Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Nuclei of Non-Muscle Cells Bind Centrosome Proteins upon Fusion with Differentiating Myoblasts

Background: In differentiating myoblasts, the microtubule network is reorganized from a centrosome-bound, radial array into parallel fibres, aligned along the long axis of the cell. Concomitantly, proteins of the centrosome relocalize from the pericentriolar material to the outer surface of the nucleus. The mechanisms that govern this relocalization are largely unknown. Methodology: In this study, we perform experiments in vitro and in cell culture indicating that microtubule nucleation at the centrosome is reduced during myoblast differentiation, while nucleation at the nuclear surface increases. We show in heterologous cell fusion experiments, between cultures of differentiating mouse myoblasts and human cells of nonmuscular origin, that nuclei from non-muscle cells recruit centrosome proteins once fused with the differentiating myoblasts. This recruitment still occurs in the presence of cycloheximide and thus appears to be independent of new protein biosynthesis. Conclusions: Altogether, our data suggest that nuclei of undifferentiated cells have the dormant potential to bin

The Mitotic Protein Kinase Haspin and Its Inhibitors

Haspin is an atypical serine/threonine protein kinase essential to mitosis. Unlike other protein kinases, its kinase domain does not require phosphorylation in order to be activated and bears very high substrate specificity and selectivity. Few substrates have been identified so far. Haspin phosphorylation on threonine 3 of Histone H3 from prophase to anaphase participates to centromeric Aurora B localization and ensures proper kinetochore-microtubule attachment. Haspin is also involved in the maintenance of centromeric cohesion and the mitotic spindle. Inhibitors have been developed and provided tools to dissect Haspin function. The kinase is now considered as a potential therapeutic target against cancer. We discuss here the latest findings on this essential mitotic protein

Use of D140 conditional-knockout cell lines to study chromosomal passenger protein function

The chromosomal passenger complex (CPC-INCENP, Aurora B kinase, Survivin and Borealin) is implicated in many mitotic processes. Here we describe how we generated DT40 conditional knockout cell lines for incenp1 and survivin1 to better understand the role of these CPC subunits in the control of Aurora B kinase activity. These lines enabled us to reassess current knowledge of Survivin function and to show that INCENP acts as a rheostat for Aurora B activity

Stability of the small ?-tubulin complex requiresHCA66, a protein of the centrosome and the nucleolus.

To investigate changes at the centrosome during the cell cycle,we analyzed the composition of the pericentriolar materialfrom unsynchronized and S-phase-arrested cells by gelelectrophoresis and mass spectrometry. We identified HCA66,a protein that localizes to the centrosome from S-phase to mitosisand to the nucleolus throughout interphase. Silencing of HCA66expression resulted in failure of centrosome duplicationand in the formation of monopolar spindles, reminiscentof the phenotype observed after ?-tubulin silencing.Immunofluorescence microscopy showed that proteins of the?-tubulin ring complex were absent from the centrosome inthese monopolar spindles. Immunoblotting revealed reducedprotein levels of all components of the ?-tubulin small complex(?-tubulin, GCP2, and GCP3) in HCA66-depleted cells. Bycontrast, the levels of ?-tubulin ring complex proteins such asGCP4 and GCP-WD/NEDD1 were unaffected. We propose thatHCA66 is a novel regulator of ?-tubulin function that plays arole in stabilizing components of the ?-tubulin small complex,which is in turn essential for assembling the larger ?-tubulinring complex

Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation.

BackgroundMuscle 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.ResultsWe 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.ConclusionOur 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

Nucleation of microtubules from salt-stripped centrosomes is restored by cytoplasmic extract of myotubes.

<p>(A) Isolated centrosomes from Jurkat cells, treated with 1M potassium iodide (KI) to strip the pericentriolar material. An immunoblot is shown, probed for gamma-tubulin in unstripped centrosomes (centr.), as well as in centrosomes after KI-treatment for 15, 30, 45, and 60 minutes. Treated centrosomes were fractionated by centrifugation, to separate the extracted pericentriolar material (supernatants, S), and the insoluble centrioles (pellets, P). (B) Purified centrosomes, centrifuged onto glass coverslips and stained for immunofluorescence of pericentrin (B1) and gamma-tubulin (B2). Centrosomes were incubated with purified tubulin at 5 mg/ml for 10 minutes, either directly (B3), or following incubation with cytoplasmic extract from undifferentiated myoblasts (B4) or from myotubes (B5). (C) Equivalent experiments as in (B), using centrosomes that had been stripped with KI for one hour. (D) The number of microtubules/aster in a focal plane, as well as the average microtubule length were quantified in each case (n≥34). Bar in (C), 10 µm.</p

Microtubule nucleation from the centrosome is reduced in the differentiation process.

<p>(A) Deconvolved optical sections of differentiated muscle cells after re-growth of microtubules for 20 seconds, stained for immunofluorescence of (left) microtubules, (middle) the centrosome protein pericentrin, and (right) stained with the DNA marker DAPI. In a myotube early after fusion, pericentrin is visible both around the nuclear surface and on the centrosomes (arrows). Microtubule re-growth is seen from these centrosomes, in addition to perinuclear and cytoplasmic sites. (B) Myotube, after cold-induced depolymerization of microtubules and re-growth for 10 seconds. Immunofluorescence of the plus-end-binding protein EB3 and of pericentrin are shown. Cytoplasmic, nuclear, and centrosomal sites of emerging EB3 comets are indicated on the left. The table indicates the percentage of EB3 comets growing from cytoplasmic, nuclear, and centrosomal locations in undifferentiated cells (n = 6), as well as in differentiating cells before fusion (mononucleate, n = 15), and after fusion (n = 12). The total number of EB3 comets per cell ranged from 39 (undifferentiated) to 1151 (differentiated, fused). (C) Left: nucleus of a myoblast at an early stage of differentiation, stained for pericentrin (top) and microtubules (bottom), recovering for 10 seconds from cold-induced depolymerization. The number of microtubules grown from the nuclear surface was quantified by counting EB3 comets as in (B). The results for each nucleus were plotted against the intensity of perinuclear pericentrin (arbitrary values, after subtraction of background) in undifferentiated cells (n = 6), and in mononucleate (n = 15) and fused differentiated cells (n = 22). Horizontal and vertical bars indicate standard deviations of pericentrin intensity and of the number of EB3 comets, respectively, in differentiated mononucleate and fused cells. Bar in (A), 10 µm; identical magnification in (B), (C).</p

Centrosome proteins are recruited to the nuclear surface of non-muscle cells, after fusion with differentiated muscle cells.

<p>(A) Co-culture of <i>H-2K</i>^b-tsA58 mouse muscle cells with human U2OS cells. Top left: phase contrast; top middle: immunofluorescence of the centrosome protein PCM-1; top right: staining of the DNA with DAPI; bottom left: immunofluorescence of the human form of the nuclear protein NuMA; bottom right: merge of PCM-1 (green) and NuMA (red) staining. Note that NuMA staining is exclusively visible in nuclei of the human U2OS cells. Mouse nuclei (m) show characteristic condensation of heterochromatin in discrete nucleoplasmic punctuate staining that is absent from the surrounding human nuclei. (B) Heterokaryon, formed by fusion of a human U2OS cell with a mouse <i>H-2K</i>^b-tsA58 cell. A cell is shown at 48 hours after induction of fusion with polyethylene glycol. Staining and microscopy as in (A). The human nucleus, as identified by NuMA immunofluorescence, shows accumulation of perinuclear PCM-1. (C) Heterokaryon, 6 hours after induction of fusion, stained for pericentrin (red), tubulin (green), and DNA (blue). “m” and “h” indicate the nuclei contributed from the mouse <i>H-2K</i>^b-tsA58 cell, or from the human U2OS cells, respectively. Insets show an enlarged area of the cytoplasm, containing remnants of the centrosomes. (D) Histograms, showing PCM-1 intensity at human nuclei in heterokaryons, as a percentage of PCM-1 immunofluorescence levels around mouse nuclei from the same heterokaryon (6 h: n = 20; 24 h: n = 45; 48 h: n = 22). Bars in (B), (C), 10 µm.</p