Panel A shows a vertical stacked bar graph depicting the percentage of cells with normal segregation, mis-segregation, or death at different MBC concentrations. The horizontal axis represents MBC concentration in micrograms per milliliter, and the vertical axis represents the percentage of cells. Panel B consists of four line graphs showing the distance between sister centromere pairs over time from sister chromatid separation (SCS) in wild-type cells and cells with 3 micrograms per milliliter MBC. The horizontal axis represents time from SCS in seconds, and the vertical axis represents sister centromere distance in micrometers. Panel C features two histograms with Gaussian fits, showing the frequency distribution of the time difference between the separation of centromeres 1 and 2, and centromeres 2 and 3 in klp5-deleted cells. The horizontal axis represents the time difference in seconds, and the vertical axis represents the percentage of cells. Panel D displays a kymograph of sister chromatid separation in a klp5-deleted cell with cen1-tdTomato and cen2-GFP markers. Panel E and F are bar graphs quantifying securin degradation rates in the presence or absence of non-degradable cyclin B and securin, respectively. The horizontal axis represents the condition, and the vertical axis represents the normalized degradation rate in inverse seconds. Panel G shows a line graph simulating released and active separase concentrations over time with high or low APC/C activity. The horizontal axis represents time in seconds, and the vertical axis represents separase concentration in micromolar. Panel H and I are line graphs of nuclear securin-GFP intensity over time in wild-type cells and mutants, with curves aligned to anaphase onset. The horizontal axis represents time from anaphase onset in minutes, and the vertical axis represents nuclear securin-GFP concentration in arbitrary units. Panel H shows the data for wild type and an APC/C mutant. Panel I shows the data for wild-type cells and cells treated with a proteasome inhibitor. Panel J is a bar graph quantifying degradation rates from the experiments in Panel H and I. The horizontal axis represents the condition, and the vertical axis represents the normalized degradation rate in inverse seconds. Panel K shows a kymograph for a proteasome inhibitor-treated cell with cnt2-tdTomato and cnt3-GFP markers. Sister centromere separation is indicated with arrowheads.
Additional analyses of the influence of microtubule perturbations and securin degradation dynamics on chromosome segregation synchrony. (A) Anaphase outcomes of cells that underwent chromosome segregation in medium containing different concentrations of MBC, shown as the percentage of all cells segregating. Outcomes were defined as either normal segregation, mis-segregation, or cell death. (B) Distance between each sister centromere pair in WT cells and cells with 3 μg/ml MBC; same cells as shown in Fig. 2 F. Distances are aligned to sister chromatid separation (SCS) of the respective chromosome at t = 0. Mean (line) ± SD (shaded area) of the cell population; n = number of cells. (C) Frequency distributions and Gaussian fit (continuous lines) of the time difference between the separation of centromeres 1 and 2 or centromeres 2 and 3 for cells with the klp5 gene deleted (klp5Δ). The fitted Gaussian distributions of WT cells (black) are shown for comparison. Mean ± SD of the fit; n = number of cells; P values from a two-sample Kolmogorov–Smirnov test. (D) Representative kymograph of sister chromatid separation in a klp5Δ cell with cen1-tdTomato and cen2-GFP markers, illustrating the misaligned chromosomes in metaphase. (E) Quantification of the securin degradation rates in the presence (ΔN-cycB, n = 22) or absence (WT, n = 31) of nondegradable cyclin B (Cdc13) (mean and SD). The corresponding securin degradation curves have been shown previously by Kamenz and Hauf (2014), Fig. 1 E. (F) Quantification of the securin degradation rates in the presence (ΔN-securin, n = 21) or absence (WT, n = 27) of nondegradable securin (mean and SD). The corresponding securin degradation curves are shown in Fig. 3 E. (G) Simulation of released separase (light blue) and active separase (darker blue) from the model with separase autoactivation (shown in Fig. 4 B) assuming high (solid lines) or low (dashed lines) APC/C activity. See Materials and methods for details. (H) Nuclear securin(Cut2)-GFP intensity was monitored by live-cell imaging of WT cells and cut9-665 temperature-sensitive mutants (APC/C mut). Anaphase onset was determined using the cen1-tdTomato marker, and individual time courses were aligned to this point (vertical dashed line). Mean (line) ± SD (shaded area) of the cell population; n = number of cells. (I) Nuclear securin(Cut2)-GFP intensity was monitored by live-cell imaging in WT cells grown in minimal medium, or cells additionally treated with 100 µM of the proteasome inhibitor velcade (bortezomib). Mean (line) ± SD (shaded area) of the cell population; n = number of cells. (J) Quantification of the degradation rates for the experiments shown in H and I after normalization of the data to the minimum and maximum values. Error bars represent the SD. (K) Kymograph for a velcade-treated cell with cnt2-tdTomato and cnt3-GFP markers. Arrowheads indicate sister centromere separation.