Organization of mitotic apparatus poles in etoposide-treated CHO-K1 cells

Analyzed in this study is the organization of mitotic spindle poles in CHO-K1 cells dividing after treatment with etoposide (1 h, 25 μM). At various periods after the treatment, we studied the following: (1) the distribution of γ-tubulin in mitotic cells by immunofluorescent staining, (2) the level of post-translational modification of α-tubulin in spindle microtubules by immunoelectron microscopy, and (3) the ultrastructure of mitotic apparatus poles by standard electron microscopy. 48 h after the addition of etoposide, disturbances in the ultrastructure of mitotic spindle poles were observed in etoposide-treated CHO-K1 cells with both bipolar and with multipolar mitotic apparatuses. The increased number of centrioles was unevenly distributed between the mitotic spindle poles; some centrioles did not take an obvious part in the mitotic spindle organization and differed in their number of outgrowing microtubules. Most centrioles were without fibrillar halos. Immunoelectron microscopy showed the differences in the staining of the poles of a multipolar spindle within one cell with antibodies to tyrosinated α-tubulin, whereas the staining of cells with antibodies to acetylated α-tubulin did not reveal such differences. Immunofluorescence staining for γ-tubulin also indicated differing organizations of poles in the same spindle. Our data findings provided the first evidence that the pattern of immunostaining and ultrastructure of mitotic apparatus poles can differ in cells dividing at various time periods after the action of etoposide.     

The reorganization of the mitotic apparatus in etoposide-treated CHO-K1 cells precedes accumulation of the apoptotic cells

Etoposide (1 h, 25 microM) causes interphase arrest in CHO-K1 after which cells resume mitotic division and die due to apoptosis after a certain time period. Accumulation of apoptotically dying cells in the culture follows a gradual increase in the number ofmultipolar mitoses. Our findings provide the first evidence that the pattern of immunostaning for alpha-tubulin, acetylated alpha-tubulin and tyrosinated alpha-tubulin in cells dividing at various periods after etoposide treatment. Moreover, some parts of the multipolar mitotic spindle differ in the presence of antigenic determinants accessible to anti-tyrosinated alpha-tubulin antibodies. It is noteworthy that these abnormalities are aggravated just before the increase in the number of apoptotically died cells. Our findings also suggest that some cells pass through at least two mitotic cycles prior to a sharp increase in the number of apoptotically died cells in the cell culture.     

The organization of the mitotic apparatus poles in etoposide-treated CHO-K1 cells

In this study, we have examined the organization of the mitotic spindle poles in CHO-K1 cells dividing after treatment with the etoposide (1 h, 25 microM). We studied at various periods after the treatment: 1) the distribution of gamma-tubulin in mitotic cells by immunofluorescent staining; 2) the level of posttranslational modification of a-tubulin in the spindle microtubules by immunoelectron microscopy; 3) the ultrastructure of the mitotic apparatus poles by standard electron microscopy. In 48 h after the addition of the agent we identified considerable changes in the ultrastructure of poles in etoposide-treated CHO-K1 cells with bipolar and multipolar spindles. The number of centrioles increased. The centrioles were unevenly distributed among the poles, and some centrioles were not explicitly involved in the organization of mitotic spindle, furthermore they can differ in the number of outgrowing microtubules. Most centrioles were without fibrillar halo. In 48 h after the addition of etoposide, electron microscopy of cells after immunoperoxidase staining with antibodies to acetylated and tyrosinated alpha-tubulin has shown that different poles of a multipolar spindle within the same cell are stained differently for tyr-tubulin but not for acet-tubulin. Immunofluorescence staining for gamma-tubulin also points to different organization of poles in the same spindle. Our findings provide the first evidence that the pattern of immunostaning and the ultrastructure of mitotic apparatus poles differ in the cells dividing at various periods after etoposide treatment.     

The solution structure of the N-terminal domain of human tubulin binding cofactor C reveals a platform for tubulin interaction

Human Tubulin Binding Cofactor C (TBCC) is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E) and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers.     

