Additional characterization of Cik1 mutants. (A) Bim1 binding is required for an efficient association of Cik1-Kar3 with microtubule plus ends. Representative images show Kar3-3xGFP signals over different cell cycle stages in indicated Cik1 mutants. Cik1^SKNN-Kar3 was still able to follow microtubule plus ends and bind to the metaphase spindle. The KLTF motif mutation abrogated plus-end localization. Scale bar is 2 µm. (B) Western blot analysis of Flag-tagged Cik1 rescue alleles. (C) Overlay of 15 line scans along the shmoo tip microtubule bundle of Kar3-3xGFP in Cik1^wt and cik1^4A genetic backgrounds. The KLTF motif mutation leads to a fourfold decrease in recruitment to the microtubule bundle. (D) Residual colocalization between Kar3-3xGFP and Stu2-mRuby2 in the Cik1-4A mutant. Microscopy images of two consecutive frames of α-factor–arrested cells are shown. Scale bar in panels 1–4 are 1 µm; scale bar for magnification is 0.5 µm. (E) Analytical SEC experiment showing that the Cik1-4A mutation in the KLTF motif prevents stable association between Cik1-Kar3 and Bim1. (F) Purification and subsequent Bim1 pull-down experiment with Cik1–Kar3 complexes containing either Cik1 wild type, Cik1-4A mutant, Cik1-SKAA mutant, or Cik1-SKAA+4A mutant. Note that only Cik1-SKAA+4A fully eliminates Bim1 binding in the pull-down assay. (G) Analysis of shmoo tip bundle dynamics in Kar3^ND2 cells. Kar3-AID cells expressing the Kar3^ND2 mutant and the plus-end markers Bik1-3xGFP and Stu2-mRuby2 were imaged in α-factor–arrested cells in the absence (control) and presence of auxin. Parameters of MT bundle dynamics are shown on the right; deviations from wild-type parameters are highlighted in red.