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1.
Glutamylation is the major posttranslational modification of neuronal and axonemal tubulin and is restricted predominantly to centrioles in nonneuronal cells (Bobinnec, Y., M. Moudjou, J.P. Fouquet, E. Desbruyères, B. Eddé, and M. Bornens. 1998. Cell Motil. Cytoskel. 39:223–232). To investigate a possible relationship between the exceptional stability of centriole microtubules and the compartmentalization of glutamylated isoforms, we loaded HeLa cells with the monoclonal antibody GT335, which specifically reacts with polyglutamylated tubulin. The total disappearance of the centriole pair was observed after 12 h, as judged both by immunofluorescence labeling with specific antibodies and electron microscopic observation of cells after complete thick serial sectioning. Strikingly, we also observed a scattering of the pericentriolar material (PCM) within the cytoplasm and a parallel disappearance of the centrosome as a defined organelle. However, centriole disappearance was transient, as centrioles and discrete centrosomes ultimately reappeared in the cell population.During the acentriolar period, a large proportion of monopolar half-spindles or of bipolar spindles with abnormal distribution of PCM and NuMA were observed. However, as judged by a quasinormal increase in cell number, these cells likely were not blocked in mitosis.Our results suggest that a posttranslational modification of tubulin is critical for long-term stability of centriolar microtubules. They further demonstrate that in animal cells, centrioles are instrumental in organizing centrosomal components into a structurally stable organelle.  相似文献   

2.
A monoclonal antibody (GT335) directed against polyglutamylated tubulin was obtained by immunization with a synthetic peptide which mimics the structure of the polyglutamylated site of alpha-tubulin. This peptide corresponds to the C-terminal sequence Glu441-Gly448 and was chemically modified by the addition of two glutamyl units at Glu445. The specificity of GT335 was assayed by direct and competitive enzyme-linked immunosorbent assay (ELISA) against tubulin and several synthetic peptides differing either by the structure of the added polyglutamyl chain or by their amino acid sequence. Further characterization was carried out by immunoblotting detection after one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The epitope appears to be formed by at least two constituents: a basic motif of monoglutamylation which is retained in the polyglutamylated forms independent of their degree of glutamylation, and some elements of the polypeptide chain close to the site of glutamylation. Given the specificity of GT335 and the delineation of its epitope, our results indicate that, in addition to alpha and beta' (class III)-tubulin, other beta-tubulin isotypes are also glutamylated. This antibody has been used to analyze the cell and tissue distributions of glutamylated tubulin. In mouse brain extracts, GT335 reacts strongly with alpha-tubulin and, to a lesser extent, with beta' (class III) and beta-tubulin. The same reactivity is also observed with cultured neurons whereas astroglial cells exhibit only low levels of glutamylated tubulin. In non-nervous mouse tissues such as spleen, lung or testis, glutamylation was shown to involve only beta-tubulin, but at far lower levels than in brain.  相似文献   

3.
The centrosome is positioned at the cell center by pushing and pulling forces transmitted by microtubules (MTs). Centrosome decentering is often considered to result from asymmetric, cortical pulling forces exerted in particular by molecular motors on MTs and controlled by external cues affecting the cell cortex locally. Here we used numerical simulations to investigate the possibility that it could equally result from the redistribution of pushing forces due to a reorientation of MTs. We first showed that MT gliding along cell edges and pivoting around the centrosome regulate MT rearrangement and thereby direct the spatial distribution of pushing forces, whereas the number, dynamics, and stiffness of MTs determine the magnitude of these forces. By modulating these parameters, we identified different regimes, involving both pushing and pulling forces, characterized by robust centrosome centering, robust off-centering, or “reactive” positioning. In the last-named conditions, weak asymmetric cues can induce a misbalance of pushing and pulling forces, resulting in an abrupt transition from a centered to an off-centered position. Taken together, these results point to the central role played by the configuration of the MTs on the distribution of pushing forces that position the centrosome. We suggest that asymmetric external cues should not be seen as direct driver of centrosome decentering and cell polarization but instead as inducers of an effective reorganization of the MT network, fostering centrosome motion to the cell periphery.  相似文献   

