Massive Chromosome Missegregation Research Articles

Epistasis, aneuploidy, and functional mutations underlie evolution of resistance to induced microtubule depolymerization.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation.

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore-microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1-Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome-spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1-Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

Targeted TPX2 increases chromosome missegregation and suppresses tumor cell growth in human prostate cancer.

Prostate cancer is a complex disease that can be relatively harmless or extremely aggressive. Although androgen-deprivation therapy is a commonly used treatment for men with prostate cancer, the adverse effects can be detrimental to patient health and quality of life. Therefore, identifying new target genes for tumor growth will enable the development of novel therapeutic intervention. TPX2 plays a critical role in chromosome segregation machinery during mitosis. Low rates of chromosome missegregation can promote tumor development, whereas higher levels might promote cell death and suppress tumorigenesis. Hence, the strategy of promoting cell death by inducing massive chromosome missegregation has been a therapeutic application for selectively eliminating highly proliferating tumor cells. RNAi was used for TPX2 protein expression knockdown, and a clonogenic assay, immunostaining, double thymidine block, image-cytometry analysis, and tumor spheroid assay were used to analyze the role of TPX2 in tumor cell growth, cell cycle progression, multinuclearity, ploidy, and tumorigenicity, respectively; finally, Western blotting was used to analyze anticancer mechanisms in TPX2 targeting. We demonstrated that targeting TPX2 reduced cell cycle regulators and chromosome segregation genes, resulting in increased cell micronucleation. Moreover, TPX2 depletion led to prostate cancer cell growth inhibition, increased apoptosis, and reduced tumorigenesis. These results confirmed the therapeutic potential of targeting TPX2 in prostate cancer treatment. Moreover, we found that TPX2 silencing led to deregulation of CDK1, cyclin B, securin, separase, and aurora A proteins; by contrast, p21 mRNA was upregulated. We also determined the molecular mechanisms for TPX2 targeting in prostate cancer cells. In conclusion, our study illustrates the power of TPX2 as a potential novel target gene for prostate cancer treatment.

Kinetochore-microtube attachments in cancer therapy.

The process of cell division represents an extraordinary target to develop antitumor therapies. Indeed, a large number of clinically relevant anti-cancer drugs, such as taxanes and vinca alkaloids, target mitosis by stimulating or inhibiting microtubule (MT) polymerization. During the past decades anti-tubulin drugs have proven very effective against a wide range of tumors. However, collateral effects, such as myelosuppression and MT disruption in non-dividing tissues, including brain, are common. Recently, the increased understanding of the cell division process and the identification of several signaling pathways controlling mitosis have provided novel opportunities for cancer drug discovery. Consequently, mitotic proteins have become attractive targets to develop molecular cancer therapeutics. In this scenario, kinetochores (KTs) represent an attractive therapeutic target in light of their fundamental role in driving chromosome segregation and controlling chromosome segregation errors. Indeed, cells require a fine regulation of the kinetochore-microtubule (KT-MT) attachment stability to prevent chromosome instability, and KT-MT attachment dynamics is often deregulated in tumour cells [2]. Chromosome instability is commonly accepted as a driving force in the development of cancer, but more recent work has demonstrated that extensive chromosome missegregation may be detrimental to cancer cells and act as a tumor suppression mechanism [3]. In light of this double role of chromosome instability in cancer, we have explored the hypothesis that interfering with KT-MT attachment dynamics could drive massive chromosome missegregation and kill tumor cells. Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the evolutionary conserved Ndc80 complex, the molecular connector between KTs and MTs. Among the subunits of the Ndc80 complex, Hec1 directly interacts with MTs and regulates KT-MT dynamics and attachment stability [3]. Importantly, Hec1 is frequently overexpressed in cancer. We previously demonstrated that expression of Hec1 fused with the enhanced green fluorescent protein (EGFP) tag at its N-terminus (EGFP-Hec1), the protein domain that regulates MT attachment dynamics, led to a strong accumulation of this modified protein, which acted as a dominant negative mutant over the endogenous Hec1. Mitotic cells expressing a N-terminus tagged Hec1 accumulated lateral KT-MT attachments and underwent a spindle assembly checkpoint (SAC) dependent mitotic arrest associated with the formation of multipolar spindles [4]. We further showed that expression of an inducible N-terminus modified Hec1 completely abolished in vitro growth of EGFP-Hec1 expressing HeLa cells but had no effects on untransformed human fibroblasts or epithelial cells [5]. These in vitro cell-based data were validated in vivo by showing that inducible EGFP-Hec1 expression strongly inhibited tumor growth in a HeLa xenograft mouse model [5]. Strikingly, in both in vitro and in vivo models, EGFP-Hec1 expressing cells were permanently arrested in mitosis and produced multipolar spindles. Live imaging of EGFP-Hec1 expressing cells demonstrated that impaired chromosome segregation within multipolar spindles induced mitotic catastrophe, identified by the induction of apoptotic death from mitosis, or cytokinesis failure and multinucleation. Finally, measurements of MT flux rates and turnover at KT demonstrated that EGFP-Hec1 increased KT-MT attachment stability, suggesting that stabilizing KT-MT attachment dynamics represents a promising therapeutic approach [5]. Consistent with KT-MT attachment dynamics being the molecular target of the anticancer effect, expression of Hec1 fused with EGFP at its C-terminus, which does not affect KT-MT attachment dynamics, did not significantly affect cancer cell proliferation [5]. Collectively, our results demonstrate that massive chromosome missegregation within multipolar spindles can be used to kill tumor cells by activating a mitotic catastrophe process. In our experimental model, induction of multipolarity is caused by the extended time cells spend in prometaphase, which promotes cohesion fatigue (uncoordinated centromeric cohesion release) and concomitant centriole disengagement by leaky separase activation as depicted in Figure ​Figure11 [6,7]. Cancer cell death induced by cohesion fatigue-dependent multipolarity has been demonstrated following depletion of proteins controlling SAC silencing or after inhibition of the Anaphase Promoting Complex/cdc20 (APC/C) activity (Figure ​(Figure1)1) and some of these treatments have been found more efficient than MT inhibitors in avoiding mitotic slippage and producing cancer cell death (7,8). These studies, together with our work, demonstrate that stimulation of spindle multipolarity can be used as an anti-cancer strategy through the activation of mitotic catastrophe after a multipolar mitosis. Moreover, they indicate that targeting the machineries involved in the regulation of KT-MT attachment dynamics, in the correction of KT-MT misattachments or in the silencing of the spindle assembly checkpoint may be a new frontier in the development of anticancer strategies. Figure 1 Induction of multipolarity by interfering with kinetochore-microtubule attachment dynamics as an anticancer strategy

