Bifurcation of the mitotic checkpoint pathway in budding yeast - PubMed (original) (raw)

Bifurcation of the mitotic checkpoint pathway in budding yeast

R Li. Proc Natl Acad Sci U S A. 1999.

Abstract

The coordination of mitotic events is ensured through the spindle assembly checkpoint. BFA1 is required for this checkpoint in budding yeast because its disruption abolishes the mitotic arrest when spindle assembly is inhibited. Analysis of the genetic interaction of BFA1 with known mitotic checkpoint genes suggest that Bfa1 functions in the same pathway with Bub2 but not with Mad1 or Mad2. Both Bfa1 and Bub2 localize to spindle poles, and overexpression of Bfa1 arrests the cell cycle in anaphase. These findings suggest a bifurcation of the spindle assembly checkpoint: whereas one branch of the pathway, consisting of Mad1-3, Bub1 and 3, and Mps1, may prevent premature disjunction of sister chromosomes, the other, consisting of Bfa1 and Bub2, may function at spindle poles to prevent cytokinesis before the completion of chromosome segregation.

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Figures

Figure 1

Figure 1

Bfa1 is a mitotic checkpoint protein acting in the same pathway as Bub2. (A) Cultures of RLY576 (WT, wild type), RLY683 (Δbfa1), RLY720 (Δmad1), RLY718 (Δmad2), and RLY716 (Δbub2) were grown to same density. Ten-fold serial dilutions were made and spotted onto YPD plates without (− ben) or with (+ ben) 10 μg/ml benomyl. Photographs were taken after 3 (− ben) and 5 (+ ben) days of growth at room temperature. (B) RLY576 and RLY683 cells were grown to 2 × 106 per ml and arrested with 5 μg/ml α-mating factor for 2.5 hours at room temperature. The cells were washed four times with water and resuspended in YPD containing 15 μg/ml nocodazol. After 5 hour growth at room temperature, cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclear DNA. (C) The same fixed RLY683 cells were treated with 0.2 mg/ml zymolyase to remove the cell wall and double stained with DAPI and an anti-tubulin antibody as previously described (37). (D) Exponential cultures of RLY576, RLY683, and RLY716 cells were shifted to media with or without 15 μg/ml nocodazol (Noc) for 5 hours at room temperature. Total cell extracts were prepared and analyzed by immunoblotting using anti-Clb2 (38) and anti-actin (25) (as a loading control) antibodies. (E) RLY576, RLY683, RLY716, RLY718, RLY720, RLY684 (Δbfa1Δbub2), RLY686 (Δbfa1Δmad2), RLY736 (Δbub2Δmad2), and RLY737 (Δmad1Δmad2) cells were arrested in G1 with α-factor as described in B and released into media with or without 15 μg/ml nocodazol. For all strains used, >95% were arrested as unbudded cells after the α-factor treatment, and the rates at which budding occurred were identical after the release into media with or without nocodazol (budded cells always started to appear around 40 minutes after release and reached 85–90% at 60 min). Cells were fixed every 30 minutes and spotted on a microscope slide. The percentage of cells that had initiated a second round of budding was determined and plotted over time after release from the G1 arrest. The nuclear morphology was also examined for both wild-type and mutant cells obtained from the time points when the second round of budding had occurred. Most (>89%) of the mutant cells that had budded a second time in the presence of nocodazol had the morphology as shown in B (data not shown). In the absence of nocodazol, the newly budded mother cells stayed together with their former bud after cells were spotted into the glass slide. This was observed for both wild-type and various mutant strains of the W303 background, indicating a delay in cell separation or cell stickiness for this strain background. However, the nuclear morphology, as shown in B, was never observed in the absence of nocodazol (i.e., all cells with two buds had at least two distinct DAPI stained masses, data not shown). [Bar = 10 μm (C).]

Figure 2

Figure 2

(A) The mitotic delay induced by Mps1 overexpression is independent of Bfa1 and Bub2. RLY700 (WT), RLY724 (Δbfa1), RLY727 (Δbub2), RLY729 (Δmad1), and RLY731 (Δmad2) strains, which all contain the same integrated copy of Gal-Mps1 (14), were cultured overnight in media without galactose (YPR) and then shifted to media containing 2% galactose (YPG) for 6 hours. A control stain, RLY569, which contains the vector alone, was also included. (a) Cells were fixed, stained with DAPI, and the percentage of large budded cells (bud size greater than 2/3 of the mother size) with undivided DNA mass were determined and shown as histograms. (b) Total cell extracts were prepared and analyzed by immunoblotting using anti-Clb2 and anti-actin antibodies. Lane 1, RLY569; lane 2, RLY700; lane 3, RLY724; lane 4, RLY727; lane 5, RLY729; lane 6, RLY731. (B) Overexpression of Bfa1 induces an anaphase cell cycle arrest that is independent of the other mitotic checkpoint proteins. (a) Strains bearing vector alone (RLY569) or Gal-Bfa1 were streaked onto a plate containing 2% galactose. The photograph was taken after 3 days at 30°C. Sector 1, RLY569; sector 2, RLY690 (WT); sector 3, RLY688 (Δbub2); sector 4, RLY691 (Δmad1); sector 5, RLY693 (Δmad2). (b) The strains in B, a were cultured overnight in media without galactose (YPR) and then shifted to media containing 2% galactose (YPG) for 6 hours. Cells were fixed and stained with DAPI and an anti-tubulin (MT) antibody. The images show the arrest morphology of RLY690 cells. The same arrest morphology was also observed for the other Gal-Bfa1 bearing strains. (c) The percentages of cells exhibiting the arrest morphology, as shown in b, were determined for each of the strains in the experiment described in B, b. (d) Total extracts were prepared from each of the cell cultures in the experiment described in B, b and analyzed by immunoblotting by using anti-Clb2 and anti-actin antibodies. [Bar = 10 μm(B, b).]

Figure 3

Figure 3

Localization of Bfa1 and Bub2 to the spindle pole bodies. Strains expressing Bfa1-GFP (RLY603) or Bub2-GFP (RLY735) at the endogenous level were constructed as described in Materials and Methods. (A) Live cells observed directly under a Nikon Eclipse E600 fluorescence microscopy equipped with an 100/1.40 oil DIC objective and a EXHQ450/50 DM480 LP/BA465LP GFP filter set. (B) Double immunofluroescence staining of fixed RLY603 cells by using a rat anti-tubulin antibody (37) and a rabbit anti-GFP antibody (a gift from P. Silver, Dana Farber Cancer Institute, Boston, MA). (Bar = 10 μm.)

Figure 4

Figure 4

A schematic diagram depicting a bifurcation of the mitotic checkpoint pathway. This model hypothesizes that defects in spindle assembly can potentially prevent two mitotic events: chromosome attachment to the spindle (a); and anaphase spindle elongation (b). A defect in a may be monitored at the kinetochores and activates a set of mitotic checkpoint proteins that prevent sister chromatid separation by blocking degradation of chromosome cohesion factors such as Pds1. A defect in b may be monitored at the spindle pole bodies through Bub2 and Bfa1, which prevent cytokinesis by blocking the degradation of mitotic cyclins such as Clb2.

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