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Cell Cycle Arrest in cdc20 Mutants of Saccharomyces cerevisiae Is Independent of Ndc10p and Kinetochore Function but Requires a Subset of Spindle Checkpoint Genes
Penny A. Tavorminaa and Daniel J. Burkeaa Department of Biology, University of Virginia, Charlottesville, Virginia 22903
Corresponding author: Daniel J. Burke, Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903, djb6t{at}virginia.edu (E-mail).
Communicating editor: M. JOHNSTON
| ABSTRACT |
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The spindle checkpoint ensures accurate chromosome segregation by inhibiting anaphase onset in response to altered microtubule function and impaired kinetochore function. In this study, we report that the ability of the anti-microtubule drug nocodazole to inhibit cell cycle progression in Saccharomyces cerevisiae depends on the function of the kinetochore protein encoded by NDC10. We examined the role of the spindle checkpoint in the arrest in cdc20 mutants that arrest prior to anaphase with an aberrant spindle. The arrest in cdc20 defective cells is dependent on the BUB2 checkpoint and independent of the BUB1, BUB3, and MAD spindle checkpoint genes. We show that the lesion recognized by Bub2p is not excess microtubules, and the cdc20 arrest is independent of kinetochore function. We show that Cdc20p is not required for cyclin proteolysis at two points in the cell cycle, suggesting that CDC20 is distinct from genes encoding integral proteins of the anaphase promoting complex.
THE accurate transmission of genetic information is governed by feedback mechanisms to ensure the strict order of events during the cell cycle. Checkpoint controls prevent late events in the cell cycle from being initiated until the completion of earlier events. Checkpoint genes are defined empirically by mutations that relieve dependency relationships, thereby uncoupling mitotic processes (![]()
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Temperature-sensitive cdc20 mutants are defective in the microtubule-dependent processes of karyogamy, nuclear transit, and chromosome segregation (![]()
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-tubulin immunofluorescence indicates that the cdc20 arrest, before chromosome segregation, is coincident with a short, intensely stained spindle, suggesting a role for Cdc20p in modulating microtubule function (![]()
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The separation of chromatids and the exit from mitosis requires ubiquitin-mediated proteolysis (![]()
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In this study, we show that arrest in cdc20 mutants is independent of kinetochore function. We show that arrest is under control of the BUB2 gene but independent of the other MAD and BUB checkpoint genes. We report that B-cyclin protein, Clb2p, does not require Cdc20p for degradation at two points in the cell cycle, suggesting that Cdc20p is not required for mitotic cyclin proteolysis. We discuss the possible role of C DC20 in regulating the metaphase to anaphase transition in the context of these and other recent results.
| MATERIALS AND METHODS |
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Strains and media:
The yeast strains and plasmids used in this study are listed in Table 1 and Table 2, respectively. Strains were constructed by standard genetic techniques using rich (Y M-1 and Y EPD) and synthetic media (SC) (![]()
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Strain construction:
Strain 1906 (cdc20-1 mad1::HIS3) was constructed by transforming strain 1739-9-2 (cdc20 his3) with EcoRV and Sac I digested plasmid pKH149 (![]()
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GFP sister chromatid separation assay:
Cells that were grown to mid-logarithmic phase in Y M-1 were resuspended in SC-HIS medium supplemented with 10 mM 3-aminotriazole for 30 min to induce the HIS3 promoter. The cells were washed by centrifugation and resuspended in Y M-1 containing 15 µg of nocodazole per ml nocodazole and incubated at the restrictive temperature of 37°. Samples were removed every 30 min and fixed in 3.7% formaldehyde for 15 min at room temperature. The cells were washed in 65 mM NaPO4, 5 mM MgCl2, mounted on slides, and GFP staining was visualized using standard FITC filters.