RNA pol II transcript abundance controls condensin accumulation at mitotically up‐regulated and heat‐shock‐inducible genes in fission yeast

Condensin plays fundamental roles in chromosome dynamics. In this study, we determined the binding sites of condensin on fission yeast (Schizosaccharomyces pombe) chromosomes at the level of nucleotide sequences using chromatin immunoprecipitation (ChIP) and ChIP sequencing (ChIP-seq). We found that condensin binds to RNA polymerase I-, II- and III-transcribed genes during both mitosis and interphase, and we focused on pol II constitutive and inducible genes. Accumulation sites for condensin are distinct from those of cohesin and DNA topoisomerase II. Using cell cycle stage and heat-shock-inducible genes, we show that pol II-mediated transcripts cause condensin accumulation. First, condensin's enrichment on mitotically activated genes was abolished by deleting the sep1+ gene that encodes an M-phase-specific forkhead transcription factor. Second, by raising the temperature, condensin accumulation was rapidly induced at heat-shock protein genes in interphase and even during mid-mitosis. In interphase, condensin accumulates preferentially during the postreplicative phase. Pol II-mediated transcription was neither repressed nor activated by condensin, as levels of transcripts per se did not change when mutant condensin failed to associate with chromosomal DNA. However, massive chromosome missegregation occurred, suggesting that abundant pol II transcription may require active condensin before proper chromosome segregation.

N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore-microtubule dynamics.