4.
In vertebrate cells, the centrosome consists of a pair of centrioles and surrounding pericentriolar material. Using anti-Golgi 58K protein antibodies that recognize formiminotransferase cyclodeaminase (FTCD), we investigated its localization to the centrosome in various cultured cells and human oviductal secretory cells by immunohistochemistry. In addition to the Golgi apparatus, FTCD was localized to the centrosome, more abundantly around the mother centriole. The centrosome localization of FTCD continued throughout the cell cycle and was not disrupted after Golgi fragmentation, which was induced by colcemid and brefeldin A. Centriole microtubules are polyglutamylated and stable against tubulin depolymerizing drugs. FTCD in the centrosome may be associated with polyglutamylated residues of centriole microtubules and may play a role in providing centrioles with glutamate produced by cyclodeaminase domains of FTCD.  相似文献   

5.
Mode of centriole duplication and distribution   总被引:8,自引:8,他引:8       下载免费PDF全文
Centriole stability and distribution during the mammalian cell cycle was studied by microinjecting biotinylated tubulin into early G1 cells and analyzing the pattern of incorporation into centrioles. Cells were extracted and cold treated to depolymerize labile microtubules, allowing the fluorescent microscopic visualization of the stable centrioles. The ability to detect single centrioles was confirmed by use of correlative electron microscopy. Indirect hapten and immunofluorescent labeling of biotinylated and total tubulin permitted us to distinguish newly formed from preexisting centrioles. Daughter centrioles incorporated biotinylated tubulin, and at mitosis each cell received a centrosome containing one new and one old centriole. We conclude that in each cell cycle tubulin incorporation into centrioles is conservative, and centriole distribution is semiconservative.  相似文献   

6.
The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.  相似文献   

7.
Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of approximately 50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function.  相似文献   

8.
Microtubule-organizing centers recruit α- and β-tubulin polypeptides for microtubule nucleation. Tubulin synthesis is complex, requiring five specific cofactors, designated tubulin cofactors (TBCs) A–E, which contribute to various aspects of microtubule dynamics in vivo. Here, we show that tubulin cofactor D (TBCD) is concentrated at the centrosome and midbody, where it participates in centriologenesis, spindle organization, and cell abscission. TBCD exhibits a cell-cycle-specific pattern, localizing on the daughter centriole at G1 and on procentrioles by S, and disappearing from older centrioles at telophase as the protein is recruited to the midbody. Our data show that TBCD overexpression results in microtubule release from the centrosome and G1 arrest, whereas its depletion produces mitotic aberrations and incomplete microtubule retraction at the midbody during cytokinesis. TBCD is recruited to the centriole replication site at the onset of the centrosome duplication cycle. A role in centriologenesis is further supported in differentiating ciliated cells, where TBCD is organized into “centriolar rosettes”. These data suggest that TBCD participates in both canonical and de novo centriolar assembly pathways.  相似文献   

9.
In animal somatic cells, bipolar spindle formation requires separation of the centrosome-based spindle poles. Centrosome separation relies on multiple pathways, including cortical forces and antiparallel microtubule (MT) sliding, which are two activities controlled by the protein kinase aurora A. We previously found that depletion of the human kinetochore protein Mcm21RCENP-O results in monopolar spindles, raising the question as to whether kinetochores contribute to centrosome separation. In this study, we demonstrate that kinetochores promote centrosome separation after nuclear envelope breakdown by exerting a pushing force on the kinetochore fibers (k-fibers), which are bundles of MTs that connect kinetochores to centrosomes. This force is based on poleward MT flux, which incorporates new tubulin subunits at the plus ends of k-fibers and requires stable k-fibers to drive centrosomes apart. This kinetochore-dependent force becomes essential for centrosome separation if aurora A is inhibited. We conclude that two mechanisms control centrosome separation during prometaphase: an aurora A–dependent pathway and a kinetochore-dependent pathway that relies on k-fiber–generated pushing forces.  相似文献   

10.
Centrosome duplication in mammalian cells is a highly regulated process, occurs in coordination of other cell cycle events. However, molecular exploration of this important cellular process had been difficult due to unavailability of a simple assay system. Here, using centrosomes loosely associated with nuclei isolated from cultured cells, we developed a cell-free centriole (duplication unit of the centrosome) duplication system: unduplicated centrosomes bound to the nuclei are able to undergo duplication in the presence of G1/S extracts. We show that the ability of G1/S extracts to induce centriole duplication in vitro depends on the presence of active CDK2/cyclin E. It has been shown that dissociation of centrosomal nucleophosmin (NPM)/B23 triggered by CDK2/cyclin E-mediated phosphorylation is required for initiation of centrosome duplication. We show that centriole duplication is blocked when nuclei were preincubated with the anti-NPM/B23 antibody that prevents phosphorylation of NPM/B23 by CDK2/cyclin E. These studies provide not only direct evidence for the requirement of CDK2/cyclin E and phosphorylation of NPM/B23 for centrosomes to initiate duplication, but a valuable experimental system for further exploration of the molecular regulation of centrosome duplication in somatic cells of higher animals.  相似文献   

11.
Formation of a bipolar spindle is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, defects in centrosome number and structural organization can lead to a loss of bipolarity. In addition, microtubule-mediated pulling and pushing forces acting on centrosomes and chromosomes are also important for bipolar spindle formation. Polo-like kinase 1 (Plk1) is a highly conserved Ser/Thr kinase that has essential roles in the formation of a bipolar spindle with focused poles. However, the mechanism by which Plk1 regulates spindle-pole formation is poorly understood. Here, we identify a novel centrosomal substrate of Plk1, Kizuna (Kiz), depletion of which causes fragmentation and dissociation of the pericentriolar material from centrioles at prometaphase, resulting in multipolar spindles. We demonstrate that Kiz is critical for establishing a robust mitotic centrosome architecture that can endure the forces that converge on the centrosomes during spindle formation, and suggest that Plk1 maintains the integrity of the spindle poles by phosphorylating Kiz.  相似文献   

12.
Centrosome duplication in mammalian cells is a highly regulated process, occurs in coordination of other cell cycle events. However, molecular exploration of this important cellular process had been difficult due to unavailability of a simple assay system. Here, using centrosomes loosely associated with nuclei isolated from cultured cells, we developed a cell-free centriole (duplication unit of the centrosome) duplication system: unduplicated centrosomes bound to the nuclei are able to undergo duplication in the presence of G1/S extracts. We show that the ability of G1/S extracts to induce centriole duplication in vitro depends on the presence of active CDK2/cyclin E. It has been shown that dissociation of centro-somal nucleophosmin (NPM)/B23 triggered by CDK2/cyclin E-mediated phosphorylation is required for initiation of centrosome duplication. We show that centriole duplication is blocked when nuclei were preincubated with the anti-NPM/B23 antibody that prevents phosphorylation of NPM/B23 by CDK2/cyclin E. These studies provide not only direct evidence for the requirement of CDK2/cyclin E and phosphorylation of NPM/B23 for centrosomes to initiate duplication, but a valuable experimental system for further exploration of the molecular regulation of centrosome duplication in somatic cells of higher animals.  相似文献   

13.
The position of the centrosome is actively maintained at the cell center, but the mechanisms of the centering force remain largely unknown. It is known that centrosome positioning requires a radial array of cytoplasmic microtubules (MTs) that can exert pushing or pulling forces involving MT dynamics and the activity of cortical MT motors. It has also been suggested that actomyosin can play a direct or indirect role in this process. To examine the centering mechanisms, we introduced an imbalance of forces acting on the centrosome by local application of an inhibitor of MT assembly (nocodazole), and studied the resulting centrosome displacement. Using this approach in combination with microinjection of function-blocking probes, we found that a MT-dependent dynein pulling force plays a key role in the positioning of the centrosome at the cell center, and that other forces applied to the centrosomal MTs, including actomyosin contractility, can contribute to this process.  相似文献   

