The core proteins of the spindle assembly checkpoint (SAC), Mads, Bubs, and Mps1, first identified in the budding yeast, are thought to be functionally and structurally conserved through evolution. We found that fission yeast Bub3 is dispensable for SAC, as bub3 null mutants blocked mitotic progression when spindle formation was disrupted. Consistently, the bub3 mutation only weakly affected the stability of minichromosome Ch16 compared with other SAC mutants. Fission yeast Rae1 has sequence homology with Bub3. The bub3 rae1 double mutant and rae1 single mutant did not have defective SAC, suggesting that these genes do not have overlapping roles for SAC. Observations of living cells revealed that the duration of the mitotic prometaphase/metaphase was longer in the bub3 mutant and was Mad2 dependent. Further, the bub3 mutant was defective in sister centromere association during metaphase. Together, these findings suggest that fission yeast Bub3 is required for normal spindle dynamics, but not for SAC.

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THE spindle assembly checkpoint (SAC) ensures the accurate segregation of chromosomes in mitosis by monitoring the presence of kinetochores that are not properly linked to spindle microtubules and blocking mitotic progression until all sister kinetochores are attached to kinetochore microtubules (kMTs) in a bioriented manner. The core SAC proteins, Mad1, Mad2, BubR1 (Mad3 in yeast), Bub1, Bub3, and Mps1 (Mph1 in fission yeast), were originally identified in budding yeast, and are evolutionarily conserved in a wide range of eukaryotes (LEW and BURKE 2003; MAY and HARDWICK 2006; MUSACCHIO and SALMON 2007).

We reported that a fission yeast -tubulin mutant requires these SAC genes for near-normal mitosis, but the requirement differs greatly between individual genes (TANGE and NIWA 2007). By isolating novel mad2 alleles, we demonstrated that the SAC gene functions required for mitosis in the -tubulin mutant are genetically discernible from those required for mitotic arrest when spindle formation is grossly perturbed. Thus, SAC genes may function in different modes in response to various mitotic defects. From this point of view, we examined why a deletion mutation in bub3 did not affect the growth of the -tubulin mutant.

The BUB3 gene was first identified in budding yeast as a high-copy suppressor of a bub1 mutant (HOYT et al. 1991). The Bub3 protein physically interacts with Bub1 in vivo in several organisms (ROBERTS et al. 1994; TAYLOR et al. 1998; VANOOSTHUYSE et al. 2004). This interaction is required for the localization of these proteins to the kinetochores in both perturbed and unperturbed mitoses (TAYLOR et al. 1998; KADURA et al. 2005). Bub3 is a stoichiometric component of the mitotic checkpoint complex (HARDWICK et al. 2000; SUDAKIN et al. 2001; PODDAR et al. 2005). Bub3 is required for SAC function not only in budding yeast, but also in other organisms, including flies and mammals (HOYT et al. 1991; BABU et al. 2003; LOPES et al. 2004). Bub3 is a WD repeat-containing protein whose structural features are conserved in orthologs from diverse species (WILSON et al. 2005). The fission yeast genome contains two protein-coding sequences that have significant homology to the budding yeast BUB3 (WOOD et al. 2002). One is rae1 (SPBC16A3.05c according to the systematic naming of genes) and the other is bub3 (SPAC23H3.08c). Because bub3-deleted cells are hypersensitive to microtubule-destabilizing drugs and also because the bub3 mutant containing a cold-sensitive β-tubulin mutation loses viability at a restrictive temperature, fission yeast bub3⁺ is postulated to be a SAC gene (VANOOSTHUYSE et al. 2004). Consistent with this idea, the fission yeast Bub3 interacts with Bub1 and Mad3, and this interaction is also required for kinetochore localization (MILLBAND and HARDWICK 2002; VANOOSTHUYSE et al. 2004; KADURA et al. 2005). In this study, however, we found that fission yeast Bub3 was dispensable for SAC function.

Strains and general genetic methods:

The strains are listed in supplemental Table 1. Details of the genetic methods were previously described in TANGE and NIWA (2007). The mad3⁺ gene and bub1⁺ gene on Ch16 were disrupted by the G418-resistant gene (kan^r) and the nourseothricin-resistant gene (nat^r), respectively (BAHLER et al. 1998; SATO et al. 2005). Nourseothricin (clonNAT) was purchased from Werner BioAgents (Jena, Germany) and used at 100 µg/ml. For the minichromosome stability assay, log phase culture of cells containing a minichromosome was prepared in a synthetic medium lacking adenine and appropriate dilutions were made before plating on YE plates. The green fluorescent protein (GFP)-tagged rad21⁺ gene was obtained from M. Yanagida via the Yeast Genetic Resource Center. A temperature-sensitive rad21 mutant (rad21-K1) was obtained from H. Ikeda (TATEBAYASHI et al. 1998). The bub3::ura4⁺ mutant used in previous studies (VANOOSTHUYSE et al 2004) was obtained from K. Hardwick (University of Edinburgh) via T. Matsumoto (Kyoto University). We verified that the minichromosome stability and mitotic-arrest activity of this bub3 mutant were identical with that of the bub3 mutant that we created and used in this study (see supplemental Table 2). A rae1 temperature-sensitive mutant strain, h⁺ ade6-216 rae1-167, was obtained from R. Dhar (National Institutes of Health) via T. Tani (Kumamoto University). This mutant formed extremely poor colonies at 30°. ade6⁺ cells had similar temperature sensitivity. Thus, we set 28° as the semipermissive temperature for the minichromosome stability assay and for all other assays.

Chromatin immunoprecipitation:

Chromatin immunoprecipitation (ChIP) was performed essentially as described by SAITOH et al. (1997). Rad21-GFP was immunoprecipitated with an anti-GFP antibody (Living Colors full length A.v. polyclonal antibody; Clontech, Mountain View, CA) and Dynabeads protein G (Invitrogen, Oslo). Coprecipitated DNA fragments were quantified by real-time polymerase chain reaction (PCR) using the Applied Biosystems (Foster City, CA) 7500 Real-Time-PCR system. PCR primers used were for inner centromere sequences (cnt and imr), outer centromere sequences (dg and dh), lys1, and mes1, which were described in YOKOBAYASHI et al. (2003). Where indicated, the cell culture was synchronized by the hydroxyurea treatment-release method, as described in TANGE and NIWA (2007).

Other methods:

Microscopy methods, including staining with 4',6-diamidino-2-phenylindole and live observation of cells, are described in detail in TANGE and NIWA (2007). Cdc2 kinase activity assay is also described in detail in TANGE and NIWA (2007), but the reaction in this study was performed at 25° for 25 min.

Minichromosome stability in SAC-related mutants:

We compared the stability of the minichromosome Ch16 in different SAC mutants. The minichromosome stabilities in different mutants determined by the half-sector method (ALLSHIRE et al. 1995) are shown in Table 1 together with published data. Ch16 stability was the most impaired in mph1 and bub1, followed by mad1, mad2, mad3, and bub3. Because Ch16 contains the mad3⁺ and bub1⁺ genes (CHIKASHIGE et al. 2007), we also tested the stability of Ch16 with both of these genes deleted. This minichromosome was three times more unstable in the bub3 background than in wild-type cells, although it was still more stable than in mad3 cells and in other SAC mutants (Table 1). When we used Ch16 with only bub1⁺ deleted, the minichromosome loss rate in bub3 cells was approximately twice that in wild type (supplemental Table 3). Thus, the presence of one extra copy of the bub1⁺ gene ameliorated the Ch16 instability induced by the the lack of Bub3. The extra copy of mad3⁺ might also contribute to this improvement. Further, disruption of the mad3⁺ and bub1⁺ genes on Ch16 did not affect the minichromosome stability in mad2 cells. It remains to be investigated how Bub1 and Mad3, when produced from extra genes, recover chromosome stability in the absence of the Bub3 protein.

