Volume 28, Issue 1, Pages 28-37.e7 (January 2018) Cells Escape an Operational Mitotic Checkpoint through a Stochastic Process  Paolo Bonaiuti, Elena Chiroli, Fridolin Gross, Andrea Corno, Claudio Vernieri, Martin Štefl, Marco Cosentino Lagomarsino, Michael Knop, Andrea Ciliberto  Current Biology  Volume 28, Issue 1, Pages 28-37.e7 (January 2018) DOI: 10.1016/j.cub.2017.11.031 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Clb2 Degradation during Adaptation Takes Place When Mad2 Is Localized Cells expressing Mad2-GFP and Clb2-mCherry (yAC3538) were released from G1 phase in YEPD medium containing nocodazole. One hour after the release, α factor was added to the medium at 12.5 μg/mL. Cells were filmed for 700 min. (A) Example of a cell where Clb2 degradation starts with localized Mad2. (B) Time series of images of the cell in (A). Time starts from Clb2 accumulation. (C) Scatterplot of Mad2 localization index at Clb2 degradation. Cells with values above threshold are depicted as filled dots, empty otherwise. Histograms pointing upward refer to cells above threshold, downward to cells below threshold. Number of independent biological replicates (N): 3, number of observations (n): 314. (D) Boxplots of Mad2 localization index (green) and Clb2 (purple) at the indicated time points before and after Clb2 degradation (t = 0). In G1, we show only Mad2 localization. For each cell, Clb2 is normalized on its value when degradation starts. Only cells where Clb2 degradation starts with Mad2 localized are included. N: 3; n: 272. The edges of the boxes are the 25th and 75th percentile; inside the boxes we show the median. The whiskers extend to the most extreme data point within 1.5 times the length of the box. Dots are data points outside the whiskers. See also Figure S1 and Table S1. Current Biology 2018 28, 28-37.e7DOI: (10.1016/j.cub.2017.11.031) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Adaptation Dynamics Is Well Approximated by an Exponential Distribution (A) CLB2-mCherry MAD2-GFP (yAC3538) cells were synchronized in α factor and released into nocodazole. We plot the fraction of cells that escaped an arrest as a function of the time spent in mitosis (defined in Figure 1A). The experimental cumulative function of adaptation times (solid line) is fitted with an exponential (dotted line) after the delay, with only one free parameter, the rate of adaptation. N: 3, n: 272. (B) The wiring diagram for the mitotic checkpoint model. Solid circles indicate the substrate of a binding reaction, which gives rise to the product indicated by an arrow. An arrow without solid dots indicates an irreversible reaction. Mad2 and Mad3 have been lumped together in the generic variable M. (C) Wild-type cells (yAC1070) were arrested in α factor and released into nocodazole. After 120 min, we added cycloheximde (CHX) and sampled cells for western blotting. Quantification of the experiment can be found in Figure S2C. Data from a single representative experiment out of four repeats are shown. (D) Mad2, Mad3, Bub3, and Cdc23 concentrations measured by fluorescence correlation spectroscopy (FCS) (strains yAC3268, yAC2886, and yAC2940). The edges of the boxes are the 25th and 75th percentile; inside the boxes we show the median. The whiskers extend to the most extreme data point within 1.5 times the length of the box. Dots are data points outside the whiskers. (E) Upper panels: wild-type cells or cells carrying myc9-Cdc20 or Cdc23-myc9 (yAC3202, yAC3307, yAC3371) were arrested in G1 and released into nocodazole. At the indicated time points, samples were taken for western blotting. Lower panel: cells of the indicated genotypes (strains yAC3430, yAC3307, yAC3367, yAC3365, yAC3436, yAC3353, yAC3261, yAC3427, yAC3262, yAC3371) were arrested in G1 with α factor and released in nocodazole. After 110 min, samples were taken for protein extract preparation and immunoblotted with anti-myc antibodies. N: 2. (F) Simulated cumulative function of adaptation times produced by the model in (B), reactions in Table S2, parameters in Table S3. In the inset, two representative trajectories of individual cells that cross the anaphase threshold at two different times are indicated by the solid dots. The same dots are displayed in the cumulative curve (solid line), which is fitted with an exponential (dotted line) after a delay. See also Figure S2 and Tables S2 and S3. Current Biology 2018 28, 28-37.e7DOI: (10.1016/j.cub.2017.11.031) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 The Mitotic Checkpoint Is Insensitive to High Levels of Cdc20 Production (A) Steady states for the inhibited (APC/Cinhib) and the active form (APC/CCdc20) of APC/C, as a function of Cdc20 synthesis rate (ksyn) for the model in Figure 2B. The dotted line is the anaphase threshold, which needs to be crossed for cells to enter anaphase. With