BMC Cell Biol
April 13, 2009;
Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation.
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.
BMC Cell Biol
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Figure 1. 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.
Figure 2. Reorganization of microtububules and centrosome protein in cells expressing myogenic differentiation markers. Culture of mouse myoblasts containing undifferentiated (u) cells, and cells that started to differentiate (d). The centrosome 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 magnifications in A-C.
Figure 3. Perinuclear localization of centrosome proteins persists 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.
Figure 4. The centrosome protein PCM-1 localizes to dense structures on the cytoplasmic site of the nuclear envelope. (A) Deconvolved image of a nucleus from a differentiated H-2Kb-tsA58 cell, expressing GFP-lamin A (green), and stained for PCM-1 (red) and DNA (blue). (B) Nucleus from a differentiated H-2Kb-tsA58 cell, stained for PCM-1 (red), and for nuclear pore complex proteins (NPC, green). Selected areas of the nuclei in (A) and (B) are shown enlarged on the right. (C) Immuno-electron microscopy of cryosections of differentiated C2C12 cells. Different views of cross-sections of the nucleus are shown. PCM-1 is labelled with antibody and protein A, coupled to 10 nm gold. White arrows indicate the outline of a layer of electron-dense material at the outer nuclear surface. Bars in (B) and (C), 1 μm.
Figure 5. 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 treatment as specified in (A). A minimum of three experiments were performed, counting more than 100 nuclei per experiment.
Figure 6. The perinuclear matrix of centrosome proteins disassembles in mitotic egg extract. Purified nuclei from differentiated 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 sections 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.
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