Mitotic proteins are attractive targets to develop molecular cancer therapeutics due to the intimate interdependence between cell proliferation and mitosis. In this work, we have explored the therapeutic potential of the kinetochore (KT) protein Hec1 (Highly Expressed in Cancer protein 1) as a molecular target to produce massive chromosome missegregation and cell death in cancer cells. Hec1 is a constituent of the Ndc80 complex, which mediates KT-microtubule (MT) attachments at mitosis and is upregulated in various cancer types. We expressed Hec1 fused with enhanced green fluorescent protein (EGFP) at its N-terminus MT-interaction domain in HeLa cells and showed that expression of this modified Hec1, which localized at KTs, blocked cell proliferation and promoted apoptosis in tumour cells. EGFP-Hec1 was extremely potent in tumour cell killing and more efficient than siRNA-induced Hec1 depletion. In striking contrast, normal cells showed no apparent cell proliferation defects or cell death following EGFP-Hec1 expression. Live-cell imaging demonstrated that cancer cell death was associated with massive chromosome missegregation within multipolar spindles after a prolonged mitotic arrest. Moreover, EGFP-Hec1 expression was found to increase KT-MT attachment stability, providing a molecular explanation for the abnormal spindle architecture and the cytotoxic activity of this modified protein. Consistent with cell culture data, EGFP-Hec1 expression was found to strongly inhibit tumour growth in a mouse xenograft model by disrupting mitosis and inducing multipolar spindles. Taken together, these findings demonstrate that stimulation of massive chromosome segregation defects can be used as an anti-cancer strategy through the activation of mitotic catastrophe after a multipolar mitosis. Importantly, this study represents a clear proof of concept that targeting KT proteins required for proper KT-MT attachment dynamics constitutes a powerful approach in cancer therapy.

Cenp-meta is required for sustained spindle checkpoint

ABSTRACTCenp-E is a kinesin-like motor protein required for efficient end-on attachment of kinetochores to the spindle microtubules. Cenp-E immunodepletion in Xenopus mitotic extracts results in the loss of mitotic arrest and massive chromosome missegregation, whereas its depletion in mammalian cells leads to chromosome segregation defects despite the presence of a functional spindle assembly checkpoint (SAC). Cenp-meta has previously been reported to be the Drosophila homolog of vertebrate Cenp-E. In this study, we show that cenp-metaΔ mutant neuroblasts arrest in mitosis when treated with colchicine. cenp-metaΔ mutant cells display a mitotic delay. Yet, despite the persistence of the two checkpoint proteins Mad2 and BubR1 on unattached kinetochores, these cells eventually enter anaphase and give rise to highly aneuploid daughter cells. Indeed, we find that cenp-metaΔ mutant cells display a slow but continuous degradation of cyclin B, which eventually triggers the mitotic exit observed. Thus, our data provide evidence for a role of Cenp-meta in sustaining the SAC response.

Schizosaccharomyces pombe centromere protein Mis19 links Mis16 and Mis18 to recruit CENP‐A through interacting with NMD factors and the SWI/SNF complex

CENP-A is a centromere-specific variant of histone H3 that is required for accurate chromosome segregation. The fission yeast Schizosaccharomyces pombe and mammalian Mis16 and Mis18 form a complex essential for CENP-A recruitment to centromeres. It is unclear, however, how the Mis16-Mis18 complex achieves this function. Here, we identified, by mass spectrometry, novel fission yeast centromere proteins Mis19 and Mis20 that directly interact with Mis16 and Mis18. Like Mis18, Mis19 and Mis20 are localized at the centromeres during interphase, but not in mitosis. Inactivation of Mis19 in a newly isolated temperature-sensitive mutant resulted in CENP-A delocalization and massive chromosome missegregation, whereas Mis20 was dispensable for proper chromosome segregation. Mis19 might be a bridge component for Mis16 and Mis18. We isolated extragenic suppressor mutants for temperature-sensitive mis18 and mis19 mutants and used whole-genome sequencing to determine the mutated sites. We identified two groups of loss-of-function suppressor mutations in non-sense-mediated mRNA decay factors (upf2 and ebs1), and in SWI/SNF chromatin-remodeling components (snf5, snf22 and sol1). Our results suggest that the Mis16-Mis18-Mis19-Mis20 CENP-A-recruiting complex, which is functional in the G1-S phase, may be counteracted by the SWI/SNF chromatin-remodeling complex and non-sense-mediated mRNA decay, which may prevent CENP-A deposition at the centromere.

The spindle assembly checkpoint: perspectives in tumorigenesis and cancer therapy

Loss or gain of chromosomes, a condition known as aneuploidy, is a common feature of tumor cells and has therefore been proposed as the driving force for tumorigenesis. Such chromosomal instability can arise during mitosis as a result of mis-segregation of the duplicated sister chromatids to the two daughter cells. In normal cells, missegregation is usually prevented by the spindle assembly checkpoint (SAC), a sophisticated surveillance mechanism that inhibits mitotic exit until all chromosomes have successfully achieved bipolar attachment to spindle microtubules. Complete abrogation of SAC activity is lethal to normal as well as to tumor cells, as a consequence of massive chromosome mis-segregation. Importantly, many human aneuploid tumor cells exhibit a weakened SAC activity that allows them to tolerate gains or losses of a small number of chromosomes; and interfering with this SAC residual activity may constitute a suitable strategy to kill cancer cells. This review focuses on the potential link between SAC and tumorigenesis, and the therapeutic strategy to target the SAC for cancer treatment.