Indirect immunofluorescence and photomicroscopy:
Antitubulin immunofluorescence and DNA staining with DAPI were performed essentially as described previously with the exception of a shorter fixation time (![]()
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Histone H1 kinase activity:
Histone H1 kinase activity was measured in crude protein extracts as the ability to phosphorylate histone H1 (Boehringer Mannheim Corp., Indianapolis, IN) as described by ![]()
Clb2p stability:
We enriched for unbudded cells by growth to stationary phase in SC-URA containing raffinose for 2 days. We diluted the cells into SC-URA medium containing raffinose plus
factor and incubated the cells until 90% of the cells displayed the morphology of pheromone-arrested cells. bar1 cells were arrested in
factor (Sigma Chemical Co.) at a concentration of 0.5 µg/ml from a stock solution of 1 mg
factor per ml in PBS. The GAL10 promoter was induced by adding galactose (2%) to cells pregrown in 2% raffinose. Galactose was added to the medium, and cells were incubated at 36°, the restrictive temperature for cdc20 mutants, for 2 hr to induce Clb2p synthesis. To examine Clb2p in cdc20 cells at G2/M, cells from strain 1904 were grown to midlogarithmic phase in SC-URA supplemented with 2% raffinose. The culture was divided in half, and galactose (2%) was added to one half of the culture, and additional raffinose (2%) added to the uninduced culture. The cells were incubated for 2 hr at the restrictive temperature.
Cell extracts and immunoblotting:
Crude cell extracts were prepared as described by ![]()
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| RESULTS |
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NDC10 is required for spindle checkpoint function:
Four subunits define the essential yeast centromere binding factor, CBF3 (![]()
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We determined the ability of a microtubule inhibitor to activate the spindle checkpoint in the absence of NDC10 function by analyzing DNA content in ndc10 cells treated with nocodazole. Cells from strain JK418 (ndc10-1) and strain W303 (WT) were grown to midlogarithmic phase and shifted to the restrictive temperature in the presence of nocodazole. We confirmed that the nocodazole treatment was effective in eliminating microtubules by analyzing samples of cells at each time point by antitubulin immunofluorescence (data not shown). In each case, greater than 90% of the cells showed tubulin staining only as punctate foci at the spindle pole bodies, indicative of completely effective nocodazole treatment ( ![]()
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To confirm that nocodazole was unable to prevent anaphase onset in ndc10 cells, we examined the cohesion of sister chromatids in nocodazole-treated ndc10 cells. Sister chromatid separation does not require microtubules, and cells defective for the spindle checkpoint separate sister chromatids in the presence of nocodazole (![]()
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cdc20 is epistatic to ndc10 :
We used the ndc10-1 mutation to eliminate the kinetochore-dependent checkpoint-signaling pathway to ask whether the arrest in cdc20 defective cells was dependent on kinetochore function. We constructed double mutants between cdc20 and two different alleles of ndc10 to ask if cells arrest in the absence of Ndc10p function. We tested the two different alleles of ndc10 that vary in the degree of asymmetric division observed. ndc10-169 mutants display less asymmetry in DNA distribution than ndc10-1 mutants (Y. WANG and D. BURKE, unpublished observations; ![]()
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cdc20 arrest requires BUB2 but not other spindle checkpoint genes:
The cell cycle arrest in cdc20 mutants may require spindle checkpoint genes and still be independent of kinetochore function. This seems likely given that spindle assembly is altered in cdc20 mutants that are arrested at the restrictive temperature (![]()
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Cells defective in the spindle checkpoint initiate new rounds of budding in the presence of microtubule depolymerizing drugs (![]()
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We did not observe the continued budding in cdc20 bub2 mutants until several hours after shift to the restrictive temperature. Therefore, we examined the kinetics of cell cycle arrest in cells from strain 1908 (cdc20 bub2) and strain 405-1 (cdc20). We analyzed spindle morphology over time in cells released from
-factor synchronization and grown at the restrictive temperature. Samples were harvested each hour and fixed for antitubulin immunofluorescence. We determined the percentage of cells containing short spindles at medial nuclear division (Figure 6A). By 2 hr, 75% of the cells from both strains had short spindles. Wild-type cells normally complete two cell divisions at this temperature (36°), suggesting that cdc20 bub2 mutants are competent to arrest for some time. However, over extended periods of time, the percentage of short spindles declined in strain 1908 (cdc20 bub2). In contrast, cells from strain 405-1 (cdc20) displayed a high percentage of short spindles throughout the extended incubations. These results suggest that cdc20 bub2 mutants were competent to establish a mitotic arrest and delay anaphase transiently but were unable to maintain the mitotic arrest.
