(a) Schematic of major components influencing C-CDK activity at mitosis, and in red, the pathways that do not influence C-CDKAF. The negative relationship between C-CDK activity and cell growth refers to the block of cell length extension in mitosis. PP2A opposes CDK activity by dephosphorylating CDK substrates, and also by opposing the activation of CDK at mitosis by opposing the phosphorylation of Wee1 and Cdc25. Reciprocally, CDK causes the downregulation of PP2A activity in mitosis. (b) Example cell lineage traces from time-lapse microscopy. Cell size in pixels2 is given in orange, and C-CDK-YFP fluorescence intensity is given in purple. Steep decreases in cell size traces correspond to cell division. (c) Scatter plot of mean C-CDK level vs cell size from time-lapse microscopy data. C-CDK level is a measure of C-CDK-YFP fluorescence intensity. Colours indicate density of data. Inset boxplot is mean nuclear C-CDK concentration immediately prior to degradation at anaphase. Boxes represent interquartile range, with whiskers delimiting 5th–95th percentiles. C-CDKWT n = 28, C-CDKAF n = 44 full cycles. (d) Plot of the probability of division at the next timepoint (P(Div)) vs cell length for CDKWT and CDKAF. Cells were followed through time-lapse microscopy with measurements taken each frame. P(Div) defined as the proportion of cells that undergo C-CDK degradation at anaphase by the next timepoint, given as rate per minute. Points represent cells binned by size, with points plotted at bin centre. C-CDKWT n = 685, C-CDKAF n = 961 timepoints. (e) Plot of P(Div) function vs C-CDK level for CDKWT and CDKAF. C-CDKWT n = 685, C-CDKAF n = 961 timepoints. C-CDK-YFP intensity measurements taken every frame from time-lapse microscopy, and binned by C-CDK level. (f) Schematic of Cut3 as a CDK activity reporter. Mitotic CDK-dependent phosphorylation of Cut3 on T19 results in nuclear translocation of the protein. (g) Experimental outline of block and release time-lapse experiment for panels (h, j–o). Asynchronous cells possessing an analogue sensitive (as) CDK were blocked in G2 using 1 μM 1NM-PP1 for 5 hr and then released into a range of 1NM-PP1 concentrations. Cells were then followed and monitored for their Cut3-tdTomato nuclear/cytoplasmic (N/C) ratio (C-CDK activity) and C-CDK-YFP level using fluorescence time-lapse microscopy (see Materials and methods). Data for (l–o) were acquired 15 min following release from 1NM-PP1. (h) Maximum CDK activity (normalized against maximum level, obtained by release into DMSO) against 1NM-PP1 concentration. Red points are the median of the data sets for each drug concentration (N = 324), and green point is median in DMSO. Black line is the Hill equation fit to the median data by a nonl-inear fitting algorithm (IC50 = 115.4, Hill coefficient = −1.71). Purple dashed line is Hill curve derived from Swaffer et al., 2016 dose–response data (IC50 = 133.4, Hill coefficient = −1.47). (i) Time-lapse quantification of CDK activity in asynchronous cells. Traces are aligned so that 0 min corresponds to peak Cut3-tdTomato N/C ratio. Curve smoothing could move Cut3 peak earlier/later than exactly 0 min. Trace colour indicates cell size. Red X indicates automatically defined mitotic entry point. C-CDKWTn = 23 and C-CDKAFn = 14. (j) Scatter plot of C-CDK-YFP levels against cell size. Experiment described in (g), with measurements taken before release from 1NM-PP1 block. Black points indicate binned data, bin window size 500 pixels2. n = 324. Pearson correlation coefficient: 0.55. (k) As in (j), but with C-CDKAF, n = 312. Pearson correlation coefficient: 0.62. (l) Scatter plot of peak Cut3-tdTomato level vs cell size. Experiment described in (g), with measurements taken 20 min after release from 1NM-PP1 block into DMSO. Black points indicate binned data, bin window size 500 pixels2. Points are coloured by YFP C-CDK levels at release. n = 83. R2 = 0.5040. Pearson correlation coefficient: 0.50. (m) As in (l), but with C-CDKAF, n = 81. R2 = 0.2150. Pearson correlation coefficient: 0.22. (n) Scatter plot of peak Cut3-tdTomato level vs C-CDK-YFP intensity level 20 min after release from 1NM-PP1 block into DMSO. Black points indicate binned data, bin window size 15 AU. Points are coloured by cell size at release. n = 83. R2 = 0.3668. Pearson correlation coefficient: 0.60. (o) As in (n), but with C-CDKAF, n = 81. R2 = 0.5501. Pearson correlation coefficient: 0.74.