Mis17 Is a Regulatory Module of the Mis6-Mal2-Sim4 Centromere Complex That Is Required for the Recruitment of CenH3/CENP-A in Fission Yeast

BackgroundThe centromere is the chromosome domain on which the mitotic kinetochore forms for proper segregation. Deposition of the centromeric histone H3 (CenH3, CENP-A) is vital for the formation of centromere-specific chromatin. The Mis6-Mal2-Sim4 complex of the fission yeast S. pombe is required for the recruitment of CenH3 (Cnp1), but its function remains obscure.Methodology/Principal FindingsMass spectrometry was performed on the proteins precipitated with Mis6- and Mis17-FLAG. The results together with the previously identified Sim4- and Mal2-TAP precipitated proteins indicated that the complex contains 12 subunits, Mis6, Sim4, Mal2, Mis15, Mis17, Cnl2, Fta1-4, Fta6-7, nine of which have human centromeric protein (CENP) counterparts. Domain dissection indicated that the carboxy-half of Mis17 is functional, while its amino-half is regulatory. Overproduction of the amino-half caused strong negative dominance, which led to massive chromosome missegregation and hypersensitivity to the histone deacetylase inhibitor TSA. Mis17 was hyperphosphorylated and overproduction-induced negative dominance was abolished in six kinase-deletion mutants, ssp2 (AMPK), ppk9 (AMPK), ppk15 (Yak1), ppk30 (Ark1), wis4 (Ssk2), and lsk1 (P-TEFb).ConclusionsMis17 may be a regulatory module of the Mis6 complex. Negative dominance of the Mis17 fragment is exerted while the complex and CenH3 remain at the centromere, a result that differs from the mislocalization seen in the mis17-362 mutant. The known functions of the kinases suggest an unexpected link between Mis17 and control of the cortex actin, nutrition, and signal/transcription. Possible interpretations are discussed.

Regulation of APC/C Activity in Oocytes by a Bub1-Dependent Spindle Assembly Checkpoint

Missegregation of chromosomes during meiosis in human females causes aneuploidy, including trisomy 21, and is thought also to be the major cause of age-related infertility. Most errors are thought to occur at the first meiotic division. The high frequency of errors raises questions as to whether the surveillance mechanism known as the spindle assembly checkpoint (SAC) that controls the anaphase-promoting complex or cyclosome (APC/C) operates effectively in oocytes. Experimental approaches hitherto used to inactivate the SAC in oocytes suffer from a number of drawbacks. Bub1 protein was depleted specifically in oocytes with a Zp3-Cre transgene to delete exons 7 and 8 from a floxed BUB1(F) allele. Loss of Bub1 greatly accelerates resolution of chiasmata and extrusion of polar bodies. It also causes defective biorientation of bivalents, massive chromosome missegregation at meiosis I, and precocious loss of cohesion between sister centromeres. By using a quantitative assay for APC/C-mediated securin destruction, we show that the APC/C is activated in an exponential fashion, with activity peaking 12-13 hr after GVBD, and that this process is advanced by 5 hr in oocytes lacking Bub1. Importantly, premature chiasmata resolution does not occur in Bub1-deficient oocytes also lacking either the APC/C's Apc2 subunit or separase. Finally, we show that Bub1's kinase domain is not required to delay APC/C activation. We conclude that far from being absent or ineffective, the SAC largely determines the timing of APC/C and hence separase activation in oocytes, delaying it for about 5 hr.

Fission Yeast Kinesin-8 Klp5 and Klp6 Are Interdependent for Mitotic Nuclear Retention and Required for Proper Microtubule Dynamics

Fission yeast has two kinesin-8s, Klp5 and Klp6, which associate to form a heterocomplex. Here, we show that Klp5 and Klp6 are mutually dependent on each other for nuclear mitotic localization. During interphase, they are exported to the cytoplasm. In sharp contrast, during mitosis, Klp5 and Klp6 remain in the nucleus, which requires the existence of each counterpart. Canonical nuclear localization signal (NLS) is identified in the nonkinesin C-terminal regions. Intriguingly individual NLS mutants (NLSmut) exhibit loss-of-function phenotypes, suggesting that Klp5 and Klp6 enter the nucleus separately. Indeed, although neither Klp5-NLSmut nor Klp6-NLSmut enters the nucleus, wild-type Klp6 or Klp5, respectively, does so with different kinetics. In the absence of Klp5/6, microtubule catastrophe/rescue frequency and dynamicity are suppressed, whereas growth and shrinkage rates are least affected. Remarkably, chimera strains containing only the N-terminal Klp5 kinesin domains cannot disassemble interphase microtubules during mitosis, leading to the coexistence of cytoplasmic microtubules and nuclear spindles with massive chromosome missegregation. In this strain, a marked reduction of microtubule dynamism, even higher than in klp5/6 deletions, is evident. We propose that Klp5 and Klp6 play a vital role in promoting microtubule dynamics, which is essential for the spatiotemporal control of microtubule morphogenesis.

Fission yeast dam1-A8 mutant is resistant to and rescued by an anti-microtubule agent

The Dam1/DASH outer kinetochore complex is required for high-fidelity chromosome segregation in budding and fission yeast. Unlike budding yeast, the fission yeast complex is non-essential, however it promotes bipolar microtubule attachment in conjunction with microtubule-depolymerising kinesin-8 Klp5 and Klp6. Here, we screened for dam1 temperature sensitive mutants in a klp5 null background and identified dam1-A8 that contains two amino acid substitutions in the C-terminus (H126R and E149G). dam1-A8klp5 mutant cells display massive chromosome missegregation with lagging chromosomes and monopolar attachment of sister chromatids to one SPB (spindle pole body). Unexpectedly contrary to a deletion mutant that is hypersensitive to microtubule-destabilising drugs, dam1-A8 is resistant and furthermore the temperature sensitivity of dam1-A8klp5 is rescued by addition of these drugs. This indicates that the hyper-stabilised rigidity of kinetochore-spindle mal-attachments is the primary cause of lethality. Our result shows that fine-tuning of Dam1 activity is essential for chromosome bi-orientation.

Cell-Type-Specific TEV Protease Cleavage Reveals Cohesin Functions in Drosophila Neurons

SummaryCohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the α kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postmitotic cells, where it is widely expressed, we have created fruit flies whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells, but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in postmitotic neurons is lethal. In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.

Mps1 Phosphorylates Borealin to Control Aurora B Activity and Chromosome Alignment

Maintenance of chromosomal stability relies on coordination between various processes that are critical for proper chromosome segregation in mitosis. Here we show that monopolar spindle 1 (Mps1) kinase, which is essential for the mitotic checkpoint, also controls correction of improper chromosome attachments. We report that Borealin/DasraB, a member of the complex that regulates the Aurora B kinase, is directly phosphorylated by Mps1 on residues that are crucial for Aurora B activity and chromosome alignment. As a result, cells lacking Mps1 kinase activity fail to efficiently align chromosomes due to impaired Aurora B function at centromeres, leaving improper attachments uncorrected. Strikingly, Borealin/DasraB bearing phosphomimetic mutations restores Aurora B activity and alignment in Mps1-depleted cells. Mps1 thus coordinates attachment error correction and checkpoint signaling, two crucial responses to unproductive chromosome attachments.

Rapid effects of acute anoxia on spindle kinetochore interactions activate the mitotic spindle checkpoint

The dramatic chromosome instability in certain tumors might reflect a synergy of spindle checkpoint defects with hypoxic conditions. In Caenorhabditis elegans and Drosophila melanogaster, spindle checkpoint activation has been implicated in the response to acute anoxia. The activation mechanism is unknown. Our analyses in D. melanogaster demonstrate that oxygen deprivation affects microtubule organization within minutes. The rapid effects of anoxia are identical in wild-type and spindle checkpoint-deficient Mps1 mutant embryos. Therefore, the anoxia effects on the mitotic spindle are not a secondary consequence of spindle checkpoint activation. Some motor, centrosome and kinetochore proteins (dynein, Kin-8, Cnn, TACC, Cenp-C, Nuf2) are rapidly relocalized after oxygen deprivation. Kinetochores congress inefficiently into the metaphase plate and do not experience normal pulling forces. Spindle checkpoint proteins accumulate mainly within the spindle midzone and inhibit anaphase onset. In checkpoint-deficient embryos, mitosis is still completed after oxygen deprivation, although accompanied by massive chromosome missegregation. Inhibitors of oxidative phosphorylation mimic anoxia effects. We conclude that oxygen deprivation impairs the chromosome segregation machinery more rapidly than spindle checkpoint function. Although involving adenosine triphosphate (ATP)-consuming kinases, the spindle checkpoint can therefore be activated by spindle damage in response to acute anoxia and protect against aneuploidies.

