Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression (original) (raw)
Figure 5
Distinct Requirement of Cdk1 for Mitotic Entry and Mitotic Progression in Human Cells
(A) Cells selected for Cdk1 shRNA expression were synchronized in G2/M by thymidine release; mitotic cells were isolated by gentle shake-off. Mitotic cells were more than 95% MPM2 positive as analyzed by immunostaining and FACS analysis (unpublished data). Separated G2 and mitotic pools were analyzed for Cdk1 expression by Western blotting. Cdc20 protein levels serve as loading control. The percentage of remaining Cdk1 protein is indicated in the figure.
(B) Cells collected by mitotic shake-off were lysed (lanes 1 and 2) or released from nocodazole and incubated in fresh medium for 3 h, recollected, and lysed (lanes 3 and4). Differences in mitotic phosphorylation shift of APC3 (human Cdc27 ortholog) and Cdc25C, depending on the Cdk1 levels, are shown (lanes 1 and 2).
(C) The impaired phosphorylation of APC3 in Cdk1-attenuated mitotic cells (lane 1) was rescued by coexpression of a Cdk1-YFP construct containing a silent mutation in the RNAi targeting region (lane 2). Lane 3 are mitotic cells transfected with a control shRNA, revealing normal endogenous Cdk1 levels.
(D) Distribution of metaphase duration, measured as time between chromosome alignment at the metaphase plate and onset of sister chromatid separation, in Cdk1 RNAi cells (right) or Cdk1 RNAi cells rescued by coexpression of non–RNAi-sensitive Cdk1-YFP (left).
(E) Time-lapse microscopy analysis of mitotic progression after entry with normal or impaired Cdk1 levels. Bottom panels are consecutive images of tubulin-YFP in a pS-control cell in mitosis; top panels show delayed chromosome alignment (frames 2 and 3) and stalled metaphase (frames 4–6) after Cdk1 shRNA.