Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae.

Inhibition of mitosis by antimitotic drugs is thought to occur by destruction of microtubules, causing cells to arrest through the action of one or more mitotic checkpoints. We have patterned experiments in the yeast Saccharomyces cerevisiae after recent studies in mammalian cells that demonstrate the effectiveness of antimitotic drugs at concentrations that maintain spindle structure. We show that low concentrations of nocodazole delay cell division under the control of the previously identified mitotic checkpoint genes BUB1, BUB3, MAD1, and MAD2 and independently of BUB2. The same genes mediate the cell cycle delay induced in ctf13 mutants, limited for an essential kinetochore component. Our data suggest that a low concentration of nocodazole induces a cell cycle delay through checkpoint control that is sensitive to impaired kinetochore function. The BUB2 gene may be part of a separate checkpoint that responds to abnormal spindle structure.     

Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast

Accurate chromosome segregation depends on precise regulation of mitosis by the spindle checkpoint. This checkpoint monitors the status of kinetochore-microtubule attachment and delays the metaphase to anaphase transition until all kinetochores have formed stable bipolar connections to the mitotic spindle. Components of the spindle checkpoint include the mitotic arrest defective (MAD) genes MAD1-3, and the budding uninhibited by benzimidazole (BUB) genes BUB1 and BUB3. In animal cells, all known spindle checkpoint proteins are recruited to kinetochores during normal mitoses. In contrast, we show that whereas Saccharomyces cerevisiae Bub1p and Bub3p are bound to kinetochores early in mitosis as part of the normal cell cycle, Mad1p and Mad2p are kinetochore bound only in the presence of spindle damage or kinetochore lesions that interfere with chromosome-microtubule attachment. Moreover, although Mad1p and Mad2p perform essential mitotic functions during every division cycle in mammalian cells, they are required in budding yeast only when mitosis goes awry. We propose that differences in the behavior of spindle checkpoint proteins in animal cells and budding yeast result primarily from evolutionary divergence in spindle assembly pathways.     

Mutations in the yeast cyclin-dependent kinase Cdc28 reveal a role in the spindle assembly checkpoint

Anaphase onset and mitotic exit are regulated by the spindle assembly or kinetochore checkpoint, which inhibits the anaphase-promoting complex (APC), preventing the degradation of anaphase inhibitors and mitotic cyclins. As a result, cells arrest with high cyclin-dependent kinase (CDK) activity due to the accumulation of cyclins. Aside from this, a clear-cut demonstration of a direct role for CDKs in the spindle checkpoint response has been elusive. Cdc28 is the main CDK driving the cell cycle in budding yeast. In this report, mutations in cdc28 are described that confer specific checkpoint defects, supersensitivity towards microtubule poisons and chromosome loss. Two alleles encode single mutations in the N and C terminal regions, respectively (R10G and R288G), and one allele specifies two mutations near the C terminus (F245L, I284T). These cdc28 mutants are unable to arrest or efficiently prevent sister chromatid separation during treatment with nocodazole. Genetic interactions with checkpoint and apc mutants suggest Cdc28 may regulate checkpoint arrest downstream of the MAD2 and BUB2 pathways. These studies identify a C-terminal domain of Cdc28 required for checkpoint arrest upon spindle damage that mediates chromosome stability during vegetative growth, suggesting that it has an essential surveillance function in the unperturbed cell cycle.Communicated by A. Aguilera     

Defective in mitotic arrest 1/ring finger 8 is a checkpoint protein that antagonizes the human mitotic exit network

A molecular pathway homologous to the S. cerevisiae mitotic exit network (MEN) and S. pombe septation initiation network has recently been described in higher eukaryotes and involves the tumor suppressor kinase LATS1 and its subunit MOB1A. The yeast MEN/septation initiation network pathways are regulated by the ubiquitin ligase defective in mitotic arrest 1 (Dma1p), a checkpoint protein that helps maintain prometaphase arrest when cells are exposed to microtubule poisons. We identified here the RING domain protein ring finger 8 (RNF8) as the human orthologue of the yeast protein Dma1p. Like its yeast counterparts, human DMA1/RNF8 localized at the midbody and its depletion by siRNA compromised mitotic arrest of nocodazole-treated cells in a manner dependent on the MEN. Depletion of MAD2, a spindle checkpoint protein, also compromised mitotic arrest, but in a MEN-independent manner. Thus, two distinct checkpoint pathways maintain mitotic arrest in cells exposed to microtubule poisons.     

Differential kinetochore requirements for establishment and maintenance of the spindle checkpoint are dependent on the mechanism of checkpoint activation in Saccharomyces cerevisiae

The spindle checkpoint in the yeast Saccharomyces cerevisiae is an intracellular signal transduction pathway comprised of two branches that inhibit two different mitotic transitions in cells treated with benzimidazole drugs such as nocodazole. The kinetochore is an integral component of the MAD2 branch of the spindle checkpoint pathway. Current models propose that the kinetochore is required for both the establishment and maintenance of the spindle checkpoint but a role for the kinetochore in the maintenance of spindle checkpoint in yeast has never been directly tested. We used a temperature sensitive ndc10-1 mutant to inactivate kinetochores before and after arresting cells in mitosis to determine the role of kinetochores in the establishment and maintenance of the spindle checkpoint. We show that both establishment and maintenance requires kinetochore function in response to spindle damage induced by benzimidazole drugs. Excess expression of the Mps1 protein kinase causes wild type cells and ndc10-1 cells to arrest in mitosis. Unlike the spindle checkpoint arrest activated by benzimidazoles, this arrest can be maintained independently of kinetochores. The arrest induced by excess Mps1p is independent of BUB2. Therefore, mitotic arrest induced by excess Mps1p expression is due to the action of the MAD2 branch of the spindle checkpoint pathway and excess Mps1p acts downstream of the kinetochore.     

Differential Kinetochore Requirements for Establishment and Maintenance of the Spindle Checkpoint Are Dependent on the Mechanism of Checkpoint Activation in Saccharomyces cerevisiae

The spindle checkpoint in the yeast Saccharomyces cerevisiae is an intracellular signal transduction pathway comprised of two branches that inhibit two different mitotic transitions in cells treated with benzimidazole drugs such as nocodazole. The kinetochore is an integral component of the MAD2 branch of the spindle checkpoint pathway. Current models propose that the kinetochore is required for both the establishment and maintenance of the spindle checkpoint but a role for the kinetochore in the maintenance of spindle checkpoint in yeast has never been directly tested. We used a temperature sensitive ndc10-1 mutant to inactivate kinetochores before and after arresting cells in mitosis to determine the role of kinetochores in the establishment and maintenance of the spindle checkpoint. We show that both establishment and maintenance requires kinetochore function in response to spindle damage induced by benzimidazole drugs. Excess expression of the Mps1 protein kinase causes wild type cells and ndc10-1 cells to arrest in mitosis. Unlike the spindle checkpoint arrest activated by benzimidazoles, this arrest can be maintained independently of kinetochores. The arrest induced by excess Mps1p is independent of BUB2. Therefore, mitotic arrest induced by excess Mps1p expression is due to the action of the MAD2 branch of the spindle checkpoint pathway and excess Mps1p acts downstream of the kinetochore.     

Novel response to microtubule perturbation in meiosis

During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G(1) or G(2), instead of metaphase. Cells arrest in G(1) if microtubule perturbation occurs as they enter the meiotic cell cycle and in G(2) if cells are already undergoing premeiotic S phase. Concomitantly, cells down-regulate genes required for cell cycle progression, meiotic differentiation, and spore formation in a highly coordinated manner. Decreased expression of these genes is likely to be responsible for halting both cell cycle progression and meiotic development. Our results point towards the existence of a novel surveillance mechanism of microtubule integrity that may be particularly important during specialized cell cycles when coordination of cell cycle progression with a developmental program is necessary.     

Characterization of regions in hsMAD1 needed for binding hsMAD2. A polymorphic change in an hsMAD1 leucine zipper affects MAD1-MAD2 interaction and spindle checkpoint function

In eukaryotes, the mitotic spindle assembly checkpoint provides a monitor for the fidelity of chromosomal segregation. In this context, the mitotic arrest deficiency protein 2 (MAD2) censors chromosomal mis-segregation by monitoring microtubule attachment/tension, a role that requires its attachment to kinetochores. Studies in yeast have shown that binding of MAD1 to MAD2 is important for the checkpoint function of the latter. The interactions between human MAD1 (hsMAD1) and human MAD2 (hsMAD2) have, however, remained poorly characterized. Here we report that two leucine zipper domains (amino acids 501-522 and 557-571) in hsMAD1 are required for its contact with hsMAD2. Interestingly, in several cancer cell lines, we noted the frequent presence of a coding single nucleotide Arg to His polymorphism at codon 558 located within the second leucine zipper of hsMAD1. We found that hsMAD1H558 is less proficient than hsMAD1R558 in binding hsMAD2 and in enforcing mitotic arrest. We also document a first example of loss-of-heterozygosity for a spindle checkpoint gene (at the hsMAD1 558 locus) in a human breast cancer. Based on our findings, it is possible that hsMAD1H558 could be an at-risk polymorphism that contributes to attenuated spindle checkpoint function in human cells.     

Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast

The spindle assembly checkpoint prevents cells from initiating anaphase until the spindle has been fully assembled. We previously isolated mitotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that interferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response to microtubule depolymerization. MAD1 is predicted to encode a 90-kD coiled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1p is soluble. Mad1p becomes hyperphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sensitive tubulin mutant at the restrictive temperature. This modification does not occur in G1- arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis machinery, suggesting that Mad1p hyperphosphorylation is a step in the activation of the spindle assembly checkpoint. Analysis of Mad1p phosphorylation in other spindle assembly checkpoint mutants reveals that this response to microtubule- disrupting agents is defective in some (mad2, bub1, and bub3) but not all (mad3, bub2) mutant strains. We discuss the possible functions of Mad1p at this cell cycle checkpoint.     

IBD2 encodes a novel component of the Bub2p-dependent spindle checkpoint in the budding yeast Saccharomyces cerevisiae

During mitosis, genomic integrity is maintained by the proper coordination of mitotic events through the spindle checkpoint. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the incorrect orientation of the mitotic spindle. Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for proper mitotic exit. We have isolated a novel Bfa1p interacting protein named Ibd2p in the budding yeast Saccharomyces cerevisiae. We found that IBD2 (Inhibition of Bud Division 2) is not an essential gene but its deletion mutant proceeded through the cell cycle in the presence of microtubule-destabilizing drugs, thereby inducing a sharp decrease in viability. In addition, overexpression of Mps1p caused partial mitotic arrest in ibd2Delta as well as in bub2Delta, suggesting that IBD2 encodes a novel component of the spindle checkpoint downstream of MPS1. Overexpression of Ibd2p induced mitotic arrest with increased levels of Clb2p in wild type and mad2Delta, but not in deletion mutants of BUB2 and BFA1. Pds1p was also stabilized by the overexpression of Ibd2p in wild-type cells. The mitotic arrest defects observed in ibd2Delta in the presence of nocodazole were restored by additional copies of BUB2, BFA1, and CDC5, whereas an extra copy of IBD2 could not rescue the mitotic arrest defects of bub2Delta and bfa1Delta. The mitotic arrest defects of ibd2Delta were not recovered by MAD2, or vice versa. Analysis of the double mutant combinations ibd2Deltamad2Delta, ibd2Deltabub2Delta, and ibd2Deltadyn1Delta showed that IBD2 belongs to the BUB2 epistasis group. Taken together, these data demonstrate that IBD2 encodes a novel component of the BUB2-dependent spindle checkpoint pathway that functions upstream of BUB2 and BFA1.     

Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint

Kinetochore attachment to the ends of dynamic microtubules is a conserved feature of mitotic spindle organization that is thought to be critical for proper chromosome segregation. Although kinetochores have been described to transition from lateral to end-on attachments, the phase of lateral attachment has been difficult to study in yeast due to its transient nature. We have previously described a kinetochore mutant, DAM1-765, which exhibits lateral attachments and misregulation of microtubule length. Here we show that the misregulation of microtubule length in DAM1-765 cells occurs despite localization of microtubule associated proteins Bik1, Stu2, Cin8, and Kip3 to microtubules. DAM1-765 kinetochores recruit the spindle checkpoint protein Bub1, however Bub1 localization to DAM1-765 kinetochores is not sufficient to cause a cell cycle arrest. Interestingly, the DAM1-765 mutation rescues the temperature sensitivity of a biorientation-deficient ipl1-321 mutant, and DAM1-765 chromosome loss rates are similar to wild-type cells. The spindle checkpoint in DAM1-765 cells responds properly to unattached kinetochores created by nocodazole treatment and loss of tension caused by a cohesin mutant. Progression of DAM1-765 cells through mitosis therefore suggests that satisfaction of the checkpoint depends more highly on biorientation of sister kinetochores than on achievement of a specific interaction between kinetochores and microtubule plus ends.     

The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity.     

Microtubules,microtubule-interfering agents and apoptosis

Microtubules are dynamic polymers that play crucial roles in a large number of cellular functions. Their pivotal role in mitosis makes them a target for the development of anticancer drugs. Microtubule-damaging agents suppress microtubule dynamics, leading to disruption of the mitotic spindle in dividing cells, cell cycle arrest at M phase, and late apoptosis. A better understanding of the processes coupling microtubule damage to the onset of apoptosis will reveal sites of potential intervention in cancer chemotherapy. Inhibition of microtubule dynamics induces persistent modification of biological processes (M arrest) and signaling pathways (mitotic spindle assembly checkpoint activation, Bcl-2 phosphorylation, c-Jun NH₂-terminal kinase activation), which ultimately lead to apoptosis through the accumulation of signals that finally reach the threshold for the onset of apoptosis or through diminishing the threshold for engagement of cell death. Microtubules serve also as scaffolds for signaling molecules that regulate apoptosis, such as Bim and survivin, and their release from microtubules affect the activities of these apoptosis regulators. Thus, sustained modification of signaling routes and changes in the scaffolding properties of microtubules seem to constitute two major processes in the apoptotic response induced by microtubule-interfering agents.     

Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint

Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly acting BUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed a G₂ DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, including BUB2 and BUB3 and MAD1, MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal.     

Human Mps1 kinase is required for the spindle assembly checkpoint but not for centrosome duplication

Budding yeast Mps1p kinase has been implicated in both the duplication of microtubule-organizing centers and the spindle assembly checkpoint. Here we show that hMps1, the human homolog of yeast Mps1p, is a cell cycle-regulated kinase with maximal activity during M phase. hMps1 localizes to kinetochores and its activity and phosphorylation state increase upon activation of the mitotic checkpoint. By antibody microinjection and siRNA, we demonstrate that hMps1 is required for human cells to undergo checkpoint arrest in response to microtubule depolymerization. In contrast, centrosome (re-)duplication as well as cell division occur in the absence of hMps1. We conclude that hMps1 is required for the spindle assembly checkpoint but not for centrosome duplication.     

Shogaols at proapoptotic concentrations induce G<Subscript>2</Subscript>/M arrest and aberrant mitotic cell death associated with tubulin aggregation

Shogaols have been previously reported to induce cancer cell death via multiple mechanisms, among which one analog 6-shogaol has been reported to cause microtubule damage through specific reaction with sulfhydryl groups in tubulin. In this study, a series of shogaols with different side chain lengths (4-, 6-, 8- and 10-shogaol) was synthesized and evaluated for antiproliferative activity in HCT 116 colon carcinoma and SH-SY5Y neuroblastoma cells. 4- and 6-shogaol were identified as lead compounds possessing the strongest antiproliferative activity. In the soft agar assay, the lead shogaols displayed dose-dependent inhibition on cancer cell colony formation under anchorage-independent conditions. Using HCT 116 as the selected cancer cell line, the molecular events linking shogaols-induced G₂/M cell cycle arrest to apoptosis characterized by caspase 3 and PARP cleavage were investigated. At sublethal concentrations, the halt at G₂/M phase was alleviated along time and cells survived. Conversely, proapoptotic concentrations of 4- and 6-shogaol induced irreversible G₂/M arrest that was at least in part associated with down-regulation of cell cycle checkpoint proteins cdk1, cyclin B and cdc25C, as well as spindle assembly checkpoint proteins mad2, cdc20 and survivin. A dose- and time-dependent accumulation of insoluble tubulin in the insoluble fractions of cell lysates provided evidence that G₂ checkpoint failure led to disruption of microtubule turnover. In summary, our results conclude that shogaols cause apoptosis by inducing aberrant mitosis at least through the attenuation of cell cycle and spindle assembly checkpoint proteins.     

