Cellular response upon proliferation in the presence of an active mitotic checkpoint

(A) WT (yAC3997) and tub2-401 (yAC3927) cells were synchronized in G1 with α-factor and released at either 22°C or 15°C. After 24 h, the cells were sampled and stained for DNA content (DAPI) and α-tubulin Tub1 (FITC) by immunofluorescence. Scale bar: 5 μm. (B) Spotting of serial dilutions of WT (yAC3568), mad2Δ (yAC3372), tub2-401 (yAC3220), and tub2-401 mad2Δ (yAC2946) cells on YEPD plates incubated at 30°C, 22°C, and 15°C. (C) Spotting of serial dilutions of WT (yAC1001) and GAL1-MAD2 (yAC2465) cells on YEPD plate (no Mad2 overexpression) and YEPRG plate (Mad2 overexpression). (D) Evaluation of mis-segregation events. WT (yAC4013), GAL1-MAD2 (yAC4018), and tub2-401 (yAC4012) cells carrying Htb2-mCherry and ChrV-GFP were synchronized in G1 and released, respectively, in YEPRG at 30°C and YEPD at 22°C. After 24 h from G1 release, the samples were collected and imaged. Left panel: examples of mis-segregating cells (pointed by the arrow). Scale bar: 5 μm. Central panel: percentages of cells with ChrV mis-segregation for each condition. Biological replicate #1 (black circle): WT [YEPRG] 0% (0/66); GAL1-MAD2 [YEPRG] 2.20% (2/91); WT [22°C] 0% (0/67); tub2-401 [22°C] 4.65% (6/129). Biological replicate #2 (red circle): WT [YEPRG] 0% (0/105); GAL1-MAD2 [YEPRG] 2.27% (3/132); WT [22°C] 0% (0/140); tub2-401 [22°C] 6.72% (16/238). Right panel: estimation of the probability of having at least one aneuploid chromosome as a function of the ChrV mis-segregation rates. Blue line represents the theoretical function of the probability—see the Materials and Methods section for details—and dots are representative of the biological replicates in the central panel.

(A) Definition of unperturbed and SAC-active cells. The first generation (i.e., gen0) has been evaluated from α-factor release and rebudding, whereas subsequent cell cycles (i.e., gen1, gen2, …) from budding to rebudding. Unperturbed cells correspond to WT cells, whereas SAC-active refers to GAL1-MAD2 or tub2-401 cells. (B) Description of the monitored GAL1-MAD2 and tub2-401 SAC-active cells up to generation 1. Proportion of censored and uncensored event were reported for each biological replicate. (C) SAC-active tub2-401 cells are delayed in mitosis because of an active SAC. Unperturbed CLB2-GFP (yAC3491), SAC-active CLB2-GFP tub2-401 (yAC2970), and SAC-deficient CLB2-GFP tub2-401 mad2Δ (yAC3034) cells were synchronized in G1 and released at the semipermissive temperature. Mitotic length of gen0 was evaluated for each condition. N, number of observations; m, median of the distribution. Censored data (i.e., dead cells or precocious end of cell monitoring) are represented as white circles. (D) Evaluation of maximum levels of nuclear Clb2 during the mitosis of unperturbed and SAC-active cells. The maximum value of Clb2 trajectory was measured for each mitotic length of unperturbed and SAC-active cells. N: number of observations; m: median of the distribution. Significant differences between unperturbed and SAC-active cells were evaluated with a two-sample t test with unequal variances or Wilcoxon rank sum test (significance level: 0.05), respectively, according to the normality or not of the distributions. (E) Distributions of Clb2 levels after Clb2 degradation in unperturbed and SAC-active cells at gen0. For each cell, mean cytoplasmic levels of Clb2 were measured from anaphase onset to rebudding, and the minimum value within this interval was evaluated. Number of observations for GAL1-MAD2: unperturbed gen0: 54; SAC-active gen0: 45. Number of observations for tub2-401: unperturbed gen0: 72; SAC-active gen0: 78. (F, G) Evaluation of cell cycle and mitotic length of SAC-active cells clustered by the number of generations after gen0. N, number of observations; m, median of the distribution. Censored data were represented as white circles.

(A) Mitotic length of GAL1-MAD2 and GAL1-MAD2 cdh1Δ cells during the first mitotic arrest. Nuclear segregation was the readout of anaphase onset. N, number of observations; m, median of the distribution. Significant differences between GAL1-MAD2 and GAL1-MAD2 cdh1Δ cells were evaluated with a two-sample t test with unequal variances or Wilcoxon rank sum test (significance level: 0.05), respectively, according to the normality or not of the distributions. (B) Proliferation of GAL1-MAD2 (yAC3883) and GAL1-MAD2 cdh1Δ (yAC3885) cells carrying Htb2-mCherry and Clb2-GFP was monitored in microfluidic chamber, in the continuous presence of YEPR medium (i.e., without Mad2 overexpression). Clb2 trajectories were synchronized in silico at the beginning of Clb2 degradation. Individual traces of Clb2 are reported as thin lines, their mean trajectory as thick lines. N, number of trajectories. (C) Duration of anaphase-to-G1 of GAL1-MAD2 and GAL1-MAD2 cdh1Δ cells during the first mitotic arrest. Nuclear segregation was the readout of anaphase onset. N, number of observations; m, median of the distribution. Significant differences between GAL1-MAD2 and GAL1-MAD2 cdh1Δ cells were evaluated with a two-sample t test with unequal variances or Wilcoxon rank sum test (significance level: 0.05), respectively, according to the normality or not of the distributions.

