutralized by addition of 20% fetal calf serum. Differentiation was induced by serum growth medium. Following centrifugation and two starvation for one or more days, by replacing the regular washes with PBS (phosphate-buffered saline), cells were growth medium with Dulbecco's modified Eagle's medium re-suspended in buffer containing 0.2 M KCl, 0.1 M PIPES containing 5% horse serum. H-2K -tsA58 myoblasts were cul- pH 7.4, 0.2 M MgCl , 10 μM cytochalasin B, 0.1 mM phe- tured and differentiated as described [11]. Transfections of H- nylmethyl sulfonylfluoride, and 10 μg/ml of leupeptin, 2K -tsA58 cells with GFP-tagged lamin A [19] were performed pepstatin, and chymostatin. After an additional centrifu- using Lipofectamine Plus transfection agent (Invitrogen). gation and wash in the same buffer, cells were homoge- nized in a douncer with tight-fitting pestle. The material Microscopy was layered onto a 30% sucrose cushion and centrifuged Cells were grown on glass coverslips and fixed in metha- at 850 × g for 10 minutes. The pellet, containing nuclei, nol at -20°C for 10 minutes. Immunofluorescence was was re-suspended in the same buffer as used for homoge- performed using standard procedures. Antibodies used in nization, and centrifuged for 450 × g for 5 minutes. The this study were against PCM-1 (rabbit and mouse anti- resulting nuclei were stored in 50% glycerol, 250 mM PCM-1, [14]), pericentrin (rabbit anti-pericentrin, Cov- sucrose, 80 mM KCl, 20 mM NaCl, 5 mM EGTA, 15 mM Page 8 of 9 (page number not for citation purposes) BMC Cell Biology 2009, 10:28 http://www.biomedcentral.com/1471-2121/10/28 3. Holtzer H, Croop J, Dienstman S, Ishikawa H, Somlyo A: Effects of PIPES pH 7.4, 1 mM dithiothreitol, 0.5 mM spermidin, cytochalasin-B and colcemide on myogenic cultures. Proc Natl 0.2 mM spermin, 0.1 mM phenylmethyl sulfonylfluoride, Acad Sci USA 1975, 72:513-517. and 10 μg/ml of leupeptin, pepstatin, and chymostatin. 4. Antin PB, Forry-Schaudies S, Friedmann TM, Tapscott SJ, Holtzer H: Taxol induces postmitotic myoblasts to assemble interdigi- To examine the solubility of perinuclear PCM-1, extrac- tating microtubule-myosin arrays that exclude actin fila- tion of nuclei was performed for 10 minutes in the same ments. J Cell Biol 1981, 90:300-308. 5. Saitoh O, Arai T, Obinata T: Distribution of microtubules and buffer without glycerol, containing 0.1% Triton X-100, or other cytoskeletal filaments during myotube elongation as 1% Triton X-100, or 1.5 M NaCl, or 6 M urea, or 8 M urea, revealed by fluorescence microscopy. Cell Tissue Res 1988, or 2% sodium dodecyl sulfate. Subsequently, immunoflu- 252:263-273. 6. Pizon V, Gerbal F, Diaz CC, Karsenti E: Microtubule-dependent orescence was performed, following centrifugation of transport and organization of sarcomeric myosin during extracted nuclei through a cushion of 30% glycerol onto skeletal muscle differentiation. EMBO J 2005, 24:3781-3792. glass coverslips. Cell cycle experiments were performed by 7. Straube A, Merdes A: EB3 regulates microtubule dynamics at the cell cortex and is required for myoblast elongation and incubation of purified nuclei in cytostatic factor-arrested fusion. Curr Biol 2007, 17:1318-1325. extracts from Xenopus laevis eggs that were prepared as 8. Tassin AM, Maro B, Bornens M: Fate of microtubule-organizing centers during myogenesis in vitro. J Cell Biol 1985, 100:35-46. described [21]. Extracts were stimulated to cycle through 9. Connolly JA, Kiosses BW, Kalnins VI: Centrioles are lost as and incubation for S-phase by addition of 0.4 mM CaCl embryonic myoblasts fuse into myotubes in vitro. Eur J Cell approximately 40 to 60 minutes. The passage through S- Biol 1986, 39:341-345. 10. Bugnard E, Zaal KJ, Ralston E: Reorganization of microtubule phase was verified in parallel experiments by monitoring nucleation during muscle differentiation. Cell Motil Cytoskeleton DNA synthesis, by incorporating bromodeoxyuridine into 2005, 60:1-13. 11. Musa H, Orton C, Morrison EE, Peckham M: Microtubule assem- nuclei, followed by immunofluorescence [22]. After 90 bly in cultured myoblasts and myotubes following nocoda- minutes, extracts had reached prophase, and a new zole induced microtubule depolymerisation. J Muscle Res Cell mitotic state was stabilized by addition of a fresh 50% vol- Motil 2003, 24:301-308. 12. Morgan JE, Beauchamp JR, Pagel CN, Peckham M, Ataliotis P, Jat PS, ume-equivalent of cytostatic factor-arrested extract. Noble MD, Farmer K, Partridge TA: Myogenic cell lines derived Immunofluorescence of nuclei was performed following from transgenic mice carrying a thermolabile T antigen: a centrifugation onto glass coverslips as described above. model system for the derivation of tissue-specific and muta- tion-specific cell lines. Dev Biol 1994, 162:486-498. The cell cycle state of extracts was monitored in parallel 13. Kubo A, Sasaki H, Yuba-Kubo A, Tsukita S, Shiina N: Centriolar sat- samples incubated with Xenopus laevis sperm and rhod- ellites: molecular characterization, ATP-dependent move- ment toward centrioles and possible involvement in amine-labelled tubulin, to monitor chromosome conden- ciliogenesis. J Cell Biol 1999, 147:969-980. sation and spindle formation. 14. Dammermann A, Merdes A: Assembly of centrosomal proteins and microtubule organization depends on PCM-1. J Cell Biol 2002, 159:255-266. Authors' contributions 15. Young A, Dictenberg JB, Purohit A, Tuft R, Doxsey SJ: Cytoplasmic V.S. and X. F. contributed equally to this manuscript, by dynein-mediated assembly of pericentrin and gamma tubu- participating in the conception of this work, and by per- lin onto centrosomes. Mol Biol Cell 2000, 11:2047-2056. 16. Dictenberg JB, Zimmerman W, Sparks CA, Young A, Vidair C, Zheng forming microscopy and biochemical experiments. C.R. Y, Carrington W, Fay FS, Doxsey SJ: Pericentrin and gamma- performed electron microscopy experiments. R.H. and tubulin form a protein complex and are organized into a novel lattice at the centrosome. J Cell Biol 1998, 141:163-174. A.M. performed experiments on Xenopus egg extracts. 17. Doxsey SJ, Stein P, Evans L, Calarco PD, Kirschner M: Pericentrin, A.M. coordinated this study and drafted the manuscript. a highly conserved centrosome protein involved in microtu- All authors read and approved the final manuscript. bule organization. Cell 1994, 76:639-650. 18. Kubo A, Tsukita S: Non-membranous granular organelle con- sisting of PCM-1: subcellular distribution and cell-cycle- Acknowledgements dependent assembly/disassembly. J Cell Sci 2003, 116:919-928. We thank our colleagues for technical help and stimulating discussions. We 19. Ruchaud S, Korfali N, Villa P, Kottke TJ, Dingwall C, Kaufmann SH, Earnshaw WC: Caspase-6 gene disruption reveals a require- thank Dr Michelle Peckham (University of Leeds) for providing mouse H- ment for lamin A cleavage in apoptotic chromatin condensa- 2K -tsA58 myoblasts. Monoclonal antibody against embryonic myosin, tion. EMBO J 2002, 21:1967-1977. developed by Helen Blau, was obtained from the Developmental Studies 20. Slot JW, Geuze HJ: Cryosectioning and immunolabeling. Nat Hybridoma Bank developed under the auspices of the NICHD and main- Protoc 2007, 2:2480-2491. 21. Murray AW: Cell cycle extracts. Methods Cell Biol 1991, tained by the University of Iowa. We also thank Despina Xanthakis 36:581-605. (Department of Cell Biology, UMC Utrecht) for help with the immuno- 22. Merdes A, Cleveland DW: The role of NuMA in the interphase electron microscopy. The work was supported in part by a Wellcome nucleus. J Cell Sci 1998, 111:71-79. Trust Senior Research Fellowship to A.M., by a Wellcome Trust Prize Fel- lowship 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. References 1. Lassar AB, Skapek SX, Novitch B: Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr Opin Cell Biol 1994, 6:788-794. 2. Warren RH: Microtubular organization in elongating myo- genic cells. J Cell Biol 1974, 63:550-566. Page 9 of 9 (page number not for citation purposes)

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