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

Abstract

Summary 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.

Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

“…These so-called end-on attachments persist during polymerization and depolymerization of the dynamic ends of microtubules, and couple pulling forces produced by depolymerizing microtubules to chromosome movement (Akiyoshi et al, 2010;Grishchuk et al, 2005;Miller et al, 2016;Powers et al, 2009;Volkov et al, 2018). Furthermore, kinetochores control the dynamics of the plus ends, likely by balancing the action of MCAK (kinesin-13, a microtubule depolymerase) and Kif18 (kinesin 8, a microtubule stabilizer) and possibly other plus end-associated proteins Monda et al, 2017).…”

Multitasking Ska in Chromosome Segregation: Its Distinct Pools Might Specify Various Functions

“…Such biochemical property of the Ska complex might allow it to harness the force generated by the peeling protofilaments to track on the depolymerizing microtubules. This microtubule end‐tracking capacity has recently been demonstrated to require diverse tubulin‐interacting surfaces of Ska1 . Interestingly, it has also been shown that the Ska complex confers this microtubule end‐tracking capacity to the Ndc80 complex in vitro .…”

Mapping the kinetochore MAP functions required for stabilizing microtubule attachments to chromosomes during metaphase

“…Several plus‐end tip‐tracking proteins that associate with elongating MTs through the end‐binding (EB) family of proteins can simply be considered as “hitchhiker” molecules and not “autonomous” tip trackers. However, in vitro analyses indicate that the Ska1 complex is capable of tracking polymerizing MTs independent of EB1 (Monda et al, ). Monda et al observed bright GFP‐Ska fluorescence at the growing MT ends (Figure a2).…”

“…Monda et al observed bright GFP‐Ska fluorescence at the growing MT ends (Figure a2). In addition, when MT depolymerization was induced by the removal of soluble tubulin, the Ska1 complex was also enriched at the shortening MT ends (Monda et al, ). Thus, the Ska complex exhibits the remarkable ability to remain associated with both the polymerizing and depolymerizing MT plus‐ends, a property that has thus far been reported only for one other human kinetochore MAP, the CENP‐E motor (Gudimchuk et al, ).…”