Panel A shows three line graphs comparing normalized securin-GFP concentrations over time relative to sister chromatid separation. The graphs compare different conditions: endogenous securin-GFP, overexpressed securin-GFP, and overexpressed securin-GFP with two versions of separase overexpression, either from an additional copy of separase or by changing the endogenous separase promoter. Panel B shows three line graphs comparing the non-normalized securin-GFP concentrations of the same experiments as in Panel A over time. Panel C shows two histograms with Gaussian fit lines depicting the frequency distribution of the time difference between the separation of centromeres 1 and 2 or centromeres 2 and 3 for cells with securin overexpression alone. Panel D shows two histograms with Gaussian fit lines depicting the frequency distribution of the time difference between the separation of centromeres 1 and 2 or centromeres 2 and 3 for cells with securin co-overexpression with separase. Panel E shows four line graphs depicting the distance between each sister centromere pair over time from SCS in seconds for wild-type cells and cells with securin overexpression. Panel F shows four line graphs depicting the distance between each sister centromere pair over time from SCS in seconds for wild-type cells and cells with securin and separase co-overexpression.
Co-overexpression of securin and separase does not strongly change chromosome separation synchrony. (A and B) Normalized (A) and non-normalized (B) concentration measurements of securin (Cut2)-GFP in strains expressing endogenous cut2-GFP (black) or overexpressing cut2-GFP from the endogenous locus using a mutated adh1 promoter (P.adh1*, blue). Cut2-GFP is either overexpressed alone (left panel) or co-overexpressed with separase, Cut1. For separase overexpression, either a second copy with the endogenous cut1 promoter was integrated at the leu1 locus (P.cut1, center panel) or the cut1 promoter at the endogenous locus was replaced with the ark1 promoter (P.ark1, right panel). Mean (line) ± SD (shaded area) of the cell population; n = number of cells. (C and D) 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 securin overexpression alone (C), or securin co-overexpression with separase (P.cut1-cut1) (D); same experiment as in Fig. 2 C. The fitted Gaussian distributions of WT cells (C) or cells with securin overexpression alone (D) are shown for comparison in black. Mean ± SD of the fit; n = number of cells; P values from a two-sample Kolmogorov–Smirnov test. (E and F) Distance between each sister centromere pair in WT cells and cells with securin overexpression (E) or securin and separase co-overexpression (F); same cells as shown in Fig. 2 D. 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.