View this table: In this window In a new window   TABLE 1 Effect of SAC mutations on minichromosome stability

 
It was reported that Ch16 with the bub1⁺ gene deletion in bub3 cells was lost at a frequency of 0.2% (VANOOSTHUYSE et al 2004), a value different from that obtained in this study. The discrepancy might be due to differences in experimental procedures; for example, in the previous study, cells from colonies were used while in this study log phase cultures were used. Because we used the same experimental condition for every SAC mutant, it was evident that individual SAC genes have highly differential contributions to chromosome stability. Thus, different (sets of) SAC genes are required to monitor various defects occurring in unperturbed mitoses, or each SAC gene has a function in mitosis in addition to the checkpoint function, or both. It should be noted that the stability was different between mad1 and mad2, particularly between bub1 and bub3. By contrast, in budding yeast, minichromosome stability is almost identical between these respective pairs, consistent with the distinct functional complex formation from the respective pairs of the gene products (WARREN et al. 2002).

We then compared the sensitivity of each SAC mutant to thiabendazole (TBZ), a microtubule-destabilizing agent. The TBZ sensitivity of individual SAC mutants roughly paralleled that of the minichromosome instability in these mutants, with the exception of bub3 (Figure 1). This mutant was more sensitive to TBZ than mad1, mad2, and mad3, and yet it only weakly affected minichromosome stability. There were modest synergetic effects of mad2 and mad3 mutations with bub3 (Figure 1).

View larger version (60K): In this window In a new window Download PPT slide   FIGURE 1.— TBZ-sensitivity test. Log phase cultures of the indicated strains in YE medium were serially diluted (fivefold) and spotted on (a) YEA, (b) YEA + TBZ (7.5 µg/ml), and (c) YEA + TBZ (12.5 µg/ml) plates, aiming to have 10 cells in the most diluted spot. Incubation was at 30° for 3 days. All mutants had the deletion mutation. WT, wild type.

 
We examined why minichromosome stability was not impaired in bub3 as much as in other SAC mutants. In accordance with the hypersensitivity of bub3 to TBZ, Ch16 was 10-fold more unstable in bub3 than in wild-type cells in the presence of a low concentration of TBZ (Table 2). Further, when bub3 was combined with mad2, mad1, or mad3, Ch16 became more unstable than in any of the single mutants (Table 1). In addition, the Mad2 function required for chromosome stability was only weakly supplied by the mad2-56 and mad2-64 mutants in the bub3 mutant (Table 2). These mutants lack the SAC activity to arrest mitotic progression, but remain functional for chromosome stability (TANGE and NIWA 2007). Together, the results suggested that the bub3 mutation induces a certain spindle defect that only slightly decreases chromosome stability if it is appropriately monitored by SAC function.

View this table: In this window In a new window   TABLE 2 Stability of minichromosome Ch16 in bub3 under different conditions

 

Live analysis of spindle dynamics in the bub3 mutant:

To determine if there is a mitotic defect in the bub3 mutant, we performed live analyses of the spindle dynamics and chromosome behavior. Spindle dynamics in wild-type fission yeast are divided into three phases (NABESHIMA et al. 1998; MALLAVARAPU et al. 1999). In phase 1, a bipolar spindle is formed; in phase 2, which corresponds to prometaphase and metaphase, spindle length remains unchanged or slightly elongates; and in phase 3, the spindle elongates. The duration of phases 1 and 2 in wild type was 7.9 ± 1.0 min (n = 31), whereas it was 10.8 ± 1.4 min (n = 28) in the bub3 mutant (Figure 2). This 37% increase in the phase length in the bub3 mutant was almost completely abolished in the bub3 mad2 mutant (8.1 ± 1.3 min; n = 31), indicating that the increase was dependent on Mad2. This finding also suggested that the Mad2-dependent extension of phase 2 allowed for the time necessary for accurate chromosome segregation. In addition, we analyzed the spindle dynamics in a SAC defective mutant, mad3, (Figure 2, bottom right). In this mutant, the duration was 7.6 ± 2.1 min (n = 22). More samples need to be observed before drawing a conclusion, but the duration might be shorter in the mad3 mutant, similar to our previous finding that the duration was shorter in the mad2 mutant (TANGE and NIWA 2007).

