Fig 2: MAD1 is the kinetochore receptor for CCNB1. (A and B) CCNB1-mCherry (CCNB1-mCh) was immunoprecipitated (IP) with anti-mCherry antibodies from CCNB1-mCh or parental HeLa cells arrested in mitosis. Coprecipitating proteins were analyzed by mass spectrometry in three independent experiments (A) or by Western blotting (B). Intensities of proteins identified by mass spectrometry in all experiments were plotted against each other. Nonspecific components of the IPs are found equally in both samples and therefore align along the diagonal. Specific components of CCNB1 complexes cluster along the y-axis of the plot. (C) Cyclin IPs were performed from HEK293T cells cotransfected with full-length mCh-MAD1 and Myc-CCNA2, CCNB1 or CCNB2 constructs indicated. Myc IPs were Western blotted for Myc to detect precipitated cyclins, for CDK1, and for mCh to detect coprecipitated MAD1. Asterisks mark the antibody light chain detected in the CDK1 blots for IP samples. (D) Prometaphase localization of CCNB1 in control (siControl) and MAD1 (siMAD1) depleted HeLa CCNB1-GFP cells arrested with 20 µM MG132 for 30 min, then stained for MAD1 and kinetochores (CREST). Kinetochore fluorescence of MAD1 and CCNB1 normalized to the respective mean signal in control cells and SEM are plotted in the bar graphs (15 kinetochores per cell and 12 cells in each of three independent experiments). (E) Prometaphase localization of MPS1 in control (siControl) and MAD1 (siMAD1) depleted HeLa MPS1-mCherry cells stained for MAD1 and kinetochores (CREST). Kinetochore fluorescence of MAD1 and MPS1 normalized to the respective mean signal in control cells and SEM are plotted in the bar graphs (n = 19, P value for MPS1-mCherry < 0.0001, Student’s t test).

Fig 3: Depletion of Septin 9 results in abnormal connection between chromosome kinetochores and spindle microtubules and sustained spindle assembly checkpoint (SAC) activation. (A) Chromosome spread showing the activation of SAC-Mad1 in Sept 9 siRNA at 9.5 h. SAC-Mad1 and DNA were stained with Mad1 antibody (Red) and DAPI (Blue), respectively. Scale bar: 5 µm. The bargraph represents the relative immunofluorescence intensity of Mad1 quantified in NC and Sept 9 siRNA oocytes at 6 and 9.5 h following release from IBMX. (B) Chromosome spread showing the activation of SAC-BubR1 in Sept 9 siRNA at 9.5 h. SAC-BubR1 and DNA were stained with BubR1 antibody (Green) and DAPI (Blue), respectively. Scale bar: 5 µm. The bargraph represents the relative immunofluorescence intensity of BubR1 quantified in NC and Sept 9 siRNA oocytes at 6 and 9.5 h following release from IBMX. (C) The percentage of PBE of NC and Sept 9 siRNA oocytes after release from IBMX were incubated in M2 media containing 0.5 µM REVERSINE. (D) Confocal microscopy showing the spindle structures and distribution of chromosomes in Sept 9 siRNA at 8 h after cold treatment. Spindle and DNA were stained with a-tubulin-FITC antibody and DAPI, respectively. Scale bar: 5 µm. Enlarged panels show high magnification views of the boxed areas. Arrows indicate the kinetochores. Scale bar: 1 µm (enlarged panels). The bargraph represents the quantitative analysis of k-MT attachments in NC and Sept 9 siRNA oocytes. ACA, anti-centromeric antibodies. **p < 0.01. Data are mean ± sem. All of the experiments were repeated at least three times, and representative results are shown.

Fig 4: PP2A-B55 opposes CCNB1 recruitment to kinetochores. (A) Checkpoint signaling and CCNB1 localization were followed in control (siControl) and PP2A-B55 (siB55) depleted HeLa CCNB1-GFP cells. Cells were arrested for 2.5 h with 20 µM MG132, and then either fixed immediately (+MG132) or treated with 3 µM nocodazole for 5 min (+Noc) or with 5 µM flavopiridol for 1 min followed by addition of 3 µM nocodazole for 5 min (+CDK-i +Noc). MAD1 and kinetochores (CREST) were detected using antibodies, and CCNB1 using GFP fluorescence. (B) Mean kinetochore intensity ± SEM of CCNB1 and MAD1 in control (siCon) and B55-depleted (siB55) cells are plotted (15 kinetochores per cell for =5 cells in each of three independent experiments). (C) A schematic of the checkpoint response assay. (D) Control (siControl) and PP2A-B55 (siB55) depleted HeLa cells arrested in mitosis for 3 h with 100 µM monastrol were washed into fresh growth medium for 25 min to allow spindle formation. At that point, 0 min, or after a further 10 or 20 min, cells were challenged with 3 µM nocodazole for 5 min to test for the checkpoint response, fixed, and then stained for MAD1 and tubulin. (E) Graphs show the fraction of checkpoint silenced cells at different times after monastrol washout, before nocodazole addition. Bars indicate the SEM (for siControl: 0/10/20 min n = 263/293/277 and for siB55: n = 296/294/277). (F) Graphs show the fraction of checkpoint active cells with unseparated sister chromatids (for siControl: 0/10/20min n = 232/302/297 and for siB55: n = 277/257/306) and MAD1 signal at kinetochores (for siControl: 0/10/20min n = 64/48/65 and for siB55: n = 46/62/49); error bars indicate the SEM. (G) CCNB1 and MPS1 localization are shown for the 20-min time point challenged with 3 µm nocodazole (+Noc) in control (siCon) or PP2A-B55 (siB55) depleted HeLa CCNB1-GFP/MPS1-mCherry cells. MPS1 intensity is plotted relative to the cytoplasmic CCNB1 signal for individual kinetochores with the mean and SD (15 kinetochores per cell in 26 [siCon] or 27 [siB55] cells).

Fig 5: 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).