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1.
Centrosomes mature as cells enter mitosis, accumulating gamma-tubulin and other pericentriolar material (PCM) components. This occurs concomitant with an increase in the number of centrosomally organized microtubules (MTs). Here, we use RNA-mediated interference (RNAi) to examine the role of the aurora-A kinase, AIR-1, during centrosome maturation in Caenorhabditis elegans. In air-1(RNAi) embryos, centrosomes separate normally, an event that occurs before maturation in C. elegans. After nuclear envelope breakdown, the separated centrosomes collapse together, and spindle assembly fails. In mitotic air-1(RNAi) embryos, centrosomal alpha-tubulin fluorescence intensity accumulates to only 40% of wild-type levels, suggesting a defect in the maturation process. Consistent with this hypothesis, we find that AIR-1 is required for the increase in centrosomal gamma-tubulin and two other PCM components, ZYG-9 and CeGrip, as embryos enter mitosis. Furthermore, the AIR-1-dependent increase in centrosomal gamma-tubulin does not require MTs. These results suggest that aurora-A kinases are required to execute a MT-independent pathway for the recruitment of PCM during centrosome maturation.  相似文献   

2.
The nucleation of microtubules requires protein complexes containing γ-tubulin, which are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. We have previously shown that these interactions require the γ-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional γ-tubulin to the centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. However, the molecular details of this important pathway are unknown and a Plk1 substrate that controls γ-tubulin recruitment has not been identified. Here we show that Plk1 associates with GCP-WD in mitosis and Plk1 activity contributes to phosphorylation of GCP-WD. Plk1 depletion or inhibition prevents accumulation of GCP-WD at mitotic centrosomes, but GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit γ-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal γ-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of γ-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates.  相似文献   

3.
Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.  相似文献   

4.
Centrosomes consist of a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Proteins that contain a Pericentrin/AKAP450 centrosomal targeting (PACT) domain have been implicated in recruiting several proteins to the PCM. We show that the only PACT domain protein in Drosophila (the Drosophila pericentrin-like protein [D-PLP]) is associated with both the centrioles and the PCM, and is essential for the efficient centrosomal recruitment of all six PCM components that we tested. Surprisingly, however, all six PCM components are eventually recruited to centrosomes during mitosis in d-plp mutant cells, and mitosis is not dramatically perturbed. Although viable, d-plp mutant flies are severely uncoordinated, a phenotype usually associated with defects in mechanosensory neuron function. We show that the sensory cilia of these neurons are malformed and the neurons are nonfunctional in d-plp mutants. Moreover, the flagella in mutant sperm are nonmotile. Thus, D-PLP is essential for the formation of functional cilia and flagella in flies.  相似文献   

5.
A female-sterile zebrafish maternal-effect mutation in cellular atoll (cea) results in defects in the initiation of cell division starting at the second cell division cycle. This phenomenon is caused by defects in centrosome duplication, which in turn affect the formation of a bipolar spindle. We show that cea encodes the centriolar coiled-coil protein Sas-6, and that zebrafish Cea/Sas-6 protein localizes to centrosomes. cea also has a genetic paternal contribution, which when mutated results in an arrested first cell division followed by normal cleavage. Our data supports the idea that, in zebrafish, paternally inherited centrosomes are required for the first cell division while maternally derived factors are required for centrosomal duplication and cell divisions in subsequent cell cycles. DNA synthesis ensues in the absence of centrosome duplication, and the one-cycle delay in the first cell division caused by cea mutant sperm leads to whole genome duplication. We discuss the potential implications of these findings with regards to the origin of polyploidization in animal species. In addition, the uncoupling of developmental time and cell division count caused by the cea mutation suggests the presence of a time window, normally corresponding to the first two cell cycles, which is permissive for germ plasm recruitment.  相似文献   

6.
Centrosomes are composed of a centriolar core surrounded by a pericentriolar material (PCM) matrix that docks microtubule-nucleating γ-tubulin complexes. During mitotic entry, the PCM matrix increases in size and nucleating capacity in a process called centrosome maturation. Polo-like kinase 1 (PLK1) is recruited to centrosomes and phosphorylates PCM matrix proteins to drive their self-assembly, which leads to PCM expansion. Here, we show that in addition to controlling PCM expansion, PLK1 independently controls the generation of binding sites for γ-tubulin complexes on the PCM matrix. Selectively preventing the generation of PLK1-dependent γ-tubulin docking sites led to spindle defects and impaired chromosome segregation without affecting PCM expansion, highlighting the importance of phospho-regulated centrosomal γ-tubulin docking sites in spindle assembly. Inhibiting both γ-tubulin docking and PCM expansion by mutating substrate target sites recapitulated the effects of loss of centrosomal PLK1 on the ability of centrosomes to catalyze spindle assembly.  相似文献   

7.
CDK5RAP2 is a centrosomal protein known to be involved in the regulation of the γ-tubulin ring complex and thus the organization of microtubule arrays. However, the mechanism by which CDK5RAP2 is itself recruited to centrosomes is poorly understood. We report here that CDK5RAP2 displays highly dynamic attachment to centrosomes in a microtubule-dependent manner. CDK5RAP2 associates with the retrograde transporter dynein-dynactin and contains a sequence motif that binds to dynein light chain 8. Significantly, disruption of cellular dynein-dynactin function reduces the centrosomal level of CDK5RAP2. These results reveal a key role of the dynein-dynactin complex in the dynamic recruitment of CDK5RAP2 to centrosomes.  相似文献   

8.
Human Cep192 is required for mitotic centrosome and spindle assembly   总被引:5,自引:0,他引:5  
As cells enter mitosis, centrosomes dramatically increase in size and ability to nucleate microtubules. This process, termed centrosome maturation, is driven by the accumulation and activation of gamma-tubulin and other proteins that form the pericentriolar material on centrosomes during G2/prophase. Here, we show that the human centrosomal protein, Cep192 (centrosomal protein of 192 kDa), is an essential component of the maturation machinery. Specifically, we have found that siRNA depletion of Cep192 results in a complete loss of functional centrosomes in mitotic but not interphase cells. In mitotic cells lacking Cep192, microtubules become organized around chromosomes but rarely acquire stable bipolar configurations. These cells contain normal numbers of centrioles but cannot assemble gamma-tubulin, pericentrin, or other pericentriolar proteins into an organized PCM. Alternatively, overexpression of Cep192 results in the formation of multiple, extracentriolar foci of gamma-tubulin and pericentrin. Together, our findings support the hypothesis that Cep192 stimulates the formation of the scaffolding upon which gamma-tubulin ring complexes and other proteins involved in microtubule nucleation and spindle assembly become functional during mitosis.  相似文献   

9.
Centrosomes consist of a centriole pair surrounded by pericentriolar material (PCM). Previous work suggested that centrioles are required to organize PCM to form a structurally stable organelle. Here, we characterize SAS-4, a centriole component in Caenorhabditis elegans. Like tubulin, SAS-4 is incorporated into centrioles during their duplication and remains stably associated thereafter. In the absence of SAS-4, centriole duplication fails. Partial depletion of SAS-4 results in structurally defective centrioles that contain reduced levels of SAS-4 and organize proportionally less PCM. Thus, SAS-4 is a centriole-associated component whose amount dictates centrosome size. These results provide novel insight into the poorly understood role of centrioles as centrosomal organizers.  相似文献   

10.
J Fu  DM Glover 《Open biology》2012,2(8):120104
The increase in centrosome size in mitosis was described over a century ago, and yet it is poorly understood how centrioles, which lie at the core of centrosomes, organize the pericentriolar material (PCM) in this process. Now, structured illumination microscopy reveals in Drosophila that, before clouds of PCM appear, its proteins are closely associated with interphase centrioles in two tube-like layers: an inner layer occupied by centriolar microtubules, Sas-4, Spd-2 and Polo kinase; and an outer layer comprising Pericentrin-like protein (Dplp), Asterless (Asl) and Plk4 kinase. Centrosomin (Cnn) and γ-tubulin associate with this outer tube in G2 cells and, upon mitotic entry, Polo activity is required to recruit them together with Spd-2 into PCM clouds. Cnn is required for Spd-2 to expand into the PCM during this maturation process but can itself contribute to PCM independently of Spd-2. By contrast, the centrioles of spermatocytes elongate from a pre-existing proximal unit during the G2 preceding meiosis. Sas-4 is restricted to the microtubule-associated, inner cylinder and Dplp and Cnn to the outer cylinder of this proximal part. γ-Tubulin and Asl associate with the outer cylinder and Spd-2 with the inner cylinder throughout the entire G2 centriole. Although they occupy different spatial compartments on the G2 centriole, Cnn, Spd-2 and γ-tubulin become diminished at the centriole upon entry into meiosis to become part of PCM clouds.  相似文献   

11.
Microtubules are essential structures that organize the cytoplasm and form the mitotic spindle. Their number and orientation depend on the rate of nucleation events and their dynamics. Microtubules are often, but not always, nucleated off a single cytoplasmic element, the centrosome. One microtubule-associated protein, XMAP215, is also a resident centrosomal protein. In this study, we have found that XMAP215 is a key component for the microtubule-nucleating activity of centrosomes. We show that depletion of XMAP215 from Xenopus egg extracts impairs their ability to reconstitute the microtubule nucleation potential of salt-stripped centrosomes. We also show that XMAP215 immobilized on polymer beads induces the formation of microtubule asters in egg extracts as well as in solutions of pure tubulin. Formation of asters by XMAP215 beads indicates that this protein is able to anchor nascent microtubules via their minus ends. The aster-forming activity of XMAP215 does not require gamma-tubulin in pure tubulin solutions, but it is gamma-tubulin-dependent in egg extracts. Our results indicate that XMAP215, a resident centrosomal protein, contributes to the microtubule-nucleating activity of centrosomes, suggesting that, in vivo, the formation of asters by centrosomes requires factors additional to gamma-tubulin.  相似文献   

12.
Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.  相似文献   

13.
14.
Microtubule‐nucleation activity and structural integrity of the centrosome are critical for various cellular functions. The γ‐tubulin ring complexes (γTuRCs) localizing to the pericentriolar matrix (PCM) of the centrosome are major sites of microtubule nucleation. The PCM is thought to be created by two cognate large coiled‐coil proteins, pericentrin/kendrin and CG‐NAP/AKAP450, and its stabilization by Kizuna is essential for bipolar spindle formation. However, the mechanisms by which these proteins are recruited and organized into a proper structure with microtubule‐organizing activity are poorly understood. Here we identify a centrosomal protein Cep72 as a Kizuna‐interacting protein. Interestingly, Cep72 is essential for the localization of CG‐NAP and Kizuna. Cep72 is also involved in γTuRC recruitment to the centrosome and CG‐NAP confers the microtubule‐nucleation activity on the γTuRCs. During mitosis, Cep72‐mediated microtubule organization is important for converging spindle microtubules to the centrosomes, which is needed for chromosome alignment and tension generation between kinetochores. Our findings show that Cep72 is the key protein essential for maintaining microtubule‐organizing activity and structural integrity of the centrosome.  相似文献   

15.
The centrosomal protein, CDK5RAP2, is a microcephaly protein that regulates centrosomal maturation by recruitment of a γ-tubulin ring complex (γ-TuRC) onto centrosomes. In this report, we identified a novel human centrosomal protein, Cep169, as a binding partner of CDK5RAP2, a member of microtubule plus-end-tracking proteins (+TIPs). Cep169 interacts directly with CDK5RAP2 through CM1, an evolutionarily conserved domain, and colocalizes at the pericentriolar matrix (PCM) around centrioles with CDK5RAP2. In addition, Cep169 interacts with EB1 through SxIP-motif responsible for EB1 binding, and colocalizes with CDK5RAP2 at the microtubule plus-end. EB1-binding–deficient Cep169 abolishes EB1 interaction and microtubule plus-end attachment, indicating Cep169 as a novel member of +TIPs. We further show that ectopic expression of either Cep169 or CDK5RAP2 induces microtubule bundling and acetylation in U2OS cells, and depletion of Cep169 induces microtubule depolymerization in HeLa cells, although Cep169 is not required for assembly of γ-tubulin onto centrosome by CDK5RAP2. These results show that Cep169 targets microtubule tips and regulates stability of microtubules with CDK5RAP2.  相似文献   

16.
Microtubules are highly dynamic structures, composed of alpha/beta-tubulin heterodimers. Biosynthesis of the functional dimer involves the participation of several chaperones, termed cofactors A-E, that act on folding intermediates downstream of the cytosolic chaperonin CCT (1, 2). We show that cofactor D is also a centrosomal protein and that overexpression of either the full-length protein or either of two centrosome localization domains leads to the loss of anchoring of the gamma-tubulin ring complex and of nucleation of microtubule growth at centrosomes. In contrast, depletion of cofactor D by short interfering RNA results in mitotic spindle defects. Because none of these changes in cofactor D activity produced a change in the levels of alpha-or beta-tubulin, we conclude that these newly discovered functions for cofactor D are distinct from its previously described role in tubulin folding. Thus, we describe a new role for cofactor D at centrosomes that is important to its function in polymerization of tubulin and organization of the mitotic spindle.  相似文献   