Identification and characterization of INMAP, a novel interphase nucleus and mitotic apparatus protein that is involved in spindle formation and cell cycle progression

A novel protein that associates with interphase nucleus and mitotic apparatus (INMAP) was identified by screening HeLa cDNA expression library with an autoimmune serum followed by tandem mass spectrometry. Its complete cDNA sequence of 1.818 kb encodes 343 amino acids with predicted molecular mass of 38.2 kDa and numerous phosphorylation sites. The sequence is identical with nucleotides 1-1800 bp of an unnamed gene (GenBank accession no. 7022388) and highly homologous with the 3′-terminal sequence of POLR3B. A monoclonal antibody against INMAP reacted with similar proteins in S. cerevisiae, Mel and HeLa cells, suggesting that it is a conserved protein. Confocal microscopy using either GFP-INMAP fusion protein or labeling with the monoclonal antibody revealed that the protein localizes as distinct dots in the interphase nucleus, but during mitosis associates closely with the spindle. Double immunolabeling using specific antibodies showed that the INMAP co-localizes with α-tubulin, γ-tubulin, and NuMA. INMAP also co-immunoprecipitated with these proteins in their native state. Stable overexpression of INMAP in HeLa cell lines leads to defects in the spindle, mitotic arrest, formation of polycentrosomal and multinuclear cells, inhibition of growth, and apoptosis. We propose that INMAP is a novel protein that plays essential role in spindle formation and cell-cycle progression.     

Phospho-Bcl-xL(Ser62) influences spindle assembly and chromosome segregation during mitosis

Functional analysis of a series of phosphorylation mutants reveals that Bcl-xL(Ser62Ala) influences cell entry into anaphase and mitotic exit in taxol-exposed cells compared with cells expressing wild-type Bcl-xL or a series of other phosphorylation mutants, an effect that appears to be independent of its anti-apoptotic activity. During normal mitosis progression, Bcl-xL(Ser62) is strongly phosphorylated by PLK1 and MAPK14/SAPKp38α at the prometaphase, metaphase, and the anaphase boundaries, while it is de-phosphorylated at telophase and cytokinesis. Phospho-Bcl-xL(Ser62) localizes in centrosomes with γ-tubulin and in the mitotic cytosol with some spindle-assembly checkpoint signaling components, including PLK1, BubR1, and Mad2. In taxol- and nocodazole-exposed cells, phospho-Bcl-xL(Ser62) also binds to Cdc20- Mad2-, BubR1-, and Bub3-bound complexes, while Bcl-xL(Ser62Ala) does not. Silencing Bcl-xL expression and expressing the phosphorylation mutant Bcl-xL(Ser62Ala) lead to an increased number of cells harboring mitotic spindle defects including multipolar spindle, chromosome lagging and bridging, aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h. Together, the data indicate that during mitosis, Bcl-xL(Ser62) phosphorylation impacts on spindle assembly and chromosome segregation, influencing chromosome stability. Observations of mitotic cells harboring aneuploidy with micro-, bi-, or multi-nucleated cells, and cells that fail to resolve undergo mitosis within 6 h were also made with cells expressing the phosphorylation mutant Bcl-xL(Ser49Ala) and dual mutant Bcl-xL(Ser49/62Ala).     

Colocalization of the cyclin B homologue P56 and β-tubulin during the cell cycle in a unicellular eucaryote dinoflagellate

We provide evidence for an unusual behavior of the cyclin B homologue, p56, in the dinoflagellate Crypthecodinium cohnii. p56, of which we previously demonstrated the presence in this original eukaryotic protist, is present all along the cell cycle progression, and is exclusively cytoplasmic as revealed after immunofluorescence labeling with anti-p56 Ab and counterstaining with Dapi. It was never found in the nucleus as is the case in higher eukaryotic cells. During motosis, p56 was essentially associated with the mitotic apparatus: centrosomes and mitotic spindle, as shown after double immunofluorescence labeling with anti p56 and anti β-tubulin Ab. Using high pressure freeze fixation, we clearly detected in transmission electron microscopy (TEM) the localization of p56 cyclin B homologue and β-tubulin: single immunogold labeling demonstrated that p56 is localized along the whole cell cortex, along the cleavage furrow of anaphase to cytokinesis cells and into cytoplasmic channels passing throughout the mitotic nucleus where is located the mitotic spindle. Double immunogold labeling realized with anti-p56 and anti-β-tubulin antibodies confirm that p56 antigens colocalize with β-tubulin in many sites. The significance of the exclusively cytoplasmic localization of the cyclin B homologue is discussed.     

Remodeling of Centrosomes in Intraspecies and Interspecies Nuclear Transfer Porcine Embryos

Remodeling of donor cell centrosomes and the centrosome-associated cytoskeleton is crucially important for nuclear cloning as centrosomes are the main microtubule organizing centers that play a significant role in cell division and embryo development. Centrosome dysfunctions have been implicated in various diseases including cancer and metabolic disorders and may also play a role in developmental abnormalities that are frequently seen in cloned animals. In the present studies we investigated microtubule organization and the reorganization and fate of the integral centrosome protein γ-tubulin and the centrosome-associated protein centrin in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using antibodies to γ-tubulin or GFP-centrin transfected mouse fibroblasts as donor cells. Microtubules were stained with antibodies to α-tubulin. In-vitro-fertilized oocytes and nuclear transfer (NT) reconstructed oocytes were sequentially analyzed at different developmental stages. Epi-fluorescence results revealed mitotic spindle abnormalities in NT embryos during the first cell cycle (39.4%, 13/33) which were significantly higher than those in IVF embryos (17.0%, 7/41). The abnormalities in IVF embryos are due to polyspermy while the abnormalities in NT embryos are due to donor cell centrosome dysfunctions. In the NT embryos with abnormal microtubule and centrosome organization, γ-tubulin staining revealed multipolar centrosome foci while DAPI staining showed misalignment of chromosomes. In intraspecies and interspecies embryos the GFP-centrin signal was detected until 3 hrs after fusion. GFP-centrin was not detected at 8 hrs after NT which is consistent with previous results using anti-centrin antibody staining in intraspecies NT porcine embryos. These data indicate that 1) abnormalities in microtubule and centrosome organization are associated with nuclear cloning at a higher rate than observed in IVF embryos; 2) centrosome and cytoskeletal abnormalities in IVF embryos are due to polyspermy while centrosome and cytoskeletal abnormalities in NT embryos are due to donor cell centrosome dysfunctions; and 3) GFP-centrin of the donor cell centrosome provides a reliable marker to follow its fate in intraspecies reconstructed embryos.     

The STARD9/Kif16a kinesin associates with mitotic microtubules and regulates spindle pole assembly

During cell division, cells form the microtubule-based mitotic spindle, a highly specialized and dynamic structure that mediates proper chromosome transmission to daughter cells. Cancer cells can show perturbed mitotic spindles and an approach in cancer treatment has been to trigger cell killing by targeting microtubule dynamics or spindle assembly. To identify and characterize proteins necessary for spindle assembly, and potential antimitotic targets, we performed a proteomic and genetic analysis of 592 mitotic microtubule copurifying proteins (MMCPs). Screening for regulators that affect both mitosis and apoptosis, we report the identification and characterization of STARD9, a kinesin-3 family member, which localizes to centrosomes and stabilizes the pericentriolar material (PCM). STARD9-depleted cells have fragmented PCM, form multipolar spindles, activate the spindle assembly checkpoint (SAC), arrest in mitosis, and undergo apoptosis. Interestingly, STARD9-depletion synergizes with the chemotherapeutic agent taxol to increase mitotic death, demonstrating that STARD9 is a mitotic kinesin and a potential antimitotic target.     

