Kinesin-14 family proteins are minus-end directed motors that cross-link microtubules and play key roles during spindle assembly. We showed previously that the Xenopus Kinesin-14 XCTK2 is regulated by Ran via the association of a bipartite NLS in the tail of XCTK2 with importin ␣/␤, which regulates its ability to cross-link microtubules during spindle formation. Here we show that mutation of the nuclear localization signal (

Summary The mitotic spindle is a macromolecular structure utilized to properly align and segregate sister chromatids to two daughter cells. During mitosis the spindle maintains a constant length, even though the spindle microtubules (MTs) are constantly undergoing polymerization and depolymerization [1]. Members of the Kinesin-8 family are important for the regulation of spindle length and for chromosome positioning [2–9]. Kinesin-8 proteins are length specific, plus-end directed motors that are proposed to be either MT depolymerases [3, 4, 8, 10, 11] or MT capping proteins [12]. How Kif18A uses its destabilization activity to control spindle morphology is not known. We found that Kif18A controls spindle length independently of its role in chromosome positioning. The ability of Kif18A to control spindle length is mediated by an ATP-independent MT binding site at the C-terminal end of the Kif18A tail that has a strong affinity for MTs in vitro and in cells. We used computational modeling to ask how modulating the motility or binding properties of Kif18A would affect its activity. Our modeling predicts that both fast motility and a low off-rate from the MT end are important for Kif18A function. In addition our studies provide new insight into how depolymerizing and capping enzymes can lead to MT destabilization.

SUMMARY Successful cytokinesis is critical for maintaining genome stability [1, 2], and requires the assembly of a robust central spindle to specify the cleavage furrow position [3], to prevent separated chromosomes from coming back together [4], and to contribute to midbody abscission [5, 6]. A proper central spindle is assembled and maintained by a number of microtubule-associated and molecular motor proteins that sort microtubules into bundles with their plus-ends overlapping at the center [1, 2]. The mechanisms by which different factors organize the central spindle microtubules remain unclear. We found that perturbation of the minus-end directed Kinesin-14 HSET increased the frequency of polyploid cells, which resulted from a failure in cytokinesis. In addition, HSET knockdown resulted in severe midzone microtubule organization, most notably at microtubule minus ends as well as mislocalization of several midbody-associated proteins. Biochemical analysis showed that both human HSET and Xenopus XCTK2 co-fractionated with the γ-tubulin ring complexes on sucrose gradients and that XCTK2 associated with γ-tubulin and Xgrip109 by immunoprecipitation. Our data reveal the novel finding that a minus-end directed motor contributes to the organization and stability of the central spindle, which is needed for proper cytokinesis.

No abstract