Panel A shows histograms and Gaussian fits of time differences between centromere separations after non-degradable cyclin B expression, compared with wild-type distributions under similar conditions. Panel B shows kymographs of sister chromatid separation in cells expressing non-degradable cyclin B, comparing a cell undergoing normal nuclear division and one that is failing mitotic progression. Panel C shows a schematic model illustrating a proposed positive feedback loop where separase activity enhances securin degradation, promoting its own activation during mitosis. Panel D shows a computational model of APC/C-mediated securin degradation and separase activation, including diagrams and simulation curves comparing system behavior with and without the feedback mechanism. Panel E shows a line graph of securin-GFP degradation over time in wild-type and non-degradable securin-expressing cells, with normalized values and variability across populations. Panel F shows kymographs of cells undergoing securin-GFP degradation in wild-type and mutant cells that express an additional copy of non-degradable securin. The kymographs highlight differences in nuclear division but similarity in securin-GFP degradation.
Separase-mediated feedback on securin degradation is unlikely. (A) Frequency distribution and Gaussian fit (continuous lines) of the time difference between the separation of centromeres 1 and 2 after expression of nondegradable cyclin B (Cdc13), ΔN-cyclin B, in minimal medium. The fitted Gaussian distributions of WT cells grown under similar conditions (black) are shown for comparison. Mean ± SD of the fit; n = number of cells; P values from a two-sample Kolmogorov–Smirnov test. (B) Representative kymographs of sister chromatid separation in cells with cen1-tdTomato and cen2-GFP after expression of nondegradable cyclin B (Cdc13), ΔN-cyclin B. The left panel displays a cell that undergoes nuclear division and progresses through mitosis; the right panel displays a cell that fails to undergo nuclear division. (C) Schematic depicting a positive feedback loop, where separase accelerates its own activation by accelerating securin degradation. (D) Computational model for APC/C-mediated securin (Sec) degradation and separase (Sep) release with or without feedback of separase on securin degradation. Left side: diagram of the reactions in the model. Right side: simulation with or without feedback (dashed and solid lines, respectively) assuming a sigmoidal increase of APC/C activity with time. See Materials and methods for model details. (E) Securin-GFP degradation in WT cells (green) or in cells after induction of nondegradable securin (Cut2), ΔN-securin (gray). Individual time courses are normalized and aligned to the start of securin degradation at t = 0. Mean (line) ± SD (shaded area) of the cell population; n = number of cells. (F) Representative kymographs of securin-GFP degradation in a WT cell (left panel) and in a cell after induction of untagged nondegradable securin (Cut2), ΔN-securin. In the latter cell, the nucleus fails to divide.