PICH, a Centromere-Associated SNF2 Family ATPase, Is Regulated by Plk1 and Required for the Spindle Checkpoint

We identify PICH (Plk1-interacting checkpoint "helicase"), a member of the SNF2 ATPase family, as an interaction partner and substrate of Plk1. Following phosphorylation of PICH on the Cdk1 site T1063, Plk1 is recruited to PICH and controls its localization. Starting in prometaphase, PICH accumulates at kinetochores and inner centromeres. Moreover, it decorates threads that form during metaphase before increasing in length and progressively diminishing during anaphase. PICH-positive threads connect sister kinetochores and are dependent on tension, sensitive to DNase, and exacerbated in response to premature loss of cohesins or inhibition of topoisomerase II, suggesting that they represent stretched centromeric chromatin. Depletion of PICH causes the selective loss of Mad2 from kinetochores and completely abrogates the spindle checkpoint, resulting in massive chromosome missegregation. These data identify PICH as a novel essential component of checkpoint signaling. We propose that PICH binds to catenated centromere-related DNA to monitor tension developing between sister kinetochores.

Mal3, the fission yeast EB1 homologue, cooperates with Bub1 spindle checkpoint to prevent monopolar attachment

Bipolar microtubule attachment is central to genome stability. Here, we investigate the mitotic role of the fission yeast EB1 homologue Mal3. Mal3 shows dynamic inward movement along the spindle, initial emergence at the spindle pole body (SPB) and translocation towards the equatorial plane, followed by sudden disappearance. Deletion of Mal3 results in early mitotic delay, which is dependent on the Bub1, but not the Mad2, spindle checkpoint. Consistently, Bub1, but not Mad2, shows prolonged kinetochore localization. Double mutants between mal3 and a subset of checkpoint mutants, including bub1, bub3, mad3 and mph1, but not mad1 or mad2, show massive chromosome mis-segregation defects. In mal3bub1 mutants, both sister centromeres tend to remain in close proximity to one of the separating SPBs. Further analysis indicates that mis-segregated centromeres are exclusively associated with the mother SPB. Mal3, therefore, has a role in preventing monopolar attachment in cooperation with the Bub1/Bub3/Mad3/Mph1-dependent checkpoint.

Unstable microtubule capture at kinetochores depleted of the centromere-associated protein CENP-F

Centromere protein F (CENP-F) (or mitosin) accumulates to become an abundant nuclear protein in G2, assembles at kinetochores in late G2, remains kinetochore-bound until anaphase, and is degraded at the end of mitosis. Here we show that the absence of nuclear CENP-F does not affect cell cycle progression in S and G2. In a subset of CENP-F depleted cells, kinetochore assembly fails completely, thereby provoking massive chromosome mis-segregation. In contrast, the majority of CENP-F depleted cells exhibit a strong mitotic delay with reduced tension between kinetochores of aligned, bi-oriented sister chromatids and decreased stability of kinetochore microtubules. These latter kinetochores generate mitotic checkpoint signaling when unattached, recruiting maximum levels of Mad2. Use of YFP-marked Mad1 reveals that throughout the mitotic delay some aligned, CENP-F depleted kinetochores continuously recruit Mad1. Others rebind YFP-Mad1 intermittently so as to produce 'twinkling', demonstrating cycles of mitotic checkpoint reactivation and silencing and a crucial role for CENP-F in efficient assembly of a stable microtubule-kinetochore interface.

Novel Genes Required for Meiotic Chromosome Segregation Are Identified by a High-Throughput Knockout Screen in Fission Yeast

Two rounds of chromosome segregation after only a single round of DNA replication enable the production of haploid gametes from diploid precursors during meiosis. To identify genes involved in meiotic chromosome segregation, we developed an efficient strategy to knock out genes in the fission yeast on a large scale. We used this technique to delete 180 functionally uncharacterized genes whose expression is upregulated during meiosis. Deletion of two genes, sgo1 and mde2, caused massive chromosome missegregation. sgo1 is required for retention of centromeric sister-chromatid cohesion after anaphase I. We show here that mde2 is required for formation of the double-strand breaks necessary for meiotic recombination.