Genome‐wide siRNA screen reveals coupling between mitotic apoptosis and adaptation

The antimitotic anti‐cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint‐dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome‐wide small interfering RNA (siRNA) screen in taxol‐treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31^comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro‐apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.     

Single-walled carbon nanotube-induced mitotic disruption

Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 μg/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 μg/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.     

Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression

Members of the NIMA-related kinases (NRK) family are recently emerging as central regulators of various aspects of the cell cycle. However, the cellular roles of the mammalian NRK, Nek7, remain obscure. We show here that the endogenous Nek7 protein is enriched at the centrosome in a microtubule-independent manner. Overexpression of wt or kinase-defective Nek7 resulted in cells of rounder appearance, and higher proportions of multinuclear and apoptotic cells. Down-regulation of Nek7 using a small interfering RNA approach resulted in a significant increase in mitotic cells presenting multipolar spindle phenotype. These results suggest a role for Nek7 in regulating proper spindle assembly and mitotic progression.     

The POLO kinase is required at multiple stages during spermatogenesis in Drosophila melanogaster

The polo gene of Drosophila melanogaster is the founding member of the polo-like kinase family which is conserved among eukaryotes. POLO has been implicated in the organisation and function of the mitotic apparatus. Furthermore, POLO has been shown to be required for normal spermatogenesis. To characterize further the role of POLO in spermatogenesis, polo mutants were analysed by immunostaining with specific antibodies and phase contrast microscopy. Immunofluorescence shows that POLO localises to the centrosomes, the centromere/kinetochore and the spindle midzone. The meiotic phenotype of various mutant allelic combinations was also studied in detail. Observation of mutant live testes indicates cytological abnormalities in all meiotic cell types, including variable DNA content and multipolar spindles. Primary spermatocytes in polo mutant testes contain an abnormal DNA content, suggesting failure of chromosome segregation during gonial division. Immunostaining of polo mutant cells with α-tubulin shows several abnormalities of the meiotic spindle, including a significantly reduced central spindle. Our results suggest that polo has multiple functions during spermatogenesis. Received: 5 August 1998; in revised form: 3 September 1998 / Accepted: 3 September, 1998     

Depletion of CPAP by RNAi disrupts centrosome integrity and induces multipolar spindles

We previously identified a novel microtubule-destabilizing motif in CPAP that can disassemble microtubules. To examine further the CPAP function in human cells, we used siRNA to knockdown its expression. Our results showed that CPAP depletion arrested cells in mitosis and induced apoptosis. Interestingly, more than 40% of these mitotic cells had multiple spindle poles. Furthermore, inhibition of the kinesin Eg5 in CPAP-depleted cells resulted in monopolar spindles, indicating that Eg5 function is required for multipolar spindle formation in the absence of CPAP. Together, our results reveal a structural role for CPAP to maintain centrosome integrity and normal spindle morphology during cell division.     

Division of cell nuclei, mitochondria, plastids, and microbodies mediated by mitotic spindle poles in the primitive red alga Cyanidioschyzon merolae

To understand the cell cycle, we must understand not only mitotic division but also organelle division cycles. Plant and animal cells contain many organelles which divide randomly; therefore, it has been difficult to elucidate these organelle division cycles. We used the primitive red alga Cyanidioschyzon merolae, as it contains a single mitochondrion and plastid per cell, and organelle division can be highly synchronized by a light/dark cycle. We demonstrated that mitochondria and plastids multiplied by independent division cycles (organelle G1, S, G2 and M phases) and organelle division occurred before cell–nuclear division. Additionally, organelle division was found to be dependent on microtubules as well as cell–nuclear division. We have observed five stages of microtubule dynamics: (1) the microtubule disappears during the G1 phase; (2) α-tubulin is dispersed within the cytoplasm without forming microtubules during the S phase; (3) α-tubulin is assembled into spindle poles during the G2 phase; (4) polar microtubules are organized along the mitochondrion during prophase; and (5) mitotic spindles in cell nuclei are organized during the M phase. Microfluorometry demonstrated that the intensity peak of localization of α-tubulin changed in the order to spindle poles, mitochondria, spindle poles, and central spindle area, but total fluorescent intensity did not change remarkably throughout mitotic phases suggesting that division and separation of the cell nucleus and mitochondrion is mediated by spindle pole bodies. Inhibition of microtubule organization induced cell–nuclear division, mitochondria separation, and division of a single membrane-bound microbody, suggesting that similar to cell–nuclear division, mitochondrion separation and microbody division are dependent on microtubules.     

