The Kinesin-14 Tail: Dual microtubule binding domains drive spindle morphogenesis through tight microtubule cross-linking and robust sliding

Abstract Proper spindle assembly requires the Kinesin-14 family of motors to organize microtubules (MTs) into the bipolar spindle by cross-linking and sliding anti-parallel and parallel MTs through their motor and tail domains. How they mediate these different activities is unclear. We identified two MT binding domains (MBD1 and MBD2) within the Xenopus Kinesin-14 XCTK2 tail and found that MBD1 MT affinity was weaker than MBD2. Comparable to full-length GFP-XCTK2 wild-type protein (GX-WT), GFP-XCTK2 containing the MBD1 mutations (GX-MBD1mut) stimulated spindle assembly, localized moderately on the spindle, and formed narrow spindles. In contrast, GX-MBD2mut only partially stimulated spindle assembly, localized weakly on the spindle, and formed shorter spindles. Biochemical reconstitution of MT cross-linking and sliding demonstrated that GX-MBD2mut slid anti-parallel MTs faster than GX-WT and GX-MBD1mut. However, GX-WT and GX-MBD1mut statically cross-linked the majority of parallel MTs, whereas GX-MBD2mut equally slid and statically cross-linked parallel MTs without affecting their sliding velocity. These results provide a mechanism by which the two different MT binding domains in the Kinesin-14 tail balance anti-parallel MT sliding velocity (MBD1) and tight parallel MT cross-linking (MBD2), which are important for spindle assembly and localization, and provide a basis for characterizing how molecular motors organize MTs within the spindle. Significance Statement Spindle assembly and organization utilize molecular motors that cross-link and slide anti-parallel and parallel microtubules. How individual motors moderate both active sliding and static cross-linking is not understood. Using biochemical reconstitution, the authors determined that the Kinesin-14 tail contains two independent microtubule binding domains. MBD1 with weaker microtubule binding facilitates faster anti-parallel microtubule sliding, whereas the stronger MBD2 mediates tight parallel microtubule cross-linking, which was important for spindle assembly. These findings provide a mechanism for how Kinesