Fig 2: The contribution of Bub1-BubR1 heterodimerization to MCC assembly in eSAC signaling. (A) Schematic at the top displays the domain organization of human Knl1. Below is a simplified schematic displaying the pathways that recruit SAC signaling proteins to unattached kinetochores. (B) Schematic at the top displays the domain organization of human Bub1. The cartoon represents the eSAC system. (C) Dose-response data for an eSAC system that uses Bub1444-620 as the phosphodomains. Each gray circle represents a single cell (n = 1920 from two technical replicates; 139 and 155 are the lower and upper 95% confidence intervals). The blue squares represent the mean values of the dose-response data binned according to mCherry fluorescence. Vertical and horizontal lines represent SEM. The solid blue curve displays the least squares four-parameter sigmoidal fitted to the binned mean values. Hill equation fits for dose-response data for one MELT motif (MELT12, dashed red curve) and an extended Bub1231-620 phosphodomain containing the Bub3- and BubR1-binding sites (dashed blue curve) from our previous study have been superimposed for comparison (Chen et al., 2019). Note that these and the Bub1444-620 phosphodomain data were obtained using identical imaging conditions to enable their direct comparison. The bar graph on the right displays the maximal mitotic duration estimated by the fit. Error bars represent 95% confidence intervals of the fitted maximal mitotic duration. (D) Cartoon displays the simplified scheme used to simulate the generation of MCC by the eSAC system. (E) Numerical simulation of the dose-response curves by assuming the assembly of signaling complexes and ultimately the MCC as shown in D followed by a model described by He et al. (2011).

Fig 3: TAO1-depleted cells display mis-segregating lagging chromosomes. (a–c) Time-lapse analysis of HeLaHis2B-GFP;CherryTub cells transfected with TAO1-St1 or Control-St siRNA. (a) Successive frames of fluorescent images from representative live-cell movies of HeLaH2BGFP;CherryTub cells treated with siRNA as indicated. Images were acquired once every 2 min. Uncongressed chromosomes are marked with blue arrowheads and mis-segregating lagging chromosomes are marked with yellow arrowheads. Scale bar, 20 µm. (b) Cumulative distribution graph showing the time consumed from NEBD to congression of chromosomes (alignment of last chromosome prior to anaphase) in cells treated with TAO1-St1 or Control-St siRNA. T50 represents chromosome congression time in 50% of mitotic cells. (c) Graph showing percentage of mitotic cells displaying normal, defective or no segregation within the first 3 h of mitosis. (d) Immunoblots of lysates of cells treated with siRNA as indicated showing the levels of Mad2, BubR1, Mad1, MPS1, TAO1, Aurora-B, Bub3 or ?-tubulin. ?-Tubulin is used as a loading control. (e) Graph showing percentage of mitotic cells displaying normal or uncongressed metaphase chromosomes and normal or mis-segregated anaphase chromosomes following a 1 h treatment with varying concentrations of 2ME2, as indicated. n = number of cells from three independent experiments. Error bars are s.e.m. values across three experimental repeats. p-Values representing significance level were derived using the proportion test. Asterisks (*) indicate significant difference.

Fig 4: Quantification of the relative abundances of the eSAC phosphodomain, mNeonGreen-Bub1, mNeonGreen-BubR1, and Mad1-mNeonGreen in genome-edited HeLa cell lines. (A) Immunoblots showing that roughly half of BubR1 and Mad1 (left) and Bub1 (middle) proteins in the three partially genome-edited cell lines is tagged with mNeonGreen (asterisks on the right of each displayed blot mark the mNG fusion protein). Right: Immunoblot of whole-cell extracts of the three cell lines probed with anti-mNeonGreen antibodies. This experiment was performed once. (B) Average mNG-Bub1, mNG-BubR1, Mad1-mNG, and M4-KI1-KI2-mNG-2xFkbp12 signals from mitotic HeLa cells (left) and estimation of the relative protein abundance, assuming that the total protein abundance is twice as high as the abundance of the mNG-labeled species (mean ± SD; n = 50 each for mNG-Bub1, mNG-BubR1, and Mad1-mNG, n = 27 for M4-KI-mNG-2xFkbp12).

Fig 5: Characterization of the binding of the KI motifs in the eSAC phosphodomain with Bub1 and BubR1 and its contribution to eSAC signaling. (A) Left: Schematic of the two phosphodomains used to test whether the KI motifs contribute to MCC assembly mediated by the MELT motif in the eSAC phosphodomain. KI1-KI2 indicates phosphodomain with intact KI motifs; KI1*-KI2* indicates phosphodomains wherein the KI motifs are inactivated using suitable point mutations (see Materials and Methods for details). The scatter plot in the middle displays the dose-response data for the two phosphodomains (n = 1888 for KI1-KI2 [green] and n = 836 for KI1*-KI2* [magenta] from =2 technical replicates; symbol usage follows the scheme established in Figure 1C). The bar graph on the right displays the maximal mitotic duration predicted by four-parameter sigmoidal fits to the binned mean values as in Figure 1C. Vertical lines display 95% confidence intervals on the fit parameter. (B) Left: Schematic of the phosphodomains consisting of four MELT motifs and either active or inactive (indicated by *) KI motifs. Right: Immunoprecipitation of the eSAC phosphodomains using mNeonGreen-Trap beads followed by immunoblot analysis to probe for the coimmunoprecipitate of the indicated proteins. This experiment was performed once. (C) Dose-response data for the indicated phosphodomains. Only the mean values of binned data are shown for clarity. Data analysis performed as in Figure 1D (n = 1019, 1024, 666, and 3219 from =2 technical replicates for KI1-KI2, KI1-KI2*, KI1*-K2, and KI1*-KI2*, respectively). The bar graph on the right displays the maximal time in mitosis of the fitted maximal mitotic duration (error bars represent 95% confidence intervals).