differently colored dots, we mark different values of Cdc20 synthesis rate, see (B). (B) Stochastic simulations of the model in Figure 2B, for the six different values of Cdc20 synthesis rate in (A). (C) MAD2-GFP CLB2-mCherry cells carrying 1, 2, 3, and 5 copies of CDC20 (yAC3538, yAC3565, yAC3555, yAC3552) were arrested in α factor and released into nocodazole, as described for Figure 1. The cumulative distribution curves of adaptation times (solid lines) are fitted with exponential curves (dotted lines) after a delay. N: 3. (D) Kaplan-Meier estimate of the cumulative distribution function of adaptation times of diploid strains, wild-type (yAC3801), CDC20 (3X) (yAC3802), CDC20 (5X) (yAC3803), or heterozygous for CDC20 (yAC3804). Cells were grown in microfluidic devices in the presence of nocodazole, without being synchronized in G1 (see STAR Methods for details on image analysis). A black cross marks the time interval where a censoring event occurred (i.e., dead cell). N: 2. (E) Mean adaptation time as a function of synthesis rate in the model (left) and as a function of Cdc20 gene copy number in the experiment shown in (C) (right). For copy number 0.5, we used the results shown in (D), obtained with diploids, which showed that the heterozygous CDC20/cdc20 has 60% of propensity to adapt of wild-type cells, Table S4. We used a different symbol (square) and a dotted line to emphasize the fact that this result was obtained in a different experimental system. See also Figures S3 and S4 and Table S4. Current Biology 2018 28, 28-37.e7DOI: (10.1016/j.cub.2017.11.031) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Inhibited APC/C Saturates with More Than 2 Copies of CDC20 MAD2-GFP CDC23-mCherry cells (A, strains yAC2886, yAC3179, yAC3176, yAC3384) and MAD2-GFP MAD3-mCherry cells (B, strains yAC3268, yAC3183, yAC3153, yAC3380) carrying 1, 2, 3, and 5 copies of CDC20 were grown and synchronized at 23° in synthetic low-fluorescent medium containing 1% peptone. Cells were released from G1 arrest into nocodazole and measured using FCCS between 150 and 210 min. Each measurement is expressed as fold increase with respect to the wild-type strain measured on the same day. More precisely, we normalized the cross-correlation signal to the exponential of the mean value of the logarithmic transform of the wild-type. In absolute concentrations, 1 corresponds to 10.8 nM (A) and 12.9 nM (B). A Kolmogorov-Smirnov two-tail test was used for comparison. Circles indicate outliers, included in the analysis. In (A), p values are 9.25e-09, 8.82e-02, 8.65e-01. In (B), p values are 6.01e-08, 6.71e-01, 2.88e-01. The edges of the boxes are the 25th and 75th percentile; inside the boxes we show the median. The whiskers extend to the most extreme data point within 1.5 times the length of the box. Dots are data points outside the whiskers. See also Figure S5. Current Biology 2018 28, 28-37.e7DOI: (10.1016/j.cub.2017.11.031) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Cells Can Respond Either Slowly or Promptly to Pulses of Cdc20 MET3pr-CDC20 MET3pr-Venus GAL1-MAD2 HTB2-mCherry cells (yAC2341) were grown and synchronized in complete low fluorescent synthetic medium with raffinose (SCR) and released from an α factor arrest in the same medium supplied with 2% galactose, to activate the GAL1 promoter. During the experiment, cells were grown in microfluidic devices. The MET3 promoters were activated by removal of methionine (we call it “pulse,” shown as gray-shaded rectangles in A), which occurred either early or late, the time difference being 1 hr. For a schematic of the experiment, see Figure S6C. (A) Histograms of adaptation times for the four conditions. (B) Comparison between adaptation times, measured from α factor release, in cells that never switch on the MET3 promoter (No pulse) and cells that do not adapt under the early or late pulse. We only keep track of adaptation times of “slow exit” cells (blue histograms in A), those adapting after the upper border for second pulse (after t = 220 min). (C) Scatterplot of adaptation times and MET3 promoter activities in the early pulse condition of (A)—same color code. See also Figure S6. Current Biology 2018 28, 28-37.e7DOI: (10.1016/j.cub.2017.11.031) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Checkpoint Deficiency versus Adaptation Simulations of cells undergoing adaptation as compared to SAC-deficient cells. In the latter, we set the total amount of Mad2/Mad3 to zero. Left panel, in red trajectories of SAC-deficient cells, in blue of adapting cells. The color of each trajectory becomes transparent after adaptation, marked by a black dot. Right panel, the histograms of adaptation times in the two cases, with the same color code. Current Biology 2018 28, 28-37.e7DOI: (10.1016/j.cub.2017.11.031) Copyright © 2017 Elsevier Ltd Terms and Conditions