Managing the centrosome numbers game: from chaos to stability in cancer cell division

Aneuploid tumor cells can arise through multipolar mitosis caused by supernumerary centrosomes. Multipolar spindles, however, are antagonistic to cell viability. Thus, most cells derived from such an aberrant mitosis would be eliminated by apoptosis. A rare daughter cell, through chance acquisition of an appropriate chromosome complement and/or gene dosage, could survive and contribute to a clone of aneuploid tumor cells. Survival and perpetuation of the clone, however, requires an additional step - the resumption of mitotic stability through the assembly of a bipolar, not multipolar, spindle. Either selective inactivation of the extra centrosomes or their coalescence into two functional spindle poles corrects the problem of centrosome excess. Current data support coalescence as a mechanism for regulating the number of functional centrosomes in tumor cells.     

Plk1-Dependent Recruitment of γ-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components

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.     

CEP70 protein interacts with γ-tubulin to localize at the centrosome and is critical for mitotic spindle assembly

Deregulation of the mitotic spindle has been implicated in genomic instability, an important aspect of tumorigenesis and malignant transformation. To ensure the fidelity of chromosome transmission, the mitotic spindle is assembled by exquisite mechanisms and orchestrated by centrosomes in animal cells. Centrosomal proteins especially are thought to act coordinately to ensure accurate spindle formation, but the molecular details remain to be investigated. In this study, we report the molecular characterization and functional analysis of a novel centrosomal protein, Cep70. Our data show that Cep70 localizes to the centrosome throughout the cell cycle and binds to the key centrosomal component, γ-tubulin, through the peptide fragments that contain the coiled-coil domains. Our data further reveal that the centrosomal localization pattern of Cep70 is dependent on its interaction with γ-tubulin. Strikingly, Cep70 plays a significant role in the organization of both preexisting and nascent microtubules in interphase cells. In addition, Cep70 is necessary for the organization and orientation of the bipolar spindle during mitosis. These results thus report for the first time the identification of Cep70 as an important centrosomal protein that interacts with γ-tubulin and underscore its critical role in the regulation of mitotic spindle assembly.     

Mitotic centromere-associated kinesin (MCAK) mediates paclitaxel resistance

Paclitaxel has powerful anticancer activity, but some tumors are inherently resistant to the drug, whereas others are initially sensitive but acquire resistance during treatment. To deal with this problem, it will be necessary to understand the mechanisms of drug action and resistance. Recent studies indicate that paclitaxel blocks cell division by inhibiting the detachment of microtubules from centrosomes. Here, we demonstrate that mitotic centromere-associated kinesin (MCAK), a kinesin-related protein that destabilizes microtubules, plays an important role in microtubule detachment. Depletion of MCAK altered mitotic spindle morphology, increased the frequency of lagging chromosomes, and inhibited the proliferation of WT CHO cells, confirming that it is an essential protein for cell division. In contrast, MCAK depletion rescued the proliferation of mutant paclitaxel-dependent cell lines that are unable to divide because of defective spindle function resulting from altered α-tubulin or class III β-tubulin overexpression. In concert with the correction of mitotic defects, loss of MCAK reversed an aberrantly high frequency of microtubule detachment in the mutant cells and increased their sensitivity to paclitaxel. The results indicate that MCAK affects cell sensitivity to mitotic inhibitors by modulating the frequency of microtubule detachment, and they demonstrate that changes in a microtubule-interacting protein can reverse the effects of mutant tubulin expression.