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We measured the viability over extended incubations to determine if a loss of viability correlates with the cell cycle delay. Cells from strain 1908 (cdc20 bub2) and strain 1907 (cdc20) were grown to midlogarithmic phase, diluted, and spread onto Y EPD plates. The plates were incubated at 36°, and at 1 hr intervals plates were moved to the permissive temperature to determine the number of viable colonies. Figure 6B shows that cdc20 bub2 cells maintain high viability for 2 hr and then decrease in viability as compared to cdc20 cells. These data suggest that the inviability is associated with failure to arrest in the cell cycle. We conclude that the ability to maintain a cell cycle arrest in cdc20 cells is dependent on BUB2 and independent of other spindle checkpoint genes.
BUB2 does not arrest cells in response to microtubule overassembly:
The unique requirement for BUB2 in executing a cdc20 arrest prompted us to investigate whether the excess numbers of microtubules in the spindle of cdc20 cells constitutes a distinct perturbation that induces a BUB2-dependent arrest in the cell cycle. There are cold-sensitive lethal mutations in the TUB1
-tubulin gene that cause cells to arrest before anaphase with excess microtubules (![]()
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APC function is not eliminated in cdc20 mutants:
Our data show that the BUB2 -dependent arrest in cdc20 mutants is independent of most spindle assembly checkpoint genes and that BUB2 does not respond to overassembled microtubules. This suggests that the BUB2-dependent arrest in cdc20 is due to some other function that is lacking in the mutant. A role for CDC20 in ubiquitin-mediated proteolysis of cyclins has been proposed based primarily on the implication of the Drosophila homologue of CDC20, fizzy, in cyclin proteolysis (![]()
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-factor arrested cells because of APC activity (![]()
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factor. We also measured Clb2p stability in cells arrested before anaphase at the CDC20 -dependent step. Clb2p was measured in total cell protein extracts by immunoblotting with an anti-HA monoclonal antibody. Clb2p does not accumulate in wild-type cells at the
-factor step but accumulates in wild-type cells arrested prior to anaphase with nocodazole (![]()
factor and incubated at the restrictive temperature (Figure 7). Clb2p is undetectable in wild-type cells arrested in
factor (data not shown; ![]()
factor step. We attribute this low level of Clb2p to cells (19%) that escaped the
factor block and continued on to arrest as large budded cells at the cdc20 step, a point in the cell cycle where APC activity is inhibited. We conclude that CDC20 function is not required to degrade Clb2p in cells arrested with
factor.
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Although the bulk of Clb2p proteolysis occurs at the metaphase to anaphase transition, a fraction remains protected from proteolysis until the completion of Cdc15p-dependent processes in late anaphase (![]()
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factor or cells that are exiting mitosis.
| DISCUSSION |
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NDC10 and the spindle checkpoint:
The spindle checkpoint prevents the onset of anaphase in yeast cells that have been treated with antimicrotubule drugs such as nocodazole and benomyl. The effects of these benzimidazole drugs are complex. Microtubules are disassembled and the intracellular levels of tubulin dimers increase. Chromosomes become disassociated from the spindle and the spindle pole bodies collapse within the nucleus ( ![]()
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The requirement for NDC10 in the spindle checkpoint can explain the unusual phenotype of ndc10 mutants. In the absence of Ndc10p function, the spindle elongates although the DNA is not attached and the result is that the chromosomes are asymmetrically distributed to the daughter cells. DNA replication continues in some of the cells and polyploid progeny are produced (![]()
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It is possible that the different phenotypes displayed by mutants affecting CBF3 proteins are indicative of the strength of the mutations. Perhaps all of the available temperature-sensitive ctf13, skp1, and cep3 mutants retain some small amount of function in vivo. It is believed that a single unattached chromosome is capable of generating an inhibitory checkpoint signal (![]()
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CDC20 and spindle function:
In yeast, perturbing the interaction of kinetochores with microtubules or destroying spindle structure with antimicrotubule drugs triggers the MAD - and BUB -dependent spindle checkpoints (![]()
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Relationship between CDC20 and the APC:
Homologues of CDC20 have been identified in Drosophila melanogaster (fizzy), Schizosaccharomyces pombe (slp1+), and humans (p55CDC), and all appear to be required for chromosome segregation (![]()
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We found that Clb2p does not accumulate in cdc20 mutants arrested by
factor. Clbp destruction is active from anaphase onset until late G1 when Clnp accumulation inhibits the proteolysis of Clbp (![]()
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-factor arrested cells. This is consistent with the observation of ![]()
-factor block. Furthermore, we extended this observation to show that Cdc20p is not involved in the late anaphase-specific function of the APC. We found that increased levels of Clb2p did not arrest cdc20 mutants (under semipermissive conditions) in anaphase, indicating that limiting CDC20 function has different consequences than either cdc16 or cdc23, which comprise APC function. These data suggest that CDC20 is distinct from CDC16 and CDC23 and is not an integral component of the APC.
One way to reconcile the differences between our data and the observations on Drosophila fizzy is that a metaphase-anaphase-specific function of the APC requires CDC20. There is recent evidence to suggest that CDC20 regulates the metaphase to anaphase transition. High copy expression of CDC20 suppresses the temperature sensitivity of cdc28-1N, suggesting a link between CDC20 and the cell cycle machinery (![]()
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CDC20 may also be able to affect cell cycle progression in cells arrested by the spindle checkpoint. A dominant allele of CDC20 (PAC5-1/CDC20-50) was recently identified in a screen for mutants that die in the absence of the spindle motor protein encoded by CIN8 (![]()
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By the genetic definition of checkpoint control (![]()
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If CDC20 encodes a modulator of APC activity, our results raise intriguing possibilities for the role of BUB2 in checkpoint maintenance. Sequence analysis has recently revealed that Bub2p is a member of a superfamily of proteins including the tre-2 oncogene and two yeast genes encoding GTPase-activating proteins (![]()
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| ACKNOWLEDGMENTS |
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We thank M. A. HOYT, K. G. HARDWICK, R.-H. CHEN, R. LI, A. W. MURRAY, A. F. STRAIGHT, A. B. FUTCHER, J. KILMARTIN, F. SOLOMON and M. WINEY for providing strains, plasmids and/or antibodies. We also thank MARK WINEY and TOM GIDDINGS, JR., ERIC SCHOTT and ANDY HOYT and ANGELIKA AMON for personal communication of results. We thank ANDY HOYT, ERIC SCHOTT, ORNA COHEN-FIX, MITCH SMITH and our colleauges, past and present, in the Burke laboratory for helpful discussions. This work was supported by a fellowship from the Association for Research College Scientists, Inc. (to P.A.T) and Public Health Service grant GM-40334 from the National Institutes of Health (to D.J.B.).
Manuscript received September 3, 1997; Accepted for publication December 16, 1997.
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) and 1746-33A (ndc10-1 G FP-lacI::HIS3 lacO::L EU2) (
) were treated with 15 µg nocodazole per ml at the restrictive temperature. Two GFP-staining foci in the unseparated nuclei of large-budded cells were scored as having undergone sister chromatid separation. Time points were taken every 30 min for 4 hr.


3) after shifting to the restrictive temperature. (B) Budding morphology in cells from strains 1908 (cdc20 bub2) and 405-1 (cdc20) grown for 4 hr at the restrictive temperature. Bar, 5 µm.
, cdc20 bub2::URA3 and
, cdc20.





