dependent modification of the Dam1 C-terminus regulates the interaction with Bim1 A: SDS-PAGE analysis of elution fractions from analytical size exclusion chromatography.Figure 4A: SDS-PAGE analysis of elution fractions from analytical size exclusion chromatography. Binding of Bim1 to Dam1WTc and Dam1-19c was analyzed by size exclusion chromatography. The gel filtration buffer contained 400 mM NaCl. B: Representative images from live cell microscopy of Dam1WT and dam1-19 strains with Dad1-GFP. Displayed cells are in metaphase (left) or anaphase (right). Scale bar: 2 μm. C: Quantification of Dad1-GFP signal intensities at metaphase and anaphase kinetochore clusters of Dam1WT and dam1-19 strains. Bars represent the mean +/- standard deviation. Each spot represents the value measured for a single kinetochore cluster. Values were normalized to the mean value of metaphase clusters of the Dam1WT strain. p-values were obtained by a one-way ANOVA test with Sidak's multiple comparisons test. n ≥ 86 kinetochore clusters. D: Schematic representation of the Dam1 protein. Previously characterized Ipl1 and Mps1 phosphorylation sites are annotated. Dam1‑19 is truncated by a Q205Stop point mutation. The C-terminus of Dam1 including several phosphorylation sites framed by the dashed box is missing in dam1‑19. E, F: GST-Bim1185-344 pull down assays. Binding of immobilized GST-Bim1185-344 to Duo1-6xFlag was analyzed in context of (E) selective inhibition of Mps1 (mps1-737) or overexpression of Mps1 from a galactose-inducible promotor (F). For inhibition of Mps1, cells were grown at the restrictive temperature of 37 °C. Overexpression of Mps1 was achieved by growing cells in medium containing galactose. GST-Bim1185-344 immobilized on glutathione sepharose beads were incubated with soluble cells lysates of the respective strains grown under the mentioned conditions. Binding of Duo1 was analyzed by western blot. Pgk1 indicates equal protein amounts in all samples. G: Live cell microscopy of a Dad1-GFP strain under conditions of Mps1 overexpression. The localization of Dam1c was visualized by Dad1-GFP in either an Mps1WT or Mps1 overexpression setup (pGal-Mps1). Ndc80-mRuby2 was used as reference point for the localization of kinetochores. Images show representative metaphase cells from both strains. The area in the white dashed box is shown as magnification in the lower left corner. White arrowheads point at nuclear microtubules decorated by Dad1-GFP. Please note that brightness and contrast of the Dad1-GFP image in the lower row were optimized to make the nuclear microtubules clearly visible. Scale bar: 2 μm. H: Representative line scans of Dad1-GFP and Ndc80-mRuby2 signal intensities along the mitotic spindle. Displayed data was acquired from the cells shown in G(labelled with white dashed boxes). I: Quantification of Dad1-GFP cluster shape in Mps1WT and pGal-Mps1 strains. Bi-lobed cluster shape with clearly separated Dad1-GFP foci was distinguished from bar-shaped cluster without clear separation. The mean +/- standard deviation from three independent experiments are shown. p-values were calculated with a two-way ANOVA test with Sidak's test for multiple comparisons.