17.
To study the role of the centrosome in microtubule organization in interphase cells, we developed a method for obtaining cytoplasts (cells lacking a nucleus) that did or did not contain centrosomes. After drug- induced microtubule depolymerization, cytoplasts with centrosomes made from sparsely plated cells reconstituted a microtubule array typical of normal cells. Under these conditions cytoplasts without centrosomes formed only a few scattered microtubules. This difference in degree of polymerization suggests that centrosomes affect not only the distribution but the amount of microtubules in cells. To our surprise, the extent of microtubules assembled increased with the cell density of the original culture. At confluent density, cytoplasts without centrosomes had many microtubules, equivalent to cytoplasts with centrosomes. The additional microtubules were arranged peripherally and differed from the centrosomal microtubules in their sensitivity to nocodazole. These and other results suggest that the centrosome stabilizes microtubules in the cell, perhaps by capping one end. Microtubules with greater sensitivity to nocodazole arise by virtue of change in the growth state of the cell and may represent free or uncapped polymers. These experiments suggest that the spatial arrangement of microtubules may change by shifting the total tubulin concentration or the critical concentration for assembly.  相似文献   

18.
Tipulid spermatocytes form normally functioning bipolar spindles after one of the centrosomes is experimentally dislocated from the nucleus in late diakinesis (Dietz, R., 1959, Z. Naturforsch., 14b:749-752; Dietz, R., 1963, Zool. Anz. Suppl., 23:131-138; Dietz, R., 1966, Heredity, 19:161-166). The possibility that dissociated pericentriolar material (PCM) is nevertheless responsible for the formation of the spindle in these cells cannot be ruled out based on live observation. In studying serial sections of complete cells and of lysed cells, it was found that centrosome-free spindle poles in the crane fly show neither pericentriolar-like material nor aster microtubules, whereas the displaced centrosomes appear complete, i.e., consist of a centriole pair, aster microtubules, and PCM. Exposure to a lysis buffer containing tubulin resulted in an increase of centrosomal asters due to aster microtubule polymerization. Aster-free spindle poles did not show any reaction, also indicating the absence of PCM at these poles. The results favor the hypothesis of chromosome-induced spindle pole formation at the onset of prometaphase and the dispensability of PCM in Pales.  相似文献   

19.
Tight regulation of centrosome duplication is critical to ensure that centrosome number doubles once and only once per cell cycle. Superimposed onto this centrosome duplication cycle is a functional centrosome cycle in which they alternate between phases of quiescence and robust microtubule (MT) nucleation and MT-anchoring activities. In vertebrate cycling cells, interphase centrioles accumulate less pericentriolar material (PCM), reducing their MT nucleation capacity. In mitosis, centrosomes mature, accumulating more PCM to increase their nucleation and anchoring capacities to form robust MT asters. Interestingly, functional cycles of centrosomes can be altered to suit the cell's needs. Some interphase centrosomes function as a microtubule-organizing center by increasing their ability to anchor MTs to form centrosomal radial arrays. Other interphase centrosomes maintain their MT nucleation capacity but reduce/eliminate their MT-anchoring capacity. Recent work demonstrates that Drosophila cells take this to the extreme, whereby centrioles lose all detectable PCM during interphase, offering an explanation as to how centrosome-deficient flies develop to adulthood. Drosophila stem cells further modify the functional cycle by differentially regulating their two centrioles – a situation that seems important for stem cell asymmetric divisions, as misregulation of centrosome duplication in stem/progenitor cells can promote tumor formation. Here, we review recent findings that describe variations in the functional cycle of centrosomes.  相似文献   

20.
The centrosomal protein ninein has been identified as a microtubules minus end capping, centriole position, and anchoring protein, but the true physiological function remains to be determined. In this report, using immunofluorescence analysis and GFP-fusions we show that coiled-coil II domain (CCII domain, 1303-2096) co-localized with gamma-tubulin and centrin at the centrosome. We further narrow down within 83 amino acids and classify a new centrosomal targeting signal. Interestingly, antibodies raised against CCII domain reveal that ninein protein declines from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Moreover, the data also suggest that fragment 1783-1866 may be attributed to declined signal of ninein. In kinase assay, we show that CCII domain could readily be phosphorylated by AIK and PKA. Taken together, our results suggest that ninein protein contains two distinct subdomains which are required for targeting and regulating asymmetry centrosomes. Importantly, the decline of ninein during mitosis implies that this centrosomal protein may play a role to regulate the process of chromosome segregation without discrimination. The model we propose here will foster a clearer picture of how two asymmetric centrosomes could direct and ensure the correct segregation of chromosomes during the mitotic stage.  相似文献   

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