Schizosaccharomyces pombe Bub3 is dispensable for mitotic arrest following perturbed spindle formation - PubMed (original) (raw)

Schizosaccharomyces pombe Bub3 is dispensable for mitotic arrest following perturbed spindle formation

Yoshie Tange et al. Genetics. 2008 Jun.

Abstract

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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Figures

F<sc>igure</sc> 1.—

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.

F<sc>igure</sc> 2.—

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.

F<sc>igure</sc> 3.—

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).

F<sc>igure</sc> 4.—

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.

F<sc>igure</sc> 5.—

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.

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