Fig 2: Kinetochore phosphatases PP1 and PP2A-B56 antagonize PLK1 recruitment to the BUB complex. (A–C) Effect of phosphatase-binding mutants on levels of PLK1 (A), BUBR1-pT620 (B), and BUB1-pT609 (C) at unattached kinetochores in nocodazole-arrested cells. Mean kinetochore intensities from 30–50 cells, three to five experiments. (D and E) Effect of mutating the PLK1 binding site on BUBR1 (pT620) alone (D) or BUBR1 (pT620) and BUB1 (pT609; E) on PLK1 kinetochore levels in nocodazole-arrested BUBR1WT/ΔPP2A cells. Mean kinetochore intensities from 40 cells per condition, four experiments. All values in E are normalized to BUBR1-WT+BUB1-WT control. For all kinetochore intensity graphs, each dot represents a cell, and the error bars display the variation between the experimental repeats (displayed as ±SD of the experimental means). Two-tailed, nonparametric Mann-Whitney unpaired t tests were performed to compare the mean values between experimental groups. Example immunofluorescence images were chosen that most closely resemble the mean values in the quantifications. The insets show magnifications of the outlined regions. Scale bars, 5 µm. Inset size, 1.5 µm. ****, P < 0.0001; ns, nonsignificant.

Fig 3: PLK1 inhibition or removal from BUB complex restores SAC silencing in the phosphatase-binding mutants (related to Fig. 2). (A) Example immunofluorescence images of the kinetochore quantifications shown in Fig. 2 A. (B) Representative images and quantification of KNL1 (BUBR1WT/ΔPP2A cells) or YFP-KNL1 (KNL1WT/ΔPP1 cells) after MPS1 inhibition with AZ-3146 (2.5 µM), as in Fig. 2 A. Graph represents 40 cells from four experiments. (C) Representative immunofluorescence images of quantifications shown in Fig. 2 C. (D) Representative images and quantification of KNL1 (BUBR1 cells) or YFP-KNL1 (KNL1 cells) after MPS1 inhibition with AZ-3146 (2.5 µM) in combination with the PLK1 inhibitor BI-2536 (100 nM), as in Fig. 2 C. Graph represents 30 cells from three experiments. (E and F) Example immunofluorescence images of the kinetochore quantifications shown in Fig. 2 E (E) and Fig. 2 G (F). (G and H) Quantification of KNL1 levels (G) and representative images (H) of BUBR1/BUB1 Polo-box–binding mutants treated with MPS1 inhibitor (2.5 µM AZ-3146), as in Fig. 2 G. Graph shows median of 40 cells per condition from four experiments. (I) Effect of mutating both PLK1 binding sites BUBR1 (pT620) and BUB1 (pT609) on mitotic exit in nocodazole-arrested cells, treated with MPS1 inhibitor AZ-3146 (2.5 µM). Graph shows the means (±SEM) of 150–200 cells from three or four experiments. The images were chosen that most closely resemble the mean values in the quantifications. The insets show magnifications of the outlined regions. Scale bars, 5 µm. Inset size, 1.5 µm.

Fig 4: Cohesion defects do not accelerate cohesion fatigue in BUB1 patient cells.(A) Cohesion defect analysis of indicated cell lines. Per experiment, 50 metaphases were analyzed for each sample. Multiple independent experiments are shown as separate bars. Aph, aphidicolin (16 hours, 1 μM). P values were calculated using a Cochran-Mantel-Haenszel statistical test to compare the frequency of total cohesion defects per condition. (B) Cells were treated with 20 μM proTAME and 100 μM apcin, and the time from NEB to the moment at which single chromatids start escaping the metaphase plate (cohesion fatigue) was assessed by live-cell imaging. Each dot represents one cell. n ≥ 80 from at least three independent experiments. Statistical analysis was performed using one-way ANOVA.

Fig 5: Biallelic germline mutations in BUB1 cause reduced protein levels.(A) Family pedigrees (based on WES) and Sanger confirmation from primary fibroblasts. Nomenclature NM_004336.5. (B) P2 primary fibroblast cDNA was PCR-amplified using indicated primers (red arrows) and analyzed on agarose gel and by Sanger sequencing. The transcript lacks exon 21. Predicted effect on BUB1 protein is shown. (C) BUB1 mRNA was assessed in indicated cells using three different primer pairs, each in three technical replicates. (D and E) Western blot of primary fibroblasts (D) (using A300-386A to detect BUB1) and SV40-transformed fibroblasts (E) (using ab9000). CDC6 is a control for marizomib-induced protein stabilization (6 hours, 500 nM). (F) Western blot of engineered RPE1-BUB1 mutant cells and complete BUB1 KO HAP1 cells using ab9000. (G) Colchicine-arrested cells (10 μM, 2 hours) were stained for CENPC, BUB1 (using A300-386A), and DNA. Graph depicts mean BUB1 intensity within CENPC-defined regions. n = 83, 73, and 72 cells from three independent experiments. (H) Colchicine-arrested cells, treated with BUB1 kinase inhibitor BAY1816032 (4 μM, 1 hour) or dimethyl sulfoxide (DMSO), were stained, and mean pH2A-T120 intensity was assessed within DAPI-defined regions. Approximately 90 cells from three independent experiments were analyzed per condition. Each dot represents one cell; black lines indicate the means. Scale bars, 5 μm. Nonparametric Kruskal-Wallis test (Dunn’s multiple comparison test) used for statistical analysis. AU, arbitrary units; aa, amino acid.