Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes

Interfering with the activity of polo-like kinases can lead to the formation of monopolar spindles. Polo-like kinases also regulate mitotic entry, activation of the anaphase-promoting complex and the necessary preconditions for cytokinesis. Similarities between the phenotypes of the Drosophila mutants asp and polo point towards a common role in spindle pole function. The abnormal spindles of asp mutants are bipolar but have disorganized broad poles at which gamma-tubulin has an abnormal distribution. Moreover, the synergism or of polo1 aspE3 double mutants indicates a possible involvement of these genes in a common process. Asp is a microtubule-associated protein of relative molecular mass 220,000 (Mr 220K) found at the face of the centrosome that contacts spindle microtubules. In partially purified centrosomes, it is required with gamma-tubulin to organize microtubule asters. Here, we show that Asp is the previously identified Mr 220K substrate of Polo kinase. Polo phosphorylates Asp in vitro, converting it into an MPM2 epitope. Polo and Asp proteins immunoprecipitate together and exist as part of a 25-38S complex. Extracts of polo-derived embryos are unable to restore the ability of salt-stripped centrosomes to nucleate microtubule asters. This can be rescued by addition of phosphorylated Asp or active Polo kinase.     

mei-38 is required for chromosome segregation during meiosis in Drosophila females

Meiotic chromosome segregation occurs in Drosophila oocytes on an acentrosomal spindle, which raises interesting questions regarding spindle assembly and function. One is how to organize a bipolar spindle without microtubule organizing centers at the poles. Another question is how to orient the chromosomes without kinetochore capture of microtubules that grow from the poles. We have characterized the mei-38 gene in Drosophila and found it may be required for chromosome organization within the karyosome. Nondisjunction of homologous chromosomes occurs in mei-38 mutants primarily at the first meiotic division in females but not in males where centrosomes are present. Most meiotic spindles in mei-38 oocytes are bipolar but poorly organized, and the chromosomes appear disorganized at metaphase. mei-38 encodes a novel protein that is conserved in the Diptera and may be a member of a multigene family. Mei-38 was previously identified (as ssp1) due to a role in mitotic spindle assembly in a Drosophila cell line. MEI-38 protein localizes to a specific population of spindle microtubules, appearing to be excluded from the overlap of interpolar microtubules in the central spindle. We suggest MEI-38 is required for the stability of parallel microtubules, including the kinetochore microtubules.     

The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle.

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.     

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.     

Astrin regulates meiotic spindle organization,spindle pole tethering and cell cycle progression in mouse oocytes

Astrin has been described as a microtubule and kinetochore protein required for the maintenance of sister chromatid cohesion and centrosome integrity in human mitosis. However, its role in mammalian oocyte meiosis is unclear. In this study, we find that Astrin is mainly associated with the meiotic spindle microtubules and concentrated on spindle poles at metaphase I and metaphase II stages. Taxol treatment and immunoprecipitation show that Astrin may interact with the centrosomal proteins Aurora-A or Plk1 to regulate microtubule organization and spindle pole integrity. Loss-of-function of Astrin by RNAi and overexpression of Tof the coiled-coil domain results in spindle disorganization, chromosome misalignment and meiosis progression arrestT. Thr24, Ser66 or Ser447 may be the potential phosphorylated sites of Astrin by Plk1, as site-directed mutation of these sites causes oocyte meiotic arrest at HTmetaphaseTH I with highly disordered spindles and disorganized chromosomes, although mutant Astrin localizes to the spindle apparatus. Taken together, these data strongly suggest that Astrin is critical for meiotic spindle assembly and maturation in mouse oocytes.     

Acentriolar microtubule organization centers and Ran‐mediated microtubule formation pathways are both required in porcine oocytes

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran‐mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC‐ and Ran‐mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo‐like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the Ran^GTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC‐ and Ran‐mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.     

GCP-WD is a gamma-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation

The gamma-tubulin ring complex (gammaTuRC) is a large multi-protein complex that is required for microtubule nucleation from the centrosome. Here, we show that the GCP-WD protein (originally named NEDD1) is the orthologue of the Drosophila Dgrip71WD protein, and is a subunit of the human gammaTuRC. GCP-WD has the properties of an attachment factor for the gammaTuRC: depletion or inhibition of GCP-WD results in loss of the gammaTuRC from the centrosome, abolishing centrosomal microtubule nucleation, although the gammaTuRC is intact and able to bind to microtubules. GCP-WD depletion also blocks mitotic chromatin-mediated microtubule nucleation, resulting in failure of spindle assembly. Mitotic phosphorylation of GCP-WD is required for association of gamma-tubulin with the spindle, separately from association with the centrosome. Our results indicate that GCP-WD broadly mediates targeting of the gammaTuRC to sites of microtubule nucleation and to the mitotic spindle, which is essential for spindle formation.     

The abnormal spindle protein is required for germ cell mitosis and oocyte differentiation during Drosophila oogenesis

In this study, we present evidence that the asp function is required in oogenesis for germline cell divisions as well as for cyst polarity and oocyte differentiation. Consistent with previously described roles in spindle organization during Drosophila meiosis and mitosis, asp mutation leads to severe defects in spindle microtubule organization within the germarium. The mitotic spindles of the mutant cystocytes are composed by wavy microtubules and have abnormal poles that often lack gamma-tubulin. The fusome structure is also compromised. In the absence of asp function, the cystocyte divisions fail resulting in egg chamber with fewer than 16 germ cells. Moreover, the microtubule network within the developing germline cysts may assemble incorrectly in turn affecting the microtubule based transport of the specific determinants that is required during mid-oogenesis for the oocyte differentiation program.     

Maternal gamma (gamma)-tubulin is involved in microtubule reorganization during bovine fertilization and parthenogenesis

In this study, gamma-tubulin distribution was determined chronologically in conjunction with microtubule dynamics during bovine fertilization and parthenogenesis. In unfertilized bovine oocytes, gamma-tubulin was identified in the cytoplasm, mainly in the cortex and concentrated in the meiotic spindle. Following sperm penetration, gamma-tubulin in the cytoplasm was recruited by a sperm component. During pronuclear apposition, gamma-tubulin was localized as spots at the spindle poles. gamma-tubulin spots were observed in blastomeres of embryos cleaved in vitro. Following electrical stimulation, gamma-tubulin and microtubule matrix were noted in oocyte cortex. In the late pronuclear stage, considerably less gamma-tubulin and microtubules were detected in the cytoplasm. At the mitotic metaphase of parthenotes, gamma-tubulin was recruited to the condensed chromatin and concentrated in the spindle. The gamma-tubulin spots were not detected until the 8-cell stage of parthenotes. This suggests that maternal gamma-tubulin is recruited by a sperm component to reconstitute the zygotic centrosome. In the absence of sperm components, the cell cycle-related assembly of gamma-tubulin organizes microtubule nucleation for positioning the pronucleus and spindle protein of mitotic metaphase during the first cell cycle of bovine parthenotes.     

ZYG-9, A Caenorhabditis elegans Protein Required for Microtubule Organization and Function, Is a Component of Meiotic and Mitotic Spindle Poles

We describe the molecular characterization of zyg-9, a maternally acting gene essential for microtubule organization and function in early Caenorhabditis elegans embryos. Defects in zyg-9 mutants suggest that the zyg-9 product functions in the organization of the meiotic spindle and the formation of long microtubules. One-cell zyg-9 embryos exhibit both meiotic and mitotic spindle defects. Meiotic spindles are disorganized, pronuclear migration fails, and the mitotic apparatus forms at the posterior, orients incorrectly, and contains unusually short microtubules. We find that zyg-9 encodes a component of the meiotic and mitotic spindle poles. In addition to the strong staining of spindle poles, we consistently detect staining in the region of the kinetochore microtubules at metaphase and early anaphase in mitotic spindles. The ZYG-9 signal at the mitotic centrosomes is not reduced by nocodazole treatment, indicating that ZYG-9 localization to the mitotic centrosomes is not dependent upon long astral microtubules. Interestingly, in embryos lacking an organized meiotic spindle, produced either by nocodazole treatment or mutations in the mei-1 gene, ZYG-9 forms a halo around the meiotic chromosomes. The protein sequence shows partial similarity to a small set of proteins that also localize to spindle poles, suggesting a common activity of the proteins.     

Requirements for NuMA in maintenance and establishment of mammalian spindle poles

Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.     

γ-微管蛋白在猪卵母细胞成熟和活化中的分布

微管蛋白(tubulin)是一蛋白质超家族,其中α-,β-微管蛋白是主要的微管蛋白,而γ-微管蛋白主要在微管组装中起作用. 我们利用蛋白质印迹和激光共聚焦技术研究了γ-微管蛋白在猪卵母细胞成熟、受精和活化中的分布. γ-微管蛋白存在于猪卵母细胞中,并且在减数分裂成熟各个时期的量保持不变. 它聚集在微管上,特别是中期纺锤体的两极和后末期的中板. 体外受精和孤雌活化后,γ-微管蛋白聚集在雌雄原核的周围.另外它也存在于精子的顶体帽和颈部.在早期卵裂中,γ-微管蛋白聚集在胚胎的细胞核周围.实验结果表明,γ-微管蛋白在猪卵母细胞、精子和胚胎的微管组装中起重要的调节作用,在猪受精过程中,精子和卵子都向受精卵贡献中心体物质.     

How do Plants Organize Microtubules Without a Centrosome？

A microtubule nucleates from a γ-tubuUn complex, which consists of γ-tubulin, proteins from the SPC971SPC98 family, and the WD40 motif protein GCP-WD. We analyzed the phylogenetic relationships of the genes encoding these proteins and found that the components of this complex are widely conserved among land plants and other eukaryotes. By contrast, the interphase and mitotic arrays of microtubules in land plants differ from those in other eukaryotes. In the interphase cortical array, the majority of microtubules nucleate on existing microtubules in the absence of conspicuous microtubule organizing centers （MTOCs）, such as a centrosome. During mitosis, the spindle also forms in the absence of conspicuous MTOCs. Both poles of the spindle are broad, and branched structures of microtubules called microtubule converging centers form at the poles. In this review, we hypothesize that the microtubule converging centers form via microtubule-dependent microtubule nucleation, as in the case of the interphase arrays. The evolutionary insights arising from the molecular basis of the diversity in microtubule organization are discussed.     

