Hongqing LIANG1, Hong Hwa LIM1, Ashok VENKITARAMAN2, Uttam SURANA1
1 - Institute of Molecular and Cell Biology, A*STAR (Agency for Science Technology and Research), Singapore
2 - University of Cambridge and the Medical Research Council Cancer Unit, Cambridge, UK.
Published in EMBO J. 2011 Nov 4. doi: 10.1038/emboj.2011.385. [Epub ahead of print]
The spindle assembly checkpoint (SAC), an evolutionarily conserved surveillance pathway, prevents chromosome segregation in response to conditions that disrupt the kinetochore-microtubule attachment. Removal of the checkpoint-activating stimulus initiates recovery during which spindle integrity is restored, kinetochores become bi-oriented, and cells initiate anaphase. Whether recovery ensues passively after the removal of checkpoint stimulus, or requires mediation by specific effectors remains uncertain. Here, we report two unrecognized functions of yeast Cdk1 required for efficient recovery from SAC-induced arrest. We show that Cdk1 promotes kinetochore bi-orientation during recovery by restraining premature spindle elongation thereby extinguishing SAC signalling. Moreover, Cdk1 is essential for sustaining the expression of Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) required for anaphase progression. We suggest a model in which Cdk1 activity promotes recovery from SAC-induced mitotic arrest by regulating bi-orientation and APC/C activity. Our findings provide fresh insights into the regulation of mitosis and have implications for the therapeutic efficacy of anti-mitotic drugs.
Figure Legends: Cdk1 inhibition does not affect kinetochore capturing.
(A) cdc28-as1 GAL-CENIII-GFP SPC42-RFP MET-CDC20 cells were incubated in nocodazole containing YEP+Raff+Gal+Met medium and then released into three different media-conditions: 1) YEP+Gal+Met+1NMPP1 medium, 2) YEP+Glu+Met +1NMPP1 medium and 3) YEP+Glu+Met -1NMPP1 medium. Photo-micrographs depict the positions of CENIII-GFPand Spc42-RFP 30 min after release. Bar scale: 5μm. Bottom panels: bar charts showing proportion of cells with distances between CENIII-GFPand Spc42-RFP after release from nocodazole arrest. ~100 cells were analyzed for each sample. (B) Bi-orientation in cdc28-as1 slk19Δ cdc15-2 cells after release from HU arrest in absence or presence of 1NM-PP1. Photo-micrographs show the state of nuclear division, spindle staining and CENV-GFP signal 60min and 120min after release from HU arrest. Bar scale: 5μm. Graph depicts bi-orientation kinetics after the release. Experiments were performed twice and >200 cells were analyzed for each sample.
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