Bim1–Cik1 interaction mutants rely on the conserved microtubule cross-linker Ase1/PRC1 for metaphase spindle assembly. (A) Live-cell imaging of Ase1 recruitment to Cik1 wild-type or Cik1^SKNN+4A mutant cells. Consecutive frames from live-cell movies are displayed, aligned to the time of SPB separation. Scale bar is 2 µm. (B) Fluorescence micrographs showing Ase1 recruitment to metaphase spindles. Equally intensity-scaled fluorescence micrographs are shown. Scale bar is 3 µm. (C) Line scan profile quantifying Ase1-3xGFP on metaphase spindles in Cik1-AID cells treated with auxin and containing either Cik1 wild-type or no rescue allele. (D) Quantification of Ase1-3xGFP intensity on pre-anaphase spindles under the indicated conditions. 100 cells were analyzed for each condition from two technical repeats. Open circles represent mean intensity from each individual experiment. Bars denote 95% CI. Kruskal-Wallis test was done; *, P < 0.0001. (E) Simultaneous depletion of Cik1 and Ase1 is analyzed by a serial dilution assay of the indicated strains on YP-Raff/Gal at 30°C in the absence and presence of auxin. Cdc20-AID is included as a control for the depletion of an essential protein. (F) Quantification of pre-anaphase spindle length in the indicated strains. 100 cells were analyzed for each condition from two technical repeats. Bars denote 95% CI. Kruskal-Wallis test was done; *, P < 0.0001. Difference between plus aux and Cik1^SKNN+4A was not significant. (G) Fluorescence microscopy of Cik1-AID Ase1-AID codepleted cells expressing the microtubule plus-end marker Bik1-3xGFP. Scale bar is 3 µm. Inserts are 2× magnifications of the indicated areas. Scale bar is 1 µm.