The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit

At the end of the cell cycle, cyclin-dependent kinase (CDK) activity is inactivated to allow mitotic exit [1]. A protein phosphatase, Cdc14, plays a key role during mitotic exit in budding yeast by activating the Cdh1 component of the anaphase-promoting complex to degrade cyclin B (Clb) and inducing the CDK inhibitor Sic1 to inactivate Cdk1 [2]. To prevent mitotic exit when the cell cycle is arrested at G2/M, cells must prevent CDK inactivation. In the spindle checkpoint pathway, this is accomplished through Bfa1/Bub2, a heteromeric GTPase-activating protein (GAP) that inhibits Clb degradation by keeping the G protein Tem1 inactive [3-5]. Tem1 is required for Cdc14 activation. Here we show that in budding yeast, BUB2 and BFA1 are also required for the maintenance of G2/M arrest in response to DNA damage and to spindle misorientation. cdc13-1 bub2 and cdc13-1 bfa1 but not cdc13-1 mad2 double mutants rebud and reduplicate their DNA at the restrictive temperature. We also found that the delay in mitotic exit in mutants with misoriented spindles depended on BUB2 and BFA1, but not on MAD2. We propose that Bfa1/Bub2 checkpoint pathway functions as a universal checkpoint in G2/M that prevents CDK inactivation in response to cell-cycle delay in G2/M.     

The spindle checkpoint of Saccharomyces cerevisiae responds to separable microtubule-dependent events

The spindle checkpoint regulates microtubule-based chromosome segregation and helps to maintain genomic stability [1,2]. Mutational inactivation of spindle checkpoint genes has been implicated in the progression of several types of human cancer. Recent evidence from budding yeast suggests that the spindle checkpoint is complex. Order-of-function experiments have defined two separable pathways within the checkpoint. One pathway, defined by MAD2, controls the metaphase-to-anaphase transition and the other, defined by BUB2, controls the exit from mitosis [3-6]. The relationships between the separate branches of the checkpoint, and especially the events that trigger the pathways, have not been defined. We localized a Bub2p-GFP fusion protein to the cytoplasmic side of the spindle pole body and used a kar9 mutant to show that cells with misoriented spindles are arrested in anaphase of mitosis. We used a kar9 bub2 double mutant to show that the arrest is BUB2 dependent. We conclude that the separate pathways of the spindle checkpoint respond to different classes of microtubules. The MAD2 branch of the pathway responds to kinetochore microtubule interactions and the BUB2 branch of the pathway operates within the cytoplasm, responding to spindle misorientation.     

A novel yeast mutant that is defective in regulation of the Anaphase-Promoting Complex by the spindle damage checkpoint

The accurate segregation of sister chromatids at the metaphase to anaphase transition in Saccharomyces cerevisiae is regulated by the activity of the anaphase-promoting complex or cyclosome (APC/C). In the event of spindle damage or monopolar spindle attachment, the spindle checkpoint is activated and inhibits APC/C activity towards the anaphase inhibitor Pds1p, resulting in a cell cycle arrest at metaphase. We have identified a novel allele of a gene for an APC/C subunit, cdc16-183 , in S. cerevisiae. cdc16-183 mutants arrest at metaphase at 37°C, and are supersensitive to the spindle-damaging agent nocodazole, which activates the spindle checkpoint, at lower temperatures. This supersensitivity to nocodazole cannot be explained by impairment of the spindle checkpoint pathway, as cells respond normally to spindle damage with a stable metaphase arrest and high levels of Pds1p. Despite showing metaphase arrest at G2/M at 37°C, cdc16-183 mutants are able to perform tested G1 functions normally at this temperature. This is the first demonstration that a mutation in a core APC/C subunit can result in a MAD2-dependent arrest at the restrictive temperature. Our results suggest that the cdc16-183 mutant may have a novel APC/C defect(s) that mimics or activates the spindle checkpoint pathway.Communicated by C. P. Hollenberg