An essential function of the C. elegans ortholog of TPX2 is to localize activated aurora A kinase to mitotic spindles

In vertebrates, the microtubule binding protein TPX2 is required for meiotic and mitotic spindle assembly. TPX2 is also known to bind to and activate Aurora A kinase and target it to the spindle. However, the relationship between the TPX2-Aurora A interaction and the role of TPX2 in spindle assembly is unclear. Here, we identify TPXL-1, a C. elegans protein that is the first characterized invertebrate ortholog of TPX2. We demonstrate that an essential role of TPXL-1 during mitosis is to activate and target Aurora A to microtubules. Our data suggest that this targeting stabilizes microtubules connecting kinetochores to the spindle poles. Thus, activation and targeting of Aurora A appears to be an ancient and conserved function of TPX2 that plays a central role in mitotic spindle assembly.     

The Xenopus laevis centrosome aurora/Ipl1-related kinase.

The cDNA encoding the protein kinase pEg2 was originally cloned through a differential screening performed during the early development of Xenopus laevis. pEg2 orthologues were found in various organisms and were classified in a new family of oncogenic mitotic protein kinases named 'aurora/Ipl1-related kinases' after the Drosophila melanogaster gene aurora and the Saccharomyces cerevisiae gene Ipl1. The catalytic activity of pEg2 is necessary for the mitotic microtubule spindle formation in Xenopus laevis egg extracts. The addition of a dominant negative form of pEg2 to in vitro spindle assembly assays leads to monopolar spindles generated by a defect of centrosome separation. In Xenopus cultured cells, pEg2 was confined around the pericentriolar material once centrosomes were duplicated. The centrosome localization does not depend on the presence of microtubules. However, in vitro, the protein binds to taxol-stabilized microtubules independently of its kinase activity. During mitosis the location of the protein changes, in metaphase the kinase localizes on the microtubules at the poles of the mitotic spindle whereas it is not present on astral microtubules. This localization persists until the segregation of the chromosomes is completed. The presence of the kinase on the spindle may reveal another yet unknown function.     

Mitotic spindle morphogenesis in animal cells.

Assembly of the mitotic spindle is an interesting example of morphogenesis at the cellular level. The temporal control of this major event involves the periodic activation of the cyclin-cdc2 kinase complex. In this review, I report recent results that have shed some light on the temporal regulation of centrosome duplication, microtubule nucleation and microtubule dynamics. Reorganization of highly dynamic microtubules into a bipolar spindle probably requires kinesin and dynein-like motors and their role is discussed in an hypothetical model that may be applicable to all mitotic spindles.     

Gamma-tubulin complex-mediated anchoring of spindle microtubules to spindle-pole bodies requires Msd1 in fission yeast

The anchoring of microtubules to subcellular structures is critical for cell polarity and motility. Although the process of anchoring cytoplasmic microtubules to the centrosome has been studied in some detail, it is not known how spindle microtubules are anchored to the mitotic centrosome and, particularly, whether anchoring and nucleation of mitotic spindles are functionally separate. Here, we show that a fission yeast coiled-coil protein, Msd1, is required for anchoring the minus end of spindle microtubules to the centrosome equivalent, the spindle-pole body (SPB). msd1 deletion causes spindle microtubules to abnormally extend beyond SPBs, which results in chromosome missegregation. Importantly, this protruding spindle is phenocopied by the amino-terminal deletion mutant of Alp4, a component of the gamma-tubulin complex (gamma-TuC), which lacks the potential Msd1-interacting domain. We propose that Msd1 interacts with gamma-TuC, thereby specifically anchoring the minus end of microtubules to SPBs without affecting microtubule nucleation.     

XRHAMM functions in ran-dependent microtubule nucleation and pole formation during anastral spindle assembly

BACKGROUND: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. RESULTS: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with gamma-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. CONCLUSIONS: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and gamma-TuRC.     

Gamma-tubulin functions in the nucleation of a discrete subset of microtubules in the eukaryotic flagellum

gamma-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of gamma-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of gamma-tubulin in the formation of the 9 + 2 flagellum axoneme. T. brucei possesses a single flagellum and forms a new flagellum during each cell cycle. We have used an inducible RNA interference (RNAi) approach to ablate expression of gamma-tubulin, and, after induction, we observe that the new flagellum is still formed but is paralyzed, while the old flagellum is unaffected. Electron microscopy reveals that the paralyzed flagellum lacks central pair microtubules but that the outer doublet microtubules are formed correctly. These differences in microtubule nucleation mechanisms during flagellum growth provide insights into spatial and temporal regulation of gamma-tubulin-dependent processes within cells and explanations for the organization and evolution of axonemal structures such as the 9 + 0 axonemes of sensory cells and primary cilia.     

Myosin-10 and actin filaments are essential for mitotic spindle function

Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.