The EB1-Kinesin-14 complex is required for efficient metaphase spindle assembly and kinetochore bi-orientation.

Kinesin-14s are conserved molecular motors required for high-fidelity chromosome segregation, but their specific contributions to spindle function have not been fully defined. Here, we show that key functions of budding yeast Kinesin-14 Cik1-Kar3 are accomplished in a complex with Bim1 (yeast EB1). Genetic complementation of mitotic phenotypes identifies a novel KLTF peptide motif in the Cik1 N-terminus. We show that this motif is one element of a tripartite binding interface required to form a high-affinity Bim1-Cik1-Kar3 complex. Lack of Bim1-binding by Cik1-Kar3 delays cells in mitosis and impairs microtubule bundle organization and dynamics. Conversely, constitutive targeting of Cik1-Kar3 to microtubule plus ends induces the formation of nuclear microtubule bundles. Cells lacking the Bim1-Cik1-Kar3 complex rely on the conserved microtubule bundler Ase1/PRC1 for metaphase spindle organization, and simultaneous loss of plus-end targeted Kar3 and Ase1 is lethal. Our results reveal the contributions of an EB1-Kinesin-14 complex for spindle formation as a prerequisite for efficient kinetochore clustering and bi-orientation.