Figure 6. PP2A-B55 and MASTL determine... | Rockefeller University Press

Figure 6.

$Figure 6. PP2A-B55 and MASTL determine the CCNB1 threshold for checkpoint signaling. (A) CCNB1 was measured every 2 min in HeLa CCNB1-GFP cells exiting mitosis; see images. Timings are relative to chromosome segregation. (B) The graph shows mean CCNB1-GFP calibrated to a steady-state level of 140 nM CCNB1 in mitosis; bars indicate the SEM (n = 7). (C) Populations of control (siControl), MASTL-depleted (siMASTL), and PP2A-B55–depleted (siB55) HeLa CCNB1-GFP cells were released from a thymidine block for 10 h and then challenged with 3 µM nocodazole for 5 min and stained for MAD1. Checkpoint status and total CCNB1 levels were determined. The fraction of checkpoint-active cells was plotted for bins of 33 nM CCNB1 on the graph for n = 880, 895, or 657 cells for siControl, siB55, and siMASTL, respectively. Colored arrows mark the CCNB1 concentration, below which checkpoint activity became unstable, defined as the 95% threshold. (D) Simulation of spindle checkpoint reactivation at different levels of CCNB1 for the siControl (MASTL = 1.0 and B55 = 1.0), siMASTL (MASTL = 0.1 and B55 = 1.0), and siB55 (MASTL = 1.0 and B55 = 0.2) conditions. Checkpoint response, measured by generation of S281 phosphorylated MPS1 at kinetochores (pMPS1KT), is plotted as a function of time in the line graphs. (E) Simulation of CCNB1 stability in unperturbed mitotic exit (gray line) and after checkpoint reactivation (+Noc) for siControl, siMASTL (MASTL = 0.1 and B55 = 1.0), and siB55 (MASTL = 1.0 and B55 = 0.2) conditions. Numbers indicate CCNB1 level at the time of Noc addition and the corresponding output curve for CCNB1. Solid lines indicate conditions where CCNB1 was stabilized, i.e., the spindle checkpoint was stably active (SAC On), and dotted lines indicate that CCNB1 was destroyed, i.e., the checkpoint failed to become stably active (SAC Off).$

PP2A-B55 and MASTL determine the CCNB1 threshold for checkpoint signaling. (A) CCNB1 was measured every 2 min in HeLa CCNB1-GFP cells exiting mitosis; see images. Timings are relative to chromosome segregation. (B) The graph shows mean CCNB1-GFP calibrated to a steady-state level of 140 nM CCNB1 in mitosis; bars indicate the SEM (n = 7). (C) Populations of control (siControl), MASTL-depleted (siMASTL), and PP2A-B55–depleted (siB55) HeLa CCNB1-GFP cells were released from a thymidine block for 10 h and then challenged with 3 µM nocodazole for 5 min and stained for MAD1. Checkpoint status and total CCNB1 levels were determined. The fraction of checkpoint-active cells was plotted for bins of 33 nM CCNB1 on the graph for n = 880, 895, or 657 cells for siControl, siB55, and siMASTL, respectively. Colored arrows mark the CCNB1 concentration, below which checkpoint activity became unstable, defined as the 95% threshold. (D) Simulation of spindle checkpoint reactivation at different levels of CCNB1 for the siControl (MASTL = 1.0 and B55 = 1.0), siMASTL (MASTL = 0.1 and B55 = 1.0), and siB55 (MASTL = 1.0 and B55 = 0.2) conditions. Checkpoint response, measured by generation of S281 phosphorylated MPS1 at kinetochores (pMPS1^KT), is plotted as a function of time in the line graphs. (E) Simulation of CCNB1 stability in unperturbed mitotic exit (gray line) and after checkpoint reactivation (+Noc) for siControl, siMASTL (MASTL = 0.1 and B55 = 1.0), and siB55 (MASTL = 1.0 and B55 = 0.2) conditions. Numbers indicate CCNB1 level at the time of Noc addition and the corresponding output curve for CCNB1. Solid lines indicate conditions where CCNB1 was stabilized, i.e., the spindle checkpoint was stably active (SAC On), and dotted lines indicate that CCNB1 was destroyed, i.e., the checkpoint failed to become stably active (SAC Off).