Spermatozoa of the Loach Misgurnus fossilis in the Identification of New Centrosome Proteins

We studied the possibility of using the spermatozoa of the loach Misgurnus fossilis L. in the identification of centrosome proteins. It has been shown that the centrosome of the loach spermatozoa consists of a pair of centrioles of the standard structure and contains the marker protein -tubulin, cytoplasmic microtubules branch out from it, and it does not contain any additional structures characteristic of the centrosomes of spermatozoa of many other fishes. A preparation enriched with intact centrosomes was obtained from the loach spermatozoa. These centrosomes contained -tubulin, although they had lost their ability to induce the polymerization of microtubules. The preparation of loach centrosomes was successfully used to obtain a set of monoclonal antibodies against the mammalian centrosome. A new protein kinase LOSTEK was identified with the help of one of these monoclonal antibodies, SN2-3D2, which was localized in the centrosome and then on microtubules in both loach spermatozoa and cultured mammalian cells. Hence, the loach spermatozoa are a promising means to identify new proteins in the mammalian centrosome.     

Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex

Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.     

Structure of the gamma-tubulin ring complex: a template for microtubule nucleation

The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative molecular mass approximately 2.2 x 10(6) that nucleates microtubules at the centrosome. Here we use electron-microscopic tomography and metal shadowing to examine the structure of isolated Drosophila gammaTuRCs and the ends of microtubules nucleated by gammaTuRCs and by centrosomes. We show that the gammaTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated gammaTuRCs and from centrosomes. Antibodies against gamma-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the gammaTuRC.     

Human Cep192 is required for mitotic centrosome and spindle assembly

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.     

Centrosome-independent mitotic spindle formation in vertebrates

BACKGROUND: In cells lacking centrosomes, the microtubule-organizing activity of the centrosome is substituted for by the combined action of chromatin and molecular motors. The question of whether a centrosome-independent pathway for spindle formation exists in vertebrate somatic cells, which always contain centrosomes, remains unanswered, however. By a combination of labeling with green fluorescent protein (GFP) and laser microsurgery we have been able to selectively destroy centrosomes in living mammalian cells as they enter mitosis. RESULTS: We have established a mammalian cell line in which the boundaries of the centrosome are defined by the constitutive expression of gamma-tubulin-GFP. This feature allows us to use laser microsurgery to selectively destroy the centrosomes in living cells. Here we show that this method can be used to reproducibly ablate the centrosome as a functional entity, and that after destruction the microtubules associated with the ablated centrosome disassemble. Depolymerization-repolymerization experiments reveal that microtubules form in acentrosomal cells randomly within the cytoplasm. When both centrosomes are destroyed during prophase these cells form a functional bipolar spindle. Surprisingly, when just one centrosome is destroyed, bipolar spindles are also formed that contain one centrosomal and one acentrosomal pole. Both the polar regions in these spindles are well focused and contain the nuclear structural protein NuMA. The acentrosomal pole lacks pericentrin, gamma-tubulin, and centrioles, however. CONCLUSIONS: These results reveal, for the first time, that somatic cells can use a centrosome-independent pathway for spindle formation that is normally masked by the presence of the centrosome. Furthermore, this mechanism is strong enough to drive bipolar spindle assembly even in the presence of a single functional centrosome.     

Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle

Since the discovery of gamma-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of gamma-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for gamma-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2-6) required for localizing gamma-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for gamma-tubulin may lie within the spindle, where augmin and gamma-tubulin function cooperatively to amplify the number of microtubules.     

Centrosome reduction during Rhesus spermiogenesis: gamma-tubulin, centrin, and centriole degeneration

Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with gamma-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard gamma-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable gamma-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice.     

The mammalian interphase centrosome: two independent units maintained together by the dynamics of the microtubule cytoskeleton.

In mammalian cells the centrosome or diplosome is defined by the two parental centrioles observed in electron microscopy and by the pericentriolar material immunostained with several antibodies directed against various centrosomal proteins (gamma-tubulin, pericentrin, centrin and centractin). Partial destabilization of the microtubule cytoskeleton by microtubule-disassembling substances induced a splitting and a slow migration of the two diplosome units to opposite nuclear sides during most of the interphase in several mammalian cell lines. These units relocated close together following drug removal, while microtubule stabilization by nM taxol concentrations inhibited this process. Cytochalasin slowed down diplosome splitting but did not affect its relocation after colcemid washing. These results account for the apparently opposite effects induced by microtubule poisons on centriole separation. Moreover, they provide new information concerning the centrosome cycle and stability. First, the centrosome is formed by two units, distinguished only by the number of attached stable microtubules, but not by pericentrin, gamma-tubulin, centrin and centractin and their potency to nucleate microtubules. Second, the centrosomal units are independent during most of the interphase. Third, according to the cell type, these centrosomal units are localized in close proximity because they are either linked or maintained close together by the normal dynamics of the microtubule cytoskeleton. Finally, the relocalization of the centrosomal units with their centrioles in cells possessing one or two centrosomes suggests that their relative position results from the overall tensional forces involving at least partially the microtubule arrays nucleated by each of these entities.     

Spindle Dynamics and the Role of γ-Tubulin in Early Caenorhabditis elegans Embryos

gamma-Tubulin is a ubiquitous and highly conserved component of centrosomes in eukaryotic cells. Genetic and biochemical studies have demonstrated that gamma-tubulin functions as part of a complex to nucleate microtubule polymerization from centrosomes. We show that, as in other organisms, Caenorhabditis elegans gamma-tubulin is concentrated in centrosomes. To study centrosome dynamics in embryos, we generated transgenic worms that express GFP::gamma-tubulin or GFP::beta-tubulin in the maternal germ line and early embryos. Multiphoton microscopy of embryos produced by these worms revealed the time course of daughter centrosome appearance and growth and the differential behavior of centrosomes destined for germ line and somatic blastomeres. To study the role of gamma-tubulin in nucleation and organization of spindle microtubules, we used RNA interference (RNAi) to deplete C. elegans embryos of gamma-tubulin. gamma-Tubulin (RNAi) embryos failed in chromosome segregation, but surprisingly, they contained extensive microtubule arrays. Moderately affected embryos contained bipolar spindles with dense and long astral microtubule arrays but with poorly organized kinetochore and interpolar microtubules. Severely affected embryos contained collapsed spindles with numerous long astral microtubules. Our results suggest that gamma-tubulin is not absolutely required for microtubule nucleation in C. elegans but is required for the normal organization and function of kinetochore and interpolar microtubules.     

Delta-tubulin and epsilon-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function

The centrosome organizes microtubules, which are made up of alpha-tubulin and beta-tubulin, and contains centrosome-bound gamma-tubulin, which is involved in microtubule nucleation. Here we identify two new human tubulins and show that they are associated with the centrosome. One is a homologue of the Chlamydomonas delta-tubulin Uni3, and the other is a new tubulin, which we have named epsilon-tubulin. Localization of delta-tubulin and epsilon-tubulin to the centrosome is independent of microtubules, and the patterns of localization are distinct from each other and from that of gamma-tubulin. Delta-tubulin is found in association with the centrioles, whereas epsilon-tubulin localizes to the pericentriolar material. epsilon-Tubulin exhibits a cell-cycle-specific pattern of localization, first associating with only the older of the centrosomes in a newly duplicated pair and later associating with both centrosomes. epsilon-Tubulin thus distinguishes the old centrosome from the new at the level of the pericentriolar material, indicating that there may be a centrosomal maturation event that is marked by the recruitment of epsilon-tubulin.     

Human 76p: A new member of the gamma-tubulin-associated protein family

The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.     

Fodrin in Centrosomes: Implication of a Role of Fodrin in the Transport of Gamma-Tubulin Complex in Brain

Gamma-tubulin is the major protein involved in the nucleation of microtubules from centrosomes in eukaryotic cells. It is present in both cytoplasm and centrosome. However, before centrosome maturation prior to mitosis, gamma-tubulin concentration increases dramatically in the centrosome, the mechanism of which is not known. Earlier it was reported that cytoplasmic gamma-tubulin complex isolated from goat brain contains non-erythroid spectrin/fodrin. The major role of erythroid spectrin is to help in the membrane organisation and integrity. However, fodrin or non-erythroid spectrin has a distinct pattern of localisation in brain cells and evidently some special functions over its erythroid counterpart. In this study, we show that fodrin and γ-tubulin are present together in both the cytoplasm and centrosomes in all brain cells except differentiated neurons and astrocytes. Immunoprecipitation studies in purified centrosomes from brain tissue and brain cell lines confirm that fodrin and γ-tubulin interact with each other in centrosomes. Fodrin dissociates from centrosome just after the onset of mitosis, when the concentration of γ-tubulin attains a maximum at centrosomes. Further it is observed that the interaction between fodrin and γ-tubulin in the centrosome is dependent on actin as depolymerisation of microfilaments stops fodrin localization. Image analysis revealed that γ-tubulin concentration also decreased drastically in the centrosome under this condition. This indicates towards a role of fodrin as a regulatory transporter of γ-tubulin to the centrosomes for normal progression of mitosis.     

Characterization of a Drosophila centrosome protein CP309 that shares homology with Kendrin and CG-NAP

The centrosome in animal cells provides a major microtubule-nucleating site that regulates the microtubule cytoskeleton temporally and spatially throughout the cell cycle. We report the identification in Drosophila melanogaster of a large coiled-coil centrosome protein that can bind to calmodulin. Biochemical studies reveal that this novel Drosophila centrosome protein, centrosome protein of 309 kDa (CP309), cofractionates with the gamma-tubulin ring complex and the centrosome-complementing activity. We show that CP309 is required for microtubule nucleation mediated by centrosomes and that it interacts with the gamma-tubulin small complex. These findings suggest that the microtubule-nucleating activity of the centrosome requires the function of CP309.     

Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of gamma-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with gamma-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring gamma-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.     

Making microtubules and mitotic spindles in cells without functional centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.     

Molecular characterization of human ninein protein: two distinct subdomains required for centrosomal targeting and regulating signals in cell cycle

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.     

CDK5RAP2 is a pericentriolar protein that functions in centrosomal attachment of the gamma-tubulin ring complex

Microtubule nucleation and organization by the centrosome require gamma-tubulin, a protein that exists in a macromolecular complex called the gamma-tubulin ring complex (gammaTuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including gamma-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the gammaTuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for gammaTuRC attachment to the centrosome but not for gammaTuRC assembly. Perturbing CDK5RAP2 function delocalized gamma-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in gammaTuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly.     

BRCA1-dependent ubiquitination of gamma-tubulin regulates centrosome number

Proper centrosome duplication and spindle formation are crucial for prevention of chromosomal instability, and BRCA1 plays a role in this process. In this study, transient inhibition of BRCA1 function in cell lines derived from mammary tissue caused rapid amplification and fragmentation of centrosomes. Cell lines tested that were derived from nonmammary tissues did not amplify the centrosome number in this transient assay. We tested whether BRCA1 and its binding partner, BARD1, ubiquitinate centrosome proteins. Results showed that centrosome components, including gamma-tubulin, are ubiquitinated by BRCA1/BARD1 in vitro. The in vitro ubiquitination of gamma-tubulin was specific, and function of the carboxy terminus was necessary for this reaction; truncated BRCA1 did not ubiquitinate gamma-tubulin. BRCA1/BARD1 ubiquitinated lysines 48 and 344 of gamma-tubulin in vitro, and expression in cells of gamma-tubulin K48R caused a marked amplification of centrosomes. This result supports the notion that the modification of these lysines in living cells is critical in the maintenance of centrosome number. One of the key problems in understanding the biology of BRCA1 has been the identification of a specific target of BRCA1/BARD1 ubiquitination and its effect on mammary cell biology. The results of this study identify a ubiquitination target and suggest a biological impact important in the etiology of breast cancer.     

Role of mitotic motors, dynein and kinesin, in the induction of abnormal centrosome integrity and multipolar spindles in cultured V79 cells exposed to dimethylarsinic acid.

The role of microtubule-based motors in the induction of abnormal centrosome integrity by dimethylarsinic acid (DMAA) was investigated with the use of monastrol, a specific inhibitor of mitotic kinesin, and vanadate, an inhibitor of dynein ATPase. Cytoplasmic dynein co-localized with multiple foci of gamma-tubulin in mitotic cells arrested by DMAA. Disruption of microtubules caused dispersion of dynein while multiple foci of gamma-tubulin were coalesced to a single dot. Vanadate also caused dispersion of dynein, which had been co-localized with multiple foci of gamma-tubulin by DMAA, without affecting spindle organization. However, the dispersion of dynein did not prohibit the induction of abnormal centrosome integrity by DMAA. Inhibition of mitotic kinesin by monastrol resulted in monoastral cells with non-migrated centrosomes in the cell center. Monastrol, when applied to mitotic cells with abnormal centrosome integrity, rapidly reduced the incidence of cells with the centrosome abnormality. Moreover, monastrol completely inhibited reorganization of abnormal centrosomes that had been coalesced to a single dot by microtubule disruption. These results suggest that abnormal centrosome integrity caused by DMAA is not simply due to dispersion of fragments of microtubule-organizing centers, but is dependent on the action of kinesin. In addition, the results suggest that kinesin plays a role not only in the induction of mitotic centrosome abnormality, but also in maintenance.     

De novo formation of centrosomes in vertebrate cells arrested during S phase

The centrosome usually replicates in a semiconservative fashion, i.e., new centrioles form in association with preexisting "maternal" centrioles. De novo formation of centrioles has been reported for a few highly specialized cell types but it has not been seen in vertebrate somatic cells. We find that when centrosomes are completely destroyed by laser microsurgery in CHO cells arrested in S phase by hydroxyurea, new centrosomes form by de novo assembly. Formation of new centrosomes occurs in two steps: approximately 5-8 h after ablation, clouds of pericentriolar material (PCM) containing gamma-tubulin and pericentrin appear in the cell. By 24 h, centrioles have formed inside of already well-developed PCM clouds. This de novo pathway leads to the formation of a random number of centrioles (2-14 per cell). Although clouds of PCM consistently form even when microtubules are completely disassembled by nocodazole, the centrioles are not assembled under these conditions.