Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces

Abstract

The macromolecular kinetochore functions to generate interactions between chromosomal DNA and spindle microtubules [1]. To facilitate chromosome movement and segregation, kinetochores must maintain associations with both growing and shrinking microtubule ends. It is critical to define the proteins and their properties that allow kinetochores to associate with dynamic microtubules. The kinetochore-localized human Ska1 complex binds to microtubules and tracks with depolymerizing microtubule ends [2]. We now demonstrate that the Ska1 complex also autonomously tracks with growing microtubule ends in vitro, a key property that would allow this complex to act at kinetochores to mediate persistent associations with dynamic microtubules. To define the basis for Ska1 complex interactions with dynamic microtubules, we investigated the tubulin-binding properties of the Ska1 microtubule binding domain. In addition to binding to the microtubule lattice and dolastatin-induced protofilament-like structures, we demonstrate that the Ska1 microtubule binding domain can associate with soluble tubulin heterodimers and promote assembly of oligomeric ring-like tubulin structures. We generated mutations on distinct surfaces of the Ska1 microtubule binding domain that disrupt binding to soluble tubulin but do not prevent microtubule binding. These mutants display compromised microtubule tracking activity in vitro and result in defective chromosome alignment and mitotic progression in cells using a CRISPR/Cas9-based replacement assay. Our work supports a model in which multiple surfaces of Ska1 interact with diverse tubulin substrates to associate with dynamic microtubule polymers and facilitate optimal chromosome segregation. Monda and Whitney et al. demonstrate that the kinetochore-localized Ska1 complex autonomously tracks with both growing and depolymerizing microtubule ends in vitro and that this activity requires multiple microtubule binding surfaces. Ska1 mutants that bind microtubules, but are unable to tip track, result in defective mitotic progression in cells. Keywords: mitosis; kinetochore; microtubule; tubulin; chromosome segregation