Fig 2: Cyclin B1 scaffolds MAD1 at the corona to allow the SAC to tolerate MPS1 inhibition Immunofluorescence images of MAD1 and MAD1-pT716 kinetochore levels in nocodazole-arrested RPE1 cells treated with or without AZ-3146. Scale bars = 5 µM.Electron micrographs of rotary-shadowed MAD193–718:MAD2 (top row), MBP-MAD1:MAD2 (middle row) and MBP-MAD1:MAD2 in complex with GST-CDK1:Cyclin B1 (bottom row). Scale bar = 50 nm.Quantifications (top) and corresponding immunofluorescence images (underneath) of relative kinetochore MAD1 and MAD1-pT716 levels in nocodazole-arrested of MAD1-WT-C14 and MAD1-3EK-C12 cells treated with different doses of AZ-3146 for 30 min. MG132 was included at the time of AZ-3146 addition to prevent mitotic exit. Each dot represents a cell, horizontal lines indicate the median and error bars show the 95% confidence interval. Note, when these vertical bars do not overlap, the difference is considered statistically significant at a level of at least P < 0.05 (see Materials and Methods). Thirty cells from 3 experiments. Both kinetochore intensity graphs display data that is relative to the WT-C14 untreated controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines.Duration of mitotic arrest in MAD1-WT-C14 or MAD1-3EK-C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ-3146. Note, the 1.25 µM AZ-3146 data are displayed in Fig 4D, but also included here to allow comparison with other drug doses. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.

Fig 3: Inhibiting Cyclin B1:MAD1 interaction weakens the SAC A–CImmunofluorescence images (A) and quantifications (B, C) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with/without AZ-3146 for 30 min to inhibit MPS1. In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median, and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap, the difference is considered statistically significant at a level of at least P < 0.05 (see Materials and Methods). All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. Thirty cells from 3 experiments. Scale bars = 5 µM.DDuration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1.25 µM AZ-3146. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.

Fig 4: Cyclin B1 and MAD1 interact directly through an N-terminal acidic patch on MAD1 Elution profiles and SDS–PAGE for SEC runs on the indicated column of the Cyclin B1:CDK1 complex (blue profile), MBP-MAD1:MAD2 (red) and their combination (green). Note that the same Cyclin B1:CDK1 elution profile and SDS–PAGE are also displayed as reference in panels (D and F) to improve clarity. For the same reason, the MBP-MAD1:MAD2 elution profile and SDS–PAGE are also displayed in panels (A–C).Elution profiles and SDS–PAGE for SEC runs on the indicated column of CDK1 (blue), MBP-MAD1:MAD2 (red) and their combination (green).Elution profiles and SDS–PAGE for SEC runs on the indicated column of Cyclin B1 (blue), MBP-MAD1:MAD2 (red) and their combination (green).Elution profiles and SDS–PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD1?93:MAD2 (green) and their combination (red).Elution profiles and SDS–PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD11–92-SNAP (red) and their combination (green).Elution profiles and SDS–PAGE for SEC runs of Cyclin B1:CDK1 (blue), MBP-MAD13EK:MAD2 (E52K, E53K and E56K mutations; red) and their combination (green). In (A-D), individual potential binding partners were combined at a concentration of 5 µM.Alignment of the N-terminal region of Cyclin B1 that contains the MAD1-binding region. Numbering refers to the human MAD1 sequence. *conserved, negatively charged residues in MAD1 (E52K, E53K and E56K) required for MAD1:Cyclin B1 interaction.