View larger version (38K): In this window In a new window Download PPT slide   FIGURE 2.— Live observation of spindle dynamics. Spindle length was determined as the distance between two separating Sid4-GFP signals, taking the difference in focal plane into account. Living cells of indicated strains were observed every 30 sec, and at each time point seven images were taken at serial focal planes at 0.3-µm steps. The time of the onset of phase 3 was set as time 0 and all graphs obtained for each strain are assembled in a single figure. Red triangles and horizontal bars indicate average time of phase 1 onset and standard deviation, respectively.

 
To further investigate bub3 mutant mitosis, we performed a live observation of a GFP-labeled locus 5 kb apart from centromere 2 (cen2-GFP; YAMAMOTO and HIRAOKA 2003). There were two apparently abnormal features in the mutant mitosis (Figure 3A; movies are available as supplemental Figures 1–12). One was that the cen2-GFP pair frequently oscillated within a range of the spindle length during phase 2; that is, the length of the kMTs (d2 and d3 in Figure 3A) frequently changed between elongated and shortened. For a quantitative analysis, we set a 5-min window immediately before the onset of anaphase A and calculated the difference between two successive d2 and d3 values from the window. The rate of elongation/shrinkage was significantly higher in the bub3 mutant (Figure 3B). The frequency of the time intervals with difference values of more than 0.4 µm next to intervals with an opposite elongation/shrinkage phase was 5.8% (37/636) and 13.8% (155/1122) in wild type and mutant, respectively, indicating that swift switching of the elongation/shrinkage phases occurred more frequently in the mutant. The other aberrant feature was that the distances between the two cen2-GFP signals in phase 2 (d4 in Figure 3A) tended to be greater in the mutant (Figure 3C). The average distances in the same windows used above were 0.32 ± 0.14 µm (n = 340) and 0.47 ± 0.21 µm (n = 595), respectively. Perhaps related to this observation, minichromosome Ch10-CN2, a derivative of Ch16, in which both of the arms are deleted (NIWA et al. 1989), was more unstable in bub3 than in mad2 and mad3 (Table 1). These results suggest that the Schizosaccharomyces pombe Bub3 function may be more important for sister centromere association in metaphase, but less important for chromosome arms. It is also possible that sister centromeres are further apart in the mutant because they are pulled apart by a stronger force. Bub3 might directly function in the regulation of the kMT attachment/assembly. Regardless of the primary reason for the apparent defect in centromere association, the establishment and/or maintenance of bipolar spindle formation may become less efficient in the absence of Bub3, which may result in the extended phase 2 duration in a SAC-dependent manner. Consistent with this idea, fission yeast Bub3 is localized on the kinetochores until the onset of anaphase in normal mitosis (VANOOSTHUYSE et al. 2004; KADURA et al. 2005).

View larger version (43K): In this window In a new window Download PPT slide   FIGURE 3.— Live analysis of sister chromatid separation. Fission yeast cells carrying both Sid4-GFP and cen2-GFP were observed every 15 sec with 10 steps (0.2 µm) along the z-axis at each time point. (A) Distances (d1–d4) were measured and plotted. (B) Distributions of the rates of elongation and shrinkage of kMTs in phase 2 (kMT lengths,d2 and d3, changed in the 15-sec intervals) (see text for details). (C) Distributions of the distances between two cen2 signals (d4).

 

Genetic interaction of bub3 with a cohesin mutant:

Because of the possibility that the bub3 mutant had impaired centromeric cohesion, we examined whether the bub3 mutant genetically interacts with a cohesin mutant, rad21-K1 (TATEBAYASHI et al. 1998). The bub3 rad21-K1 double mutant did not make colonies at 33°, a temperature at which the rad21 mutant formed visible colonies (Figure 4). In contrast, the bub1 mutant slightly affected the restrictive temperature, but not as much as the bub3 mutant, the mad2 mutant did not affect at all (Figure 4), indicating that the deleterious effect of the bub3 mutation on the rad21 mutant was not due to a functional defect in SAC.