(A) Probability density of the cellular size at anaphase onset for gen0 unperturbed, unperturbed, gen0 SAC-active, and SAC-active cells under the two experimental conditions. Sample sizes for GAL1-MAD2: 50 for gen0 unperturbed, 65 for unperturbed, 50 for gen0 SAC-active, and 48 for SAC-active. Sample sizes for tub2-401: 66 for gen0 unperturbed, 71 for unperturbed, 96 for gen0 SAC-active, and 65 for SAC-active. (B) Scatter plot of cellular size at prometaphase versus size at the anaphase onset for unperturbed and SAC-active cells. Linear dependency was tested by evaluation of the Spearman correlation coefficient (r; significance level 0.05) and the slope of the linear regression (slope). Number of points for GAL1-MAD2: 65 for unperturbed, 48 for SAC-active. Number of points for tub2-401: 71 for unperturbed, 65 for SAC-active. (C) Scatter plot of mitotic length versus cellular size at prometaphase for unperturbed and SAC-active cells. Linear dependency was tested by performing a linear regression on data, evaluating the slope λ (significance level: 0.05). Number of points for GAL1-MAD2: 65 for unperturbed, 48 for SAC-active. Number of points for tub2-401: 71 for unperturbed, 65 for SAC-active. (D–F) Description of the filtering and smoothing procedure for size trajectories of gen0 unperturbed, unperturbed, gen0 SAC-active, and SAC-active cells—see the Material and Methods section for details. (D) For each size trajectory, the local size variation ΔA = A_i+1 − A_i was plotted as a function of the size A_i for GAL1-MAD2 (on the left) and tub2-401 (on the right). Yellow dots correspond to high density regions, whereas blue ones to low density regions. Point cloud data were then divided in three clusters (from 0 to 40 μm², from 40 to 80 μm², and from 80 to 120 μm²). For each cluster, outlier data (in the white regions) were discriminated from good data (in the green region) according to the distribution of local size variations. Number of points: 2,996 for GAL1-MAD2 and 5,652 for tub2-401. (E) An example of size trajectory adjustment. Raw data are represented by gray dots. From the thresholds that were identified in (D), outliers were detected in the original size trajectories (red points falling on the white regions). Each outlier was replaced by the mean of the two adjacent size values in the trajectory (green point). (F) An example of size trajectory smoothing. Data are represented by blueberry dots. Once filtered, size trajectories were smoothed with a moving average filter (green trajectory).

(A) Saturation of cell growth in gen0 SAC-active and SAC-active cells occurs during mitosis. Local growth rate (dA/dt|_A) in function of Clb2 mean levels. Individual measurements (dots) were binned by Clb2 levels and the mean value of each bin (squares) and their trend (thick line) were evaluated. Number of points for GAL1-MAD2: 778 for gen0 unperturbed, 297 for unperturbed, 1,640 for gen0 SAC-active, and 492 for SAC-active; number of points for tub2-401: 781 for gen0 unperturbed, 538 for unperturbed, 3,169 for gen0 SAC-active, and 1,444 for SAC-active. See the Material and Methods section for details about local growth rate measurements. (B) Local growth rate (dA/dt|_A) as a function of cell size. Individual measurements (dots) were binned by cell size and the mean value of each bin (squares) and their trend (thick line) were evaluated. Number of points for GAL1-MAD2: 778 for gen0 unperturbed, 297 for unperturbed, 1,640 for gen0 SAC-active, and 492 for SAC-active cells. Number of points for tub2-401: 781 for gen0 unperturbed, 538 for unperturbed, 3,169 for gen0 SAC-active, and 1,444 for SAC-active. See the Material and Methods section for details about local growth rate measurements. (C) Examples of morphologies of gen0 unperturbed, gen1 unperturbed, gen0 SAC-active, and gen1 SAC-active cells under the two experimental conditions. Scale bar: 5 μm. (D) Evaluation of cell size at the cell cycle entry as a function of the cycle number from checkpoint activation, assuming two alternative models of cell growth: exponential growth or exponential with linear growth (used in the simulations).

(A) Expression of the CDC20-127 allele bypasses the mitotic arrest induced by Mad2 overexpression. GAL1-MAD2 (yAC2465), GAL1-MAD2 mad3Δ (yAC436), and GAL1-MAD2 tetO₂-CDC20-127 (yAC2807) cells were synchronized in G1 with α-factor and released in the presence of galactose to induce Mad2 overexpression. Two populations of yAC2807 were monitored: one expressing and one not expressing CDC20-127 (gen0 unperturbed and gen0 SAC-active, respectively). α-factor was re-added after 1h30 min from the release and then every 2 h, to re-synchronize cells in the subsequent G1 phase. Cells were monitored for 7 h and sampled for FACS analysis to measure DNA content. The leakage from the promoter is minimal, as can be observed comparing the first round of adaptation in cells expressing or not CDC20-127 (compare GAL1-MAD2 and gen0 SAC-active cells). (B) PCA of label-free quantities (LFQ) intensities for all samples used in the proteomic analysis after batch correction. (C, D) Scatter plot of P-values of GO enrichment for down-regulated and up-regulated proteins ranked by positive log2-fold change. GO terms outside a circle with radius three are highlighted in red and reported in the figure legend.