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.     

Two Related Kinesins, klp5+ and klp6+, Foster Microtubule Disassembly and Are Required for Meiosis in Fission Yeast

The kinesin superfamily of microtubule motor proteins is important in many cellular processes, including mitosis and meiosis, vesicle transport, and the establishment and maintenance of cell polarity. We have characterized two related kinesins in fission yeast, klp5+ and klp6+,, that are amino-terminal motors of the KIP3 subfamily. Analysis of null mutants demonstrates that neither klp5+ nor klp6+, individually or together, is essential for vegetative growth, although these mutants have altered microtubule behavior. klp5Delta and klp6Delta are resistant to high concentrations of the microtubule poison thiabendazole and have abnormally long cytoplasmic microtubules that can curl around the ends of the cell. This phenotype is greatly enhanced in the cell cycle mutant cdc25-22, leading to a bent, asymmetric cell morphology as cells elongate during cell cycle arrest. Klp5p-GFP and Klp6p-GFP both localize to cytoplasmic microtubules throughout the cell cycle and to spindles in mitosis, but their localizations are not interdependent. During the meiotic phase of the life cycle, both of these kinesins are essential. Spore viability is low in homozygous crosses of either null mutant. Heterozygous crosses of klp5Delta with klp6Delta have an intermediate viability, suggesting cooperation between these proteins in meiosis.     

Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s)

We report the isolation and characterization of pds1 mutants in Saccharomyces cerevisiae. The initial pds1-1 allele was identified by its inviability after transient exposure to microtubule inhibitors and its precocious dissociation of sister chromatids in the presence of these microtubule inhibitors. These findings suggest that pds1 mutants might be defective in anaphase arrest that normally is imposed by a spindle-damage checkpoint. To further examine a role for Pds1p in anaphase arrest, we compared the cell cycle arrest of pds1 mutants and PDS1 cells after: (a) the inactivation of Cdc16p or Cdc23p, two proteins that are required for the degradation of mitotic cyclins and are putative components of the yeast anaphase promoting complex (APC); (b) the inactivation of Cdc20p, another protein implicated in the degradation of mitotic cyclins; and (c) the inactivation of Cdc13 protein or gamma irradiation, two circumstances that induce a DNA- damage checkpoint. Under all these conditions, anaphase is inhibited in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor that plays a critical role in the control of anaphase by both APC and checkpoints. We also show that pds1 mutants exit mitosis and initiate new rounds of cell division after gamma irradiation and Cdc13p inactivation but no after nocodazole-treatment or inactivation of Cdc16p, Cdc20p or Cdc23p function. Therefore, in the DNA-damage checkpoint, Pds1p is required for the inhibition of cytokinesis and DNA replication as well as anaphase. The role of Pds1 protein in anaphase inhibition and general cell cycle regulation is discussed.     

Isolation and partial characterization of conditional cell division cycle mutants inChlamydomonas

Summary We have isolated a number of temperature conditional cell division cycle mutants of the unicellular plantChlamydomonas reinhardtii that are defective in single nuclear genes. Cells grow and divide normally at the permissive temperature (21 °C), but arrest in division at the restrictive temperature (33 °C). We have characterized these mutants using DNA probes and immunofluorescence techniques to localize cytoskeletal and microtubule organizing centre proteins. We describe here 3 broad classes of cell cycle mutation which result in cell cycle arrest with: unreplicated DNA (G1 arrest), duplicated DNA (G2 arrest) and multiple nuclei due to defective cytokinesis (cytokinesis arrest). The continuation of nuclear division in mutants blocked in cytokinesis provides support of an earlier hypothesis that stage specific events in theChlamydomonas cell cycle are arranged in separate dependent sequences. The mutants isolated in the present study provide insights into the role of cytoskeletal proteins in the coordination of plant cell division and the means to investigate the molecular mechanisms whereby division by multiple fission is controlled in the unicellular plantChlamydomonas.Abbreviations BB basal bodies - EMS ethylmethane sulphonate - MT microtubule - MTOC Microtubule organizing centre - NBBC nucleus-basal body connector - PAR photosynthetically active radiation     

Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants

One hundred and forty-eight temperature-sensitive cell division cycle (cdc) mutants of Saccharomyces cerevisiae have been isolated and characterized. Complementation studies ordered these recessive mutations into 32 groups and tetrad analysis revealed that each of these groups defines a single nuclear gene. Fourteen of these genes have been located on the yeast genetic map. Functionally related cistrons are not tightly clustered.

Mutations in different cistrons frequently produce different cellular and nuclear morphologies in the mutant cells following incubation at the restrictive temperature, but all the mutations in the same cistron produce essentially the same morphology. The products of these genes appear, therefore, each to function individually in a discrete step of the cell cycle and they define collectively a large number of different steps.

The mutants were examined by time-lapse photomicroscopy to determine the number of cell cycles completed at the restrictive temperature before arrest. For most mutants, cells early in the cell cycle at the time of the temperature shift (before the execution point) arrest in the first cell cycle while those later in the cycle (after the execution point) arrest in the second cell cycle. Execution points for allelic mutations that exhibit first or second cycle arrest are rather similar and appear to be cistron-specific. Other mutants traverse several cycles before arrest, and its suggested that the latter type of response may reveal gene products that are temperature-sensitive for synthesis, whereas the former may be temperature-sensitive for function.

The gene products that are defined by the cdc cistrons are essential for the completion of the cell cycle in haploids of a and α mating type and in a/α diploid cells. The same genes, therefore, control the cell cycle in each of these stages of the life cycle.

     

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.     

Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells

Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.     

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.     

Commitment to the mitotic cell cycle in yeast in relation to meiosis

Under restrictive vegetative conditions, cells of cell-division cycle (cdc) temperature-sensitive mutants arrest at specific points in the cycle. Meiotic and mitotic behaviour of such arrested cells was examined under permissive sporulation conditions. Those mutants which were committed to mitosis at their specific point of arrest finished the cell cycle and could only then go into meiosis. It was found that commitment to mitosis occurred early in the cell cycle, prior to DNA replication, and that this commitment was dependent upon the gene function of cdc4.     

An essential role of the yeast pheromone-induced Ca2+ signal is to activate calcineurin.

Previous studies showed that, in wild-type (MATa) cells, alpha-factor causes an essential rise in cytosolic Ca2+. We show that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is one target of this Ca2+ signal. Calcineurin mutants lose viability when incubated with mating pheromone, and overproduction of constitutively active (Ca(2+)-independent) calcineurin improves the viability of wild-type cells exposed to pheromone in Ca(2+)-deficient medium. Thus, one essential consequence of the pheromone-induced rise in cytosolic Ca2+ is activation of calcineurin. Although calcineurin inhibits intracellular Ca2+ sequestration in yeast cells, neither increased extracellular Ca2+ nor defects in vacuolar Ca2+ transport bypasses the requirement for calcineurin during the pheromone response. These observations suggest that the essential function of calcineurin in the pheromone response may be distinct from its modulation of intracellular Ca2+ levels. Mutants that do not undergo pheromone-induced cell cycle arrest (fus3, far1) show decreased dependence on calcineurin during treatment with pheromone. Thus, calcineurin is essential in yeast cells during prolonged exposure to pheromone and especially under conditions of pheromone-induced growth arrest. Ultrastructural examination of pheromone-treated cells indicates that vacuolar morphology is abnormal in calcineurin-deficient cells, suggesting that calcineurin may be required for maintenance of proper vacuolar structure or function during the pheromone response.     

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     

Wip1 contributes to cell homeostasis maintained by the steady-state level of Wtp53

Wip1, a human protein Ser/Thr phosphatase also called PPM1D, stands for wild-type p53 induced phosphatase 1. Emerging evidences indicate that Wip1 can act as an oncogene largely by turning off DNA damage checkpoint responses. Here we report an unrecognized role of Wipl in normally growing cells. Wip1 can be induced by wild-type p53 under not only stressed but also non-stressed conditions. It can trigger G₂/M arrest in wild-type p53 containing cells, which was attributed to the decreased Cdc2 kinase activity resulting at least partly from a high level of inhibitory tyrosine phosphorylation on Cdc2 protein at Tyr-15. Furthermore, we also found that Wip1 not only causes G₂/M arrest but also decreases cell death triggered by microtubule assembly inhibitor in mouse fibroblasts when wild-type p53 function was restored. These results indicate that Wip1 can provide ample time for wild-type p53-containing cells to prepare entry into mitosis and avoid encountering mitotic catastrophe. Therefore, Wipl may play important roles in cell/tissue homeostasis maintained by wild-type p53 under normal conditions, enhancing our understanding of how p53 makes cell-fate decisions.Key words: p53, Wip1, cell homeostasis, cell arrest, cell death     

Inactivation of p21 by E1A leads to the induction of apoptosis in DNA-damaged cells

A major impediment to successful chemotherapy is the propensity for some tumor cells to undergo cell cycle arrest rather than apoptosis. It is well established, however, that the adenovirus E1A protein can sensitize these cells to the induction of apoptosis by anticancer agents. To further understand how E1A enhances chemosensitivity, we have made use of a human colon carcinoma cell line (HCT116) which typically undergoes cell cycle arrest in response to chemotherapeutic drugs. As seen by the analysis of E1A mutants, we show here that E1A can induce apoptosis in these cells by neutralizing the activities of the cyclin-dependent kinase inhibitor p21. E1A's ability to interact with p21 and thereby restore Cdk2 activity in DNA-damaged cells correlates with the reversal of G(1) arrest, which in turn leads to apoptosis. Analysis of E1A mutants failing to bind p300 (also called CBP) or Rb shows that they are almost identical to wild-type E1A in their ability to initially overcome a G(1) arrest in cells after DNA damage, while an E1A mutant failing to bind p21 is not. However, over time, this mutant, which can still target Rb, is far more efficient in accumulating cells with a DNA content greater than 4N but is similar to wild-type E1A and the other E1A mutants in releasing cells from a p53-mediated G(2) block following chemotherapeutic treatment. Thus, we suggest that although E1A requires the binding of p21 to create an optimum environment for apoptosis to occur in DNA-damaged cells, E1A's involvement in other pathways may be contributing to this process as well. A model is proposed to explain the implications of these findings.     

A beta-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance.

Analysis of beta-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged beta-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin beta-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.     

Action of rotenone and related respiratory inhibitors on mammalian cell division. 1 Cell kinetics and biochemical aspects.

Inhibitors of mitochondrial respiration, phosphorylation inhibitors, and uncoupling agents have been reported to delay or inhibit mitosis in cultured mammalian cells. Although the molecular mechanism by which mitosis is delayed in the presence of most respiratory inhibitors presumably involves lowered ATP production for mitotic requirements, one respiratory inhibitor, rotenone, was determined to arrest mitosis by an unrelated mechanism. Cell cycle kinetics studies, oxygen consumption measurements, and viscosity assays indicate that rotenone arrests cultured mammalian cells in mitosis by inhibiting spindle microtubule assembly by a mechanism analogous with colchicine, Colecemid and related antimitotic drugs. Amytal, which blocks electron transport at the same site as does rotenone, failed to arrest cell progression at mitosis. Rotenone delayed cell progression in all phases of the cell cycle, apparently as a direct result of respiration inhibition. Thus, rotenone appears to exert a dual function on events of the cell cycle.     

The CDC20 gene product of Saccharomyces cerevisiae, a beta-transducin homolog, is required for a subset of microtubule-dependent cellular processes.

Previous analysis of cdc20 mutants of the yeast Saccharomyces cerevisiae suggests that the CDC20 gene product (Cdc20p) is required for two microtubule-dependent processes, nuclear movements prior to anaphase and chromosome separation. Here we report that cdc20 mutants are defective for a third microtubule-mediated event, nuclear fusion during mating of G1 cells, but appear normal for a fourth microtubule-dependent process, nuclear migration after DNA replication. Therefore, Cdc20p is required for a subset of microtubule-dependent processes and functions at multiple stages in the life cycle. Consistent with this interpretation, we find that cdc20 cells arrested by alpha-factor or at the restrictive temperature accumulate anomalous microtubule structures, as detected by indirect immunofluorescence. The anomalous microtubule staining patterns are due to cdc20 because intragenic revertants that revert the temperature sensitivity have normal microtubule morphologies. cdc20 mutants have a sevenfold increase in the intensity of antitubulin fluorescence in intranuclear spindles compared with spindles from wild-type cells, yet the total amount of tubulin is indistinguishable by Western immunoblot analysis. This result suggests that Cdc20p modulates microtubule structure in wild-type cells either by promoting microtubule disassembly or by altering the surface of the microtubules. Finally, we cloned and sequenced CDC20 and show that it encodes a member of a family of proteins that share homology to the beta subunit of transducin.