Bim1–Cik1 interaction mutants display severe mitotic phenotypes. (A) Scheme of the constructed Bim1–Cik1 interface mutants. Multiple sequence alignment reveals the presence of a conserved canonical EB1 binding motif (SxIP) and the novel KLTF motif. The constructed Cik1 mutants are shown below the alignment. (B) Serial dilution assay testing viability of different Cik1 mutants in the Cik1-AID mad1Δ background. (C) Heterozygous diploid dissection of different Cik1 alleles in a mad1Δ background. The four spores from an individual tetrad are shown in the respective columns; the indicated gentoypes are marked by circles. Combined mutations in SKIP and KLTF motifs result in a severe growth defect. (D) Budding index assay of Cik1-depleted strains expressing the indicated rescue constructs. Log-phase cells were treated with auxin for 3 h. The percentage of the respective cell cycle stage is indicated in white on the graph. Average means of three independent experiments are shown. (E) Live-cell microscopy analysis of metaphase duration in Cik1-AID strains expressing a wild-type Cik1 or the Cik1^SKNN+4A allele. Galleries show consecutive frames from live-cell imaging, aligned at the time of SPB separation. Scale bar is 2 µm. Quantification of metaphase duration is from 200 individual cells (two technical repeats). Open circles represent mean duration from each individual experiment. Bars show mean values and 95% CI. χ² test was applied; *, P < 0.0001. (F) Analysis of spindle size and dynamics in Cik1-depleted cells expressing either Cik1 wild-type or Cik1^SKNN+4A. Left panel: Gallery of consecutive frames from live-cell imaging. Middle and right panels: Quantification of spindle elongation for n = 15 cells. Error bars are means with 95% CIs. Student’s two-tailed t tests were made (*, P < 0.0001; NS, P = 0.14).