View larger version (86K): In this window In a new window Download PPT slide   FIGURE 4.— Genetic interaction of rad21-K1 mutation with SAC-related mutations. Strains with the indicated genotypes were inoculated on YES plates and incubated for 4 days at the indicated temperatures.

 
We then investigated whether the amount of chromatin-bound cohesin complex was altered in the bub3 mutant. ChIP was performed using a GFP-tagged Rad21. Consistent with previous results (TOMONAGA et al. 2000; YOKOBAYASHI et al. 2003), Rad21-GFP in wild-type cells was enriched in the outer centromere regions (dg and dh sequences, see supplemental Figure 13). Rad21 enrichment in the dg and dh sequences was also observed in bub3 mutant extracts from a logarithmic phase culture, metaphase-arrested cut9 cells, and synchronized cell culture at G2 or mitotic phases produced by the hydroxyurea treatment method (supplemental Figure 13, and data not shown). Using this method, it was not possible to determine whether the amount of Rad21 protein bound to the centromeric region in the bub3 mutant relative to that bound to the arm regions differed from that in wild type. Further detailed studies are needed to determine whether Bub3 is involved in regulating the proper assembly of cohesin complexes, both temporally and spatially, in the centromeric regions, or if there is only an indirect relation between them.

SAC activity is retained in bub3:

The results described above led us to reinvestigate whether bub3 has defective SAC function. For quantitative evaluation of SAC activity, we used the cut7-24 and nda3-KM311 mutants in which mitotic spindle formation is severely impaired. Under the restrictive condition in these mutants, cells with hypercondensed chromosomes accumulate when SAC function is active, whereas those with the cut phenotype increase when the function is impaired (HE et al. 1997; KIM et al. 1998). Both the wild type and bub3 mutant were active in SAC function, whereas the mad2 and bub1 mutants almost completely lacked the activity (Table 3). To verify this result, we examined Cdc2 kinase activity. Cdc2 kinase activity increased after shifting to restrictive temperatures both in the wild type and bub3 mutants, whereas there was only a small increase in mad2 and bub1 (Figure 5A). These results indicated that the fission yeast bub3⁺ gene is dispensable for SAC function. The fission yeast Bub3 protein is required for the localization of Bub1 and Mad3 to kinetochores in perturbed mitoses (VANOOSTHUYSE et al. 2004; KADURA et al. 2005). Although this localization had been postulated to be required for SAC function, our results indicated that the Bub3-dependent localization of these SAC proteins is not necessary, at least for SAC function.

View this table: In this window In a new window   TABLE 3 Mitotic arrest activity

 

View larger version (38K): In this window In a new window Download PPT slide   FIGURE 5.— (A) The Cdc2 kinase assay. Log phase cultures of cut7 (a) and nda3 (b) were shifted to a restrictive temperature (36° in (a) and 18° in (b) at time 0. Cells were harvested at the indicated time for cell-extract preparation. 32P-Phosphorylated histone H1 is shown in gel images (a and b) and the increase of incorporated 32P from the time 0 sample is shown with arbitrary units (c and d). (B) Mitotic arrest activity. CBZ (50 µg/ml) was added to log phase cultures (in YES medium at 26°) of wild type, bub3, rae1-167, and rae1-167 bub3 at time 0 and shifted to 28°. At the indicated time, cells with hypercondensed chromosomes were scored and plotted. The histogram shows the increase in H1 kinase activity from the CBZ-treated cells. bub1 mutant is included as a control.

 
In fission yeast, the rae1⁺ gene has sequence homology with the bub3⁺ gene (29% identity and 51% similarity in >80% of the whole sequence). The rae1⁺ gene is essential for cell viability and is required for mRNA export (YOON et al. 1997). The gene is not involved in SAC function, on the basis of observations that a temperature-sensitive mutant, rae1-167, is slightly more resistant to a microtubule polymerization inhibitor, the Rae1 protein does not form any complexes with known checkpoint proteins, and the overproduction of Rae1 does not rescue a bub3 mutant phenotype (VANOOSTHUYSE et al. 2004). Both Rae1 and Bub3, however, are involved in the spindle checkpoint in mice (BABU et al. 2003). Therefore, we examined whether fission yeast Rae1 and Bub3 have overlapping roles in SAC. To this end, we investigated the rae1-167 mutant at a semipermissive temperature of 28° (see below and MATERIALS AND METHODS for temperature settings).

We determined the stability of Ch16 in rae1-167 and in rae1-167 bub3 mutants (Table 2). In both mutants, Ch16 was 10 times more unstable than in wild type, indicating that Rae1 activity was reduced at 28°. Why the minichromosome was destabilized in the rae1 mutant is not known, but the destabilization was not due to defective SAC function (see below).

The TBZ sensitivity of the rae1-167 single mutant was almost the same as that of the wild type, and the rae1 bub3 double mutant was only marginally more sensitive than the bub3 single mutant (supplemental Figure 14). Because increased sensitivity to microtubule destabilizing agents is a common property of all known SAC mutants, this finding argues that the rae1 gene is unlikely to be a SAC gene. We then directly analyzed whether the rae1⁺ gene is involved in SAC function. First, cells were treated with carbendazim (CBZ), an inhibitor of microtubule polymerization, and the frequency of cells with hypercondensed chromosomes was determined (SAITOH et al. 2005). Cells with hypercondensed chromosomes accumulated after the addition of CBZ in both rae1 single and rae1 bub3 double mutants. Importantly, the effect of CBZ was more prominent in the rae1 bub3 double mutant (Figure 5B). The Cdc2 kinase activity data were consistent with this result (Figure 5B). Thus, in the rae1 mutant where Rae1 function was sufficiently impaired to appreciably affect minichromosome stability, there was no indication of any SAC defect, even in the absence of Bub3. We therefore conclude that fission yeast bub3 is not defective in SAC function, and this is not due to the presence of Rae1, a protein structurally similar to Bub3. How can our findings be reconciled with previously published results (RAJAGOPALAN et al. 2004; TOURNIER et al. 2004; VANOOSTHUYSE et al. 2004; ASAKAWA et al. 2005, 2006; KADURA et al. 2005), when all the findings are consistent with the idea that fission yeast Bub3 is a SAC protein? One of the most crucial results leading to this conclusion was that the Bub3 activity is required for the viability of mitotically arrested β-tubulin mutant cells (VANOOSTHUYSE et al. 2004). As shown in this study, the fission yeast bub3 mutant has a defect in spindle dynamics. Therefore, it is possible that the bub3 mutation affects the recovery process or spindle reformation after mitotic arrest in the tubulin mutant. It was recently reported that the recovery process is greatly impaired in the absence of Shugoshin 2, which is not a SAC protein (VANOOSTHUYSE et al. 2007). Likewise, all of the previous results might be reevaluated without postulating Bub3 to be a SAC protein. At present, the cellular function of the S. pombe Bub3 is not known, although it is possible that Bub3 has a role in centromeric cohesion, as suggested above. The known interaction of Bub3 with Bub1 and Mad3 and its requirement for their localization to kinetochores might be intimately related to the function of Bub3. Further comparisons of the fission yeast spindle checkpoint system with those in other organisms not only should elucidate the function of Bub3 in fission yeast, but also will contribute to determining the nature of the spindle checkpoint in general.

We thank A. Yamamoto, H. Ikeda, T. Tani, R. Dhar, M. Yanagida, T. Matsumoto, and K. Hardwick for the yeast strains. We are also grateful to an anonymous reviewer who suggested the use of a Ch16 derivative containing the mad3 and bub1 deletions. This work was supported by the Kazusa DNA Research Institute and partly by a grant from Japan Society for the Promotion of Science (no. 16370083) to O.N.

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Communicating editor: P. RUSSELL

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