The novel murine calmodulin-binding protein Sha1 disrupts mitotic spindle and replication checkpoint functions in fission yeast.

The mitotic spindle checkpoint represents a signal transduction pathway that prevents the onset of anaphase until all chromosomes are properly aligned on a metaphase plate. Partial inactivation of this checkpoint allows premature separation of sister chromatids and results in aneuploidy, which might contribute to tumorigenesis. Unlike other cell cycle checkpoints, the spindle checkpoint is essential for cell viability, giving rise to the idea that the spindle checkpoint itself might represent a valuable target for anticancer therapy. We used a cell-based screen and identified the indolocarbazole compound Gö6976 as a pharmacologic inhibitor of the spindle checkpoint. Gö6976 potently overrides a spindle checkpoint-mediated mitotic arrest by abrogating the phosphorylation and kinetochore localization of several spindle checkpoint proteins. We identified the Aurora-A and Aurora-B kinases, which have been previously implicated in proper mitotic progression and spindle checkpoint function, as targets for Gö6976. Accordingly, Gö6976 treatment causes severe mitotic abnormalities and chromosome alignment defects, which are not properly detected by the drug-inactivated spindle checkpoint. This results in an aberrant progression of mitosis, leading to apoptosis in various human cancer cell lines, including spindle checkpoint-compromised cancer cells. Thus, our work describes a novel and promising strategy for anticancer treatment that targets the mitotic spindle checkpoint.

The nonerythrocyte isoform of the cytoskeletal protein 4.1R (4.1R) is associated with morphologically dynamic structures during cell division and has been implicated in mitotic spindle function. In this study, we define important 4.1R isoforms expressed in interphase and mitotic cells by RT-PCR and mini-cDNA library construction. Moreover, we show that 4.1R is phosphorylated by p34cdc2 kinase on residues Thr60 and Ser679 in a mitosis-specific manner. Phosphorylated 4.1R135 isoform(s) associate with tubulin and Nuclear Mitotic Apparatus protein (NuMA) in intact HeLa cells in vivo as well as with the microtubule-associated proteins in mitotic asters assembled in vitro. Recombinant 4.1R135 is readily phosphorylated in mitotic extracts and reconstitutes mitotic aster assemblies in 4.1R-immunodepleted extracts in vitro. Furthermore, phosphorylation of these residues appears to be essential for the targeting of 4.1R to the spindle poles and for mitotic microtubule aster assembly in vitro. Phosphorylation of 4.1R also enhances its association with NuMA and tubulin. Finally, we used siRNA inhibition to deplete 4.1R from HeLa cells and provide the first direct genetic evidence that 4.1R is required to efficiently focus mitotic spindle poles. Thus, we suggest that 4.1R is a member of the suite of direct cdc2 substrates that are required for the establishment of a bipolar spindle.

: During anaphase of mitosis, sister chromatids are separated by the dynamic mitotic spindle. Failure to equally distribute the chromosomal DNA can lead to genetic loss and aneuploidy. In eukaryotic cells, the spindle assembly checkpoint monitors the attachments between chromatids and the spindle to initiate a cell cycle delay if the attachments are defective. The fission yeast, Schizosaccharomyces pombe, is a useful system for discovering and characterizing components of this regulatory pathway because genetic approaches can be coupled with excellent cytology. Previous work in the Sazer laboratory identified the first two spindle checkpoint genes in S. pombe: mad2 and mph1. Cells in which the mphl or mad2 gene is deleted are sensitive to the microtubule inhibitor, thiabendazole (TBZ), because they fail to arrest at metaphase to repair the spindle, and therefore undergo a lethal division. Overexpression of either mad2 or mphl activates the spindle checkpoint, arrests cells prior to anaphase, and is toxic to wild type cells. mphl acts upstream of mad2, and the toxic effect of mphl overexpression is suppressed by a mad2 deletion. This project aims to discover and characterize novel spindle checkpoint genes as suppressors of mphl overexpression. The mph1 overexpression screen has yielded mutations in three known spindle checkpoint genes and a mutation, called 4101, in an apparently novel component of the spindle checkpoint pathway. 4101 mutant cells are checkpoint deficient as indicated by their failure to arrest the cell cycle in response to spindle disruption. 4101 cells also exhibit temperature sensitive lethality, and mitotic defects are observed at the restrictive temperature in the absence of TBZ. Plasmid rescue of the temperature sensitive lethality will be exploited to identify the gene mutated in strain 4101.

In a screen for second site mutations capable of reducing the restrictive temperature of the fission yeast mutant cdc2-D217N, we have isolated a novel temperature-sensitive mutant, dim1-35. When shifted to restrictive temperature, dim1-35 mutant cells arrest before entry into mitosis or proceed through mitosis in the absence of nuclear division, demonstrating an uncoupling of proper DNA segregation from other cell cycle events. Deletion of dim1 from the Schizosaccharomyces pombe genome produces a lethal G2 arrest phenotype. Lethality is rescued by overexpression of the mouse dim1 homolog, mdim1. Likewise, deletion of the Saccharomyces cerevisiae dim1 homolog, CDH1, is lethal. Both mdim1 and dim1(+) are capable of rescuing lethality in the cdh1::HIS3 mutant. Although dim1-35 displays no striking genetic interactions with various other G2/M or mitotic mutants, dim1-35 cells incubated at restrictive temperature arrest with low histone H1 kinase activity. Morevoer, dim1-35 displays sensitivity to the microtubule destabilizing drug, thiabendazole (TBZ). We conclude that Dim1p plays a fundamental, evolutionarily conserved role as a protein essential for entry into mitosis as well as for chromosome segregation during mitosis. Based on TBZ sensitivity and failed chromosome segregation in dim1-35, we further speculate that Dim1p may play a role in mitotic spindle formation and/or function.

In the presence of unaligned chromosomes or other spindle abnormalities, the mitotic spindle checkpoint blocks both chromatid separation at anaphase and exit from mitosis. The spindle checkpoint is sustained by signaling from kinetochores that are not properly attached to microtubules of the mitotic spindle. Normally, when chromosomes align at the spindle equator at metaphase, microtubule-kinetochore interactions act to extinguish checkpoint signaling. The cessation of the checkpoint signal activates the Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. The APC/C ubiquitylates the mitotic effectors, securin and cyclin B, which are then degraded by the proteasome. Securin and cyclin B degradation initiates chromatid separation at anaphase and triggers mitotic exit. We have found that long-term arrest of Hela cells with their chromosomes aligned at the metaphase plate results in chromatid separation without mitotic exit. Recently, we and others have characterized C13orf3/Ska3, a newly discovered component of the Ska (Spindle and kinetochore-associated) protein complex. We found that in mitosis Ska3 protein accumulates at kinetochores reaching maximal levels at metaphase. Upon anaphase onset, kinetochore-associated Ska3 levels decline and are eventually lost at telophase. We demonstrated that cells depleted of Ska3 by RNAi achieve metaphase alignment but fail to silence the spindle checkpoint. During this mitotic arrest with chromosomes aligned at the metaphase plate, kinetochores retain robust microtubule attachments. Kinetochores within these Ska3-depleted cells accumulate abnormally high levels of the mitotic spindle checkpoint protein, Bub1, presumably accounting for the inability to silence the checkpoint. Under extended metaphase arrest, the chromatids of individual chromosomes eventually separate, but cells remain arrested in mitosis. To test if other means of inducing metaphase arrest would also lead to chromatid separation, we treated cells in mitosis with the proteasome inhibitor, MG132. Again, after several hours at metaphase, chromatids separated. We speculate that for cells arrested at metaphase, continual pulling forces of the kinetochores on the mitotic spindle eventually overcomes the cohesin complex that holds chromatids together. This “cohesin slippage” results in abnormal chromatid separation for cells still in mid-mitosis. This response reflects a novel terminal phenotype for cells arrested in mitosis with an intact mitotic spindle. (Supported by the NIGMS and the McCasland Foundation.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3868.

The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assemble in the complete absence of centrioles, suggesting a level of centriole-independence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions.

The kinetochore is a multi-protein complex which supports chromosome segregation in mitosis by mediating the connection between centromeric DNA and the mitotic spindle. In addition, it serves as a docking platform for the spindle checkpoint proteins. The spindle checkpoint is a surveillance mechanism which monitors the proper assembly of the mitotic spindle apparatus. It allows the transition from meta- into anaphase only if all kinetochores are bipolarly attached to the spindle microtubules and if tension across the spindle is applied. The tetrameric Ndc80-complex (Ndc80, Nuf2, Spc24, Spc25) is part of the kinetochore. It is highly conserved and was shown to be required for the spindle checkpoint in S. cerevisiae (Janke et al. 2001). The kinase Mps1 has also been implicated in spindle checkpoint control (Hartwick et al. 1996). In mammalian cells, the kinetochore localisation of Mps1 depends on the presence of the Ndc80-complex (Stucke et al. 2004). Furthermore, endogenous Mps1 was found as a faint Coomassie-stained protein band in a Ndc80-complex pull down from yeast (C. Jaeger, unpublished results). Based on these observations, the goal of the present studies was to elucidate whether components of the S. cerevisiae Ndc80-complex are phosphorylated by Mps1 and if this phosphorylation has an influence on the regulation of the spindle checkpoint. The following observations were made: 1. Mps1 physically interacts with Ndc80 ∑ Mps1 weakly associates with the kinetochore by ChIP-analysis in S. cerevisiae. ∑ Mps1 physically interacts with the Ndc80-complex after spindle checkpoint activation by Mps1-overexpression. ∑ Mps1 specifically interacts with the conserved, globular N-terminus of Ndc80 in vitro. 2. Mps1 phosphorylates Ndc80 ∑ Mps1 phosphorylates the N-terminus of Ndc80 in vitro. ∑ Ndc80-phosphorylation depends on Mps1 in vivo. 3. Ndc80-phosphorylation activates the spindle assembly checkpoint ∑ Ndc80 in its non-phosphorylated state (ndc8014A) is checkpoint deficient. ∑ constitutive pseudo-phosphorylation of Ndc80 (ndc8014D) causes lethality due to a permanently activated spindle checkpoint (cell cycle arrest in mitosis). This checkpoint activation specifically depends on the checkpoint proteins Mad2 and Bub1. fi Ndc80-phosphorylation by Mps1 is an integral step in the spindle checkpoint pathway! 4. The kinetochore of ndc8014D-cells is functional ∑ The expression levels and the kinetochore localisation of the ndc8014D protein are comparable to the wild-type protein. ∑ The ndc8014D-protein is able to compete with the wild-type protein for binding-sites at the kinetochore. ∑ the spindle in ndc8014D-cells is under tension, a state which can only be achieved in the presence of an intact kinetochore. fi the observed checkpoint activation is specifically caused by the introduced point mutations rather than by a general kinetochore defect. 5. Mps1-activity is required downstream of Ndc80 ∑ The ndc8014D mutant is unable to activate the spindle checkpoint in Mps1-depleted cells. fi Mps1 has a second function in spindle checkpoint activation downstream of Ndc80-phosphorylation which might involve the phosphorylation of the checkpoint protein Mad1. The results which were obtained in the current work provide evidence that Mps1 specifically phosphorylates Ndc80 and that Ndc80-phosphorylation is a mechanism by which the spindle checkpoint is activated in the presence of unattached kinetochores in early stages of mitosis. The functional characterisation of Ndc80-phosphorylation allows for a refinement of the current model of the spindle assembly checkpoint (Figure 32).

Effect of vitrification and thawing on human oocyte ATP concentration

Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.

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.

Click to increase image sizeClick to decrease image sizeKeywords:: cell divisionfission yeastcell sizemitotic entryCdr2 kinasePom1 kinaseThis article refers to:Distinct levels in Pom1 gradients limit Cdr2 activity and localization to time and position division

The accurate segregation of chromosomes at mitosis depends on a correctly assembled bipolar spindle that exerts balanced forces on each sister chromatid. The integrity of mitotic chromosome segregation is ensured by the spindle assembly checkpoint (SAC) that delays mitosis in response to defective spindle organisation or failure of chromosome attachment. Here we describe a distinct mitotic checkpoint in the fission yeast, Schizosaccharomyces pombe, that monitors the integrity of the actin cytoskeleton and delays sister chromatid separation, spindle elongation and cytokinesis until spindle poles have been properly oriented. This mitotic delay is imposed by a stress-activated mitogen-activated protein (MAP) kinase pathway but is independent of the anaphase-promoting complex (APC).

For ordered mitotic progression, various proteins have to be regulated by an ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) with appropriate timing. Recent studies have implied that the activity of APC/C also contributes to release of mitotic checkpoint complexes (MCCs) from its target Cdc20 in the process of silencing the spindle assembly checkpoint (SAC). Here we describe a temperature-sensitive mutant (ubc11-P93L) in which cell cycle progression is arrested at mitosis. The mutant grows normally at the restrictive temperature when SAC is inactivated, suggesting that the arrest is not due to abnormal spindle assembly, but rather due to prolonged activation of SAC. Supporting this notion, MCCs remain bound to APC/C even when SAC is satisfied. The ubc11+ gene encodes one of the two E2 enzymes required for progression through mitosis in fission yeast. Remarkably, Slp1 (a fission yeast homolog of Cdc20), which is degraded in an APC/C-dependent manner, stays stable throughout the cell cycle in the ubc11-P93L mutant lacking the functional SAC. Other APC/C substrates, in contrast, were degraded on schedule. We have also found that a loss of Ubc4, the other E2 required for progression through mitosis, does not affect the stability of Slp1. We propose that each of the two E2 enzymes is responsible for collaborating with APC/C for a specific set of substrates, and that Ubc11 is responsible for regulating Slp1 with APC/C for silencing the SAC.

Since the discovery of γ-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of γ-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for γ-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2–6) required for localizing γ-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 γ-tubulin may lie within the spindle, where augmin and γ-tubulin function cooperatively to amplify the number of microtubules.

Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex