Functional analysis of the EB1-Kinesin-14 complex in chromosome segregation

Self-copying is the goal of every organism’s life. For some species, like budding yeasts, a single cell constitutes a whole organism. For every cell division the genetic material must be fully duplicated and properly segregated between inheritors. A complex microtubule-based machinery mediates faithful genome inheritance. In this system, microtubule-associated proteins drive the assembly of a supramolecular structure that constitutes the mitotic spindle.

Kinesin-14 molecular motor proteins are known to be required for error-free chromosome segregation in mitosis and meiosis and display an exceptional evolutionary conservation. A variety of functions have been assigned to kinesin-14 family members, but it is unclear how these activities contribute to chromosome segregation. The models for kinesin-14’s mode of action fall in the range from support of lateral chromosome transport along microtubules to the regulation of microtubule nucleation at MTOCs. One of the limitations that prevents a deeper characterization of kinesin-14 functions in chromosome segregation is a lack of molecular understanding outside of the well-characterized motor domain. Budding yeasts have a single catalytically active kinesin-14, termed Kar3, which performs mitotic functions in a complex with the kinesin-associated protein Cik1. Using a conditional depletion system and an unbiased mutant screening approach, I have discovered a tripartite binding interface between kinesin-14 and the EB1 homolog Bim1 in budding yeast. The “A-B-C” Bim1-binding motif system is distributed between the Cik1 and Kar3 polypeptide chains and consists of conserved peptide motifs. The resulting Bim1-Cik1-Kar3 complex regulates the assembly of a metaphase spindle of proper length, and promotes microtubule bundle organization and dynamics. Lack of Bim1 binding delays cells in mitosis similar to the absence of Cik1-Kar3 proteins. Artificial plus-end targeting of Cik1-Kar3 bypasses the requirements for Bim1 binding and is sufficient to promote bundle formation in cells. In the absence of Bim1-Cik1-Kar3 cells rely on the conserved microtubule crosslinker Ase1/PRC1 for metaphase spindle assembly and chromosome bi-orientation. Simultaneous loss of plus-end targeted Kar3 and Ase1 is lethal. In summary, I have identified a molecular binding interface between Bim1 and Cik1-Kar3 as a prerequisite for error-free chromosome segregation.


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