(A) Quantification of immunofluorescence labeling. Cytoplasmic Cdk1-P (y-axis) and cyclin B1 signal (x-axis) were quantified in the cytoplasm of unsynchronized cells. Each dot corresponds to one cell. Cells were labeled according to morphology and staining as follows: cells with cytoplasmic cyclin B1 (blue diamonds), cytoplasmic cyclin B1 and separated centrosomes (red squares), predominant cyclin B1 staining in nucleus (green triangles), condensed DNA and no distinguishable nucleus in cyclin B1 staining (light blue Xs), all chromosomes aligned on a metaphase plate (black dots), and anaphase (red Xs).
(B) Relative cyclin B1–Cdk1-P during nuclear translocation. Ratio of cytoplasmic Cdk1-P and cyclin B1 signal (y-axis) in cells with separated centrosomes (red squares) and translocated cyclin B1 (green triangles) correlated with the ratio of nuclear and cytoplasmic cyclin B1 (x-axis). The average cytoplasmic Cdk1-P/cyclin B1 ratio of early G2 cells is defined as 100%. Early G2 cells are defined as cells with cytoplasmic cyclin B1 levels between average anaphase cyclin B1 levels and lowest cyclin B1 level of cell with separated centrosomes.
(C) Average values of cytoplasmic cyclin B1 and phosphorylated Cdk1. Values are shown as percentage of the highest average. Error bars indicate SD. Neg, cells used for background subtraction; E G2, G2 cells with cyclin B1 levels between average anaphase levels and lowest value of cell with separated centrosomes; L G2, G2 cells with cyclin B1 levels above lowest value of cell with separated centrosomes; Sep, separated centrosomes; Tra, translocated cyclin B1; Pro, prometaphase; Met, metaphase; Ana, anaphase.
(D) Estimated cytoplasmic cyclin B1/Cdk1 activity of individual cells. The degree of dephosphorylation multiplied with the level of cyclin B1 (y-axis), as a function of the level of cyclin B1 (x-axis). Cells are labeled as in (A).
(E) Average values of estimated cytoplasmic cyclin B1–Cdk1 activities within the distinct phases, shown in Figure 3D, as a function of cytoplasmic cyclin B1 levels.
(F) Average values of cytoplasmic Cdk1-P/cyclin B1 ratio and estimated cytoplasmic cyclin B1–Cdk1 activities in the different phases. Values are shown as percentage of the highest average. Error bars indicate SD. Labels are as in Figure 2C.

Average ratio of Cdk1-P and cyclin B1 staining (black solid line), average estimated cyclin B1–Cdk1 activity (gray solid line), and average APC-3 S426-P (black dotted line) plotted as a function of time (x-axis). The duration of mitotic events is derived from measurements described in Figure S8. Note the decrease in total cytoplasmic activity, but not in the progression of cyclin B1–Cdk1 dephosphorylation, between centrosome separation and nuclear translocation of cyclin B1–Cdk1.

Model of relation between Cdk1 activity and mitotic progression. Cells do not enter mitosis unless a threshold concentration of active cyclin B1–Cdk1 is present (G₂ arrest). After mitotic entry, the Cdk1 activity gradually increases, which enables the cell to prepare for mitotic exit (normal G2/M). If the Cdk1 activity does not develop fully after mitotic entry, the cell is delayed before anaphase (mitotic delay).

Fluorescence images are undeconvolved maximum intensity projections of unsynchronized cells from a single cover slip. Cells are presented from G2 (left) to anaphase (right). Top row: DNA, second row: phosphorylated Cdk1, third row: cyclin B1, bottom row: merge of all three fluorescence channels.

(A) Deconvolved maximum projections of unsynchronized HeLa cells. The cell-cycle stage is indicated above the figure. Top row: merge, second row: DNA, third row: cyclin B1, bottom row, APC3 S426P.
(B) Dot plot of cytoplasmic cyclin B1 and cytoplasmic APC3 S426P staining. Each dot corresponds to one cell. Cells are arranged according to morphology and staining as in Figure 2A.
(C) Average values of cytoplasmic cyclin B1 and phosphorylated APC3. Values are shown as percentage of the highest average. Error bars indicate SD. Bars are labeled as in Figure 2C.

(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.