Figure 3. A gradient of Mps1 kinase activity continues to phosphorylate MELT motifs after chromosome biorientation.

(A) Top left: Cartoons show schematics of the two Spc105 phosphodomains tethered to kinetochore subunits using the rapamycin-induced dimerization of Fkbp12 and Frb domains. Top right: The kinetochore subunits (Spc34-C, Ndc80-C, Spc24-C and Mtw1-C) at which, the phosphodomains were tethered by rapamycin induced dimerization. Bottom: Micrographs show representative images of cycling cells wherein the phosphodomain is tethered to the C-terminus of the indicated kinetochore subunit. (n.a. - Not Applicable). (B) Left: The bar plot shows the scoring of cells with bioriented kinetochore clusters based on whether or not they visibly recruit Bub3-mCherry. (n = 71 for Spc34-C, 109 and 94 for Ndc80-C, 95 and 103 for Spc24-C, 106 and 122 for Mtw1-C, accumulated from two experimental and biological repeats (whenever possible). Right: The scatter plot on the right shows the quantification of Bub3-mCherry fluorescence from all cells with bioriented kinetochore clusters. (mean+ s.e.m. n = 47 for Spc34-C, 159 and 153 for Ndc80-C, 114 and 154 for Spc24-C, 173 and 104 for Mtw1-C, accumulated from two experimental and biological repeats wherever possible). n.s. Not significant. (C) Micrographs show the recruitment of Bub3-mCherry by the phosphosensor tethered at the Ndc80 C-terminus in cycling (before CDC20 repression) and metaphase arrested cells (after CDC20 repression). Scatterplots show the relative fluorescence signal from the GFP-tagged phosphosensor and Bub3-mCherry respectively. (mean+ s.e.m. n = 52 and 45 for rapamycin treated samples before and after Cdc20 depletion from two technical replicates). n.s. Not significant. (D) Bar graph shows fraction of cells visibly recruiting Mad1-mCherry as shown in the micrographs (n = 242 and 142 for Ask1-C and Ndc80-C respectively).

Figure 3—figure supplement 1. The distribution of the Spc105 phosphodomain in bioriented kinetochores and the position-dependent effects of tethering an additional phosphodomain within the kinetochore.

(A) The assessment of the nanoscale distribution of the unstructured phosphodomain of Spc105 using FRET. Top: Schematic displays the GFP-insertion positions within Spc105. Middle: Positions of the mCherry tagged kinetochore subunits (Dad4-C and Spc25-C) that demarcate the region that was expected to be occupied by the Spc105 phosphodomain. Bottom: Scatter plots show the proximity ratio (which is proportional to FRET) of donor GFP fused at the indicated residue in Spc105 with Spc25 C-terminus (left) and Dad4 C-terminus (right). (mean+ s.e.m. n = 74, 165, 142, 177 and 103 respectively for FRET involving Spc25-mCherry, accumulated from at least three repeats. n = 114, 134 and 224 for N, 222 and 455 respectively from three repeats for FRET involving Dad4-mCherry, accumulated from at least two repeats). n.s. Not significant. (B) Flow cytometry-based quantification of the DNA content in cells treated with rapamycin (prior to sample collection at 0 min) to tether the Spc105 phosphodomain anchored to Ask1-2xFkpb12 (which is proximal to the CH-domains) or Ndc80-2xFkbp12 (which is distal from the CH-domains) (schematic shown at the top). Bottom: Representative histogram from two trials for each strain is shown. (C) Quantification of amount of Spc105 phosphodomain tethered to bioriented kinetochore clusters (mean+ s.e.m. n = 74 and 80 for Ndc80-C, 78 and 76 for Spc24-C, 102 and 173 for Mtw1-C, pooled from at least two experimental replicates). (D) (Supports in Figure 4C) Quantification of population growth for the indicated strains in benomyl-containing media (30 µg/ml) (mean ± s.e.m., n = at least 3).