14.
In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The centrosome duplicates once per cell cycle. Polo like kinases (Plks) perform crucial functions in cell-cycle progression and during mitosis. The polo-like kinase-2, Plk2, is activated near the G1/S phase transition, and plays an important role in the reproduction of centrosomes. In this study, we show that the polo-box of Plk2 is required both for association to the centrosome and centriole duplication. Mutation of critical sites in the Plk2 polo-box prevents centrosomal localization and impairs centriole duplication. Plk2 is localized to centrosomes during early G1 phase where it only associates to the mother centriole and then distributes equally to both mother and daughter centrioles at the onset of S phase. Furthermore, our results imply that Plk2 mediated centriole duplication is dependent on Plk4 function. In addition, we find that siRNA-mediated down-regulation of Plk2 leads to the formation of abnormal mitotic spindles confirming that Plk2 may have a function in the reproduction of centrioles.  相似文献   

15.
Centrosomes contain a pair of centrioles that duplicate once during the cell cycle togive rise to two mitotic spindle poles, each containing one old and one newcentriole. Centrosome duplication initiates at the G1/S transition in mammaliancells, and is completed during S and G2 phase. The localization of a number ofprotein kinases to the centrosome has revealed the importance of proteinphosphorylation in controlling the centrosome duplication cycle. Recent studieshave shown that polo-like kinase-2 is required for centriole duplication inmammalian cells. In this article I discuss the implication of these findings to ourcurrent understanding of centrosome duplication.  相似文献   

16.
Centrioles in the cell cycle. I. Epithelial cells   总被引:20,自引:14,他引:6       下载免费PDF全文
A study was made of the structure of the centrosome in the cell cycle in a nonsynchronous culture of pig kidney embryo (PE) cells. In the spindle pole of the metaphase cell there are two mutually perpendicular centrioles (mother and daughter) which differ in their ultrastructure. An electron-dense halo, which surrounds only the mother centriole and is the site where spindle microtubules converge, disappears at the end of telophase. In metaphase and anaphase, the mother centriole is situated perpendicular to the spindle axis. At the beginning of the G1 period, pericentriolar satellites are formed on the mother centriole with microtubules attached to them; the two centrioles diverge. The structures of the two centrioles differ throughout interphase; the mother centriole has appendages, the daughter does not. Replication of the centrioles occurs approximately in the middle of the S period. The structure of the procentrioles differs sharply from that of the mature centriole. Elongation of procentrioles is completed in prometaphase, and their structure undergoes a number of successive changes. In the G2 period, pericentriolar satellites disappear and some time later a fibrillar halo is formed on both mother centrioles, i.e., spindle poles begin to form. In the cells that have left the mitotic cycle (G0 period), replication of centrioles does not take place; in many cells, a cilium is formed on the mother centriole. In a small number of cells a cilium is formed in the S and G2 periods, but unlike the cilium in the G0 period it does not reach the surface of the cell. In all cases, it locates on the centriole with appendages. At the beginning of the G1 period, during the G2 period, and in nonciliated cells in the G0 period, one of the centrioles is situated perpendicular to the substrate. On the whole, it takes a mature centriole a cycle and a half to form in PE cells.  相似文献   

17.
In an attempt to better understand the role of centrioles in vertebrate centrosomes, hydrostatic pressure was applied to isolated centrosomes as a means to disassemble centriole microtubules. Treatments of the centrosomes were monitored by analyzing their protein composition, ultrastructure, their ability to nucleate microtubules from pure tubulin, and their capability to induce parthenogenetic development of Xenopus eggs. Moderate hydrostatic pressure (95 MPa) already affected the organization of centriole microtubules in isolated centrosomes, and also impaired microtubule nucleation. At higher pressure, the protein composition of the peri-centriolar matrix (PCM) was also altered and the capacity to nucleate microtubules severely impaired. Incubation of the treated centrosomes in Xenopus egg extract could restore their capacity to nucleate microtubules after treatment at 95 MPa, but not after higher pressure treatment. However, the centriole structure was in no case restored. It is noteworthy that centrosomes treated with mild pressure did not allow parthenogenetic development after injection into Xenopus eggs, even if they had recovered their capacity to nucleate microtubules. This suggested that, in agreement with previous results, centrosomes in which centriole architecture is impaired, could not direct the biogenesis of new centrioles in Xenopus eggs. Centriole structure could also be affected by applying mild hydrostatic pressure directly to living cells. Comparison of the effect of hydrostatic pressure on cells at the G1/S border or on the corresponding cytoplasts suggests that pro-centrioles are very sensitive to pressure. However, cells can regrow a centriole after pressure-induced disassembly. In that case, centrosomes eventually recover an apparently normal duplication cycle although with some delay.  相似文献   

18.
The mammalian SPD-2 ortholog Cep192 regulates centrosome biogenesis   总被引:2,自引:0,他引:2  
Centrosomes are the major microtubule-organizing centers of mammalian cells. They are composed of a centriole pair and surrounding microtubule-nucleating material termed pericentriolar material (PCM). Bipolar mitotic spindle assembly relies on two intertwined processes: centriole duplication and centrosome maturation. In the first process, the single interphase centrosome duplicates in a tightly regulated manner so that two centrosomes are present in mitosis. In the second process, the two centrosomes increase in size and microtubule nucleation capacity through PCM recruitment, a process referred to as centrosome maturation. Failure to properly orchestrate centrosome duplication and maturation is inevitably linked to spindle defects, which can result in aneuploidy and promote cancer progression. It has been proposed that centriole assembly during duplication relies on both PCM and centriole proteins, raising the possibility that centriole duplication depends on PCM recruitment. In support of this model, C. elegans SPD-2 and mammalian NEDD-1 (GCP-WD) are key regulators of both these processes. SPD-2 protein sequence homologs have been identified in flies, mice, and humans, but their roles in centrosome biogenesis until now have remained unclear. Here, we show that Cep192, the human homolog of C. elegans and D. melanogaster SPD-2, is a major regulator of PCM recruitment, centrosome maturation, and centriole duplication in mammalian cells. We propose a model in which Cep192 and Pericentrin are mutually dependent for their localization to mitotic centrosomes during centrosome maturation. Both proteins are then required for NEDD-1 recruitment and the subsequent assembly of gamma-TuRCs and other factors into fully functional centrosomes.  相似文献   

19.
Centriole duplication initiates at the G1-to-S transition in mammalian cells and is completed during the S and G2 phases. The localization of a number of protein kinases to the centrosome has revealed the importance of protein phosphorylation in controlling the centriole duplication cycle. Here we show that the human Polo-like kinase 2 (Plk2) is activated near the G1-to-S transition of the cell cycle. Endogenous and overexpressed HA-Plk2 localize with centrosomes, and this interaction is independent of Plk2 kinase activity. In contrast, the kinase activity of Plk2 is required for centriole duplication. Overexpression of a kinase-deficient mutant under S-phase arrest blocks centriole duplication. Downregulation of endogenous Plk2 with small hairpin RNAs interferes with the ability to reduplicate centrioles. Furthermore, centrioles failed to duplicate during the cell cycle of human fibroblasts and U2OS cells after overexpression of a Plk2 dominant-negative mutant. These results show that Plk2 is a physiological centrosomal protein and that its kinase activity is likely to be required for centriole duplication near the G1-to-S phase transition.  相似文献   

20.
The presence of glutamylated tubulin, a widespread posttranslational modification of alpha- and beta-tubulin, has been investigated in Drosophila melanogaster using the specific monoclonal antibody GT335. We show here that this modification is strongly detected in brain and testis whereas other tissues analyzed did not appear to contain any glutamylated isoforms. Neuronal microtubules are glutamylated on alpha-tubulin only whereas sperm flagella showed a strong modification of both alpha- and beta-tubulin. These results argue for an essential role for glutamylation in differentiation processes that require microtubule stabilization.  相似文献   

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