The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint - PubMed (original) (raw)

The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint

S Biggins et al. Genes Dev. 2001.

Abstract

The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects. The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and we propose that Ipl1p is required to monitor tension at the kinetochore.

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Figures

Figure 1

Figure 1

ipl1 mutants do not activate the spindle checkpoint despite a chromosome segregation defect. Wild-type (SBY818) and ipl1-321 cells (SBY819) containing Pds1-myc18 were arrested in G1 with α-factor at the permissive temperature (23°C) and released to the non-permissive temperature (37°C). α-factor was added back when small buds formed to prevent cells from entering the next cell cycle. (A) Lysates were prepared at the indicated time points and immunoblotted with anti-myc antibodies to analyze Pds1-myc protein levels. Pds1p levels cycle in both wild-type cells and ipl1 mutant cells, indicating that the spindle checkpoint is not activated. Equal protein concentrations were loaded in all lanes as judged by Ponceau S staining (data not shown). (B) The percentage of budded cells in the same experiment was quantified by microscopy and shows that wild-type (filled squares) and ipl1-321 cells (open squares) undergo budding and then cytokinesis with similar kinetics. (C) In the same experiment, the percent chromosome IV segregation was monitored as the fraction of cells that had segregated two GFP-marked copies of chromosome IV to opposite poles of the spindle. Although chromosome IV segregates in wild-type cells (filled squares), there is a severe chromosome segregation defect in the ipl1-321 cells (open squares).

Figure 2

Figure 2

Ipl1p is required to maintain the GAL-MPS1 checkpoint arrest. GAL-MPS1 (SBY679) and GAL-MPS1 ipl1-321 (SBY680) cells were arrested in galactose for 3.5 h and then released to the nonpermissive temperature (35°C) to inactivate ipl1. (A) Pds1-myc protein levels were monitored by immunoblotting with anti-myc antibodies. Pds1 levels decline faster in the GAL-MPS1 ipl1-321 cells compared with the GAL-MPS1 cells, indicating that Ipl1p is required for full maintenance of the GAL-MPS1 checkpoint arrest. (B) Mps1 levels were monitored in the same experiment by immunoblotting with anti-myc antibodies. The levels of Mps1 protein decline in both strains but are unstable in the ipl1-321 cells. Equal protein concentrations were loaded in all lanes as judged by Ponceau S staining (data not shown). (C) Chromosome IV segregation was monitored by microscopy of GFP-marked chromosome IV. This chromosome segregated normally as GAL-MPS1 cells (filled squares) and GAL-MPS1 ipl1-321 cells (open squares) left the checkpoint arrest.

Figure 3

Figure 3

(A) Ipl1p is not required for the checkpoint arrest induced by nocodazole. Wild-type (SBY818), ipl1-321 (SBY819), mad2Δ (SBY920), and bub2Δ (SBY934) cells containing Pds1-myc18 were arrested in G1 with α-factor at the permissive temperature (23°C). They were released into nocodazole and benomyl at the nonpermissive temperature (37°C), and α-factor was added back when small buds appeared, to prevent cells from entering the next cell cycle. Pds1p levels were analyzed by immunoblotting and show that wild-type, ipl1-321, and bub2Δ mutant cells activate the spindle checkpoint because they maintain high Pds1 levels. Pds1p levels cycle in mad2Δ mutant cells, which lack the spindle checkpoint. Equal protein concentrations were loaded in all lanes as judged by Ponceau S staining (data not shown). (B) IPL1 does not function in the _BUB2_-dependent checkpoint pathway. Wild-type (SBY214), ipl1-321 (SBY322), and bub2Δ (SBY432) mutant cells were released into nocodazole plus benomyl at the nonpermissive temperature. The percentage of large budded cells was monitored and shows that wild-type (filled squares) and ipl1-321 cells (shaded squares) arrest as large budded cells whereas bub2Δ cells (gray squares) rebud, indicating that Ipl1p is not in the same pathway as Bub2p.

Figure 4

Figure 4

Ipl1p is required for the checkpoint arrested induced by kinetochore tension defects. (A) Cells depleted of the Cdc6 protein were grown at the permissive temperature (23°C) and arrested in G1 with α-factor. Cells were then released from G1 to the nonpermissive temperature (37°C) in glucose to keep GAL-CDC6 repressed; Pds1p levels were analyzed by immunoblotting. Pds1p levels cycle in wild-type cells (SBY818) but are stabilized in GAL-CDC6 cells grown in repressing media (SBY772). Pds1p levels are not stabilized in GAL-CDC6 mad1Δ (SBY762) and GAL-CDC6 ipl1-321 (SBY771) mutant cells, indicating that the checkpoint is not activated. (B) mcd1-1 (SBY870) and mcd1-1 ipl1-321 cells (SBY871) were arrested in G1 with α-factor at the permissive temperature. They were released to the nonpermissive temperature (37°C) in the absence of α-factor, and Pds1-myc18 protein levels were monitored by immunoblotting. There is a delay in the degradation of Pds1p in the mcd1/scc1 mutant cells that is eliminated in the mcd1-1 ipl1-321 cells, indicating that Ipl1p is required for the spindle checkpoint induced by defects in sister chromatid cohesion. Equal protein concentrations were loaded in all lanes as judged by Ponceau S staining (data not shown).

Figure 5

Figure 5

Ipl1p localizes to kinetochores at metaphase. (A) Ipl1p localizes to kinetochores during a metaphase arrest. Cdc26Δ cells containing Cse4p–myc13 and Ipl1p–HA3 (SBY961) were arrested in metaphase by shifting cells to the nonpermissive temperature (37°C ) for 3 h. Chromosome spreads were performed and stained with DAPI to recognize the DNA (left panel), and with anti-myc and anti-HA antibodies to recognize Cse4p and Ipl1p, respectively (middle panels). The merged image (right panel) shows that there is colocalization of Ipl1p and Cse4p during a metaphase arrest. (B) Ipl1p localizes to kinetochores during a checkpoint arrest. Cells containing Ndc10p–HA3 and Ipl1p–myc12 (SBY596) were arrested in nocodazole for 3 h at 23°C. Chromosome spreads were performed and stained with DAPI to recognize the DNA (left panel), and with anti-HA and anti-myc antibodies to recognize Ndc10p and Ipl1p, respectively (middle panels). The merged image (right panel) of the Ndc10p and Ipl1p images shows that there is a colocalization of Ipl1p with Ndc10p to kinetochores during a checkpoint arrest.

Figure 6

Figure 6

Model for the role of Ipl1p in monitoring tension during spindle assembly. Telocentric chromosomes are shown for simplicity in illustrating kinetochore orientation. Early in mitosis, kinetochores are neither attached to microtubules nor under tension (black-filled kinetochores). Attaching both sister chromatids to the same pole (mono-orientation) produces kinetochores that are not under tension, but are bound to microtubules (gray-filled kinetochores). Once a bioriented spindle is established, tension can be generated on the kinetochores (open kinetochores). The spindle checkpoint must monitor defects in both attachment and tension to ensure bipolar spindle assembly. Our experiments suggest that Ipl1p has a specific role in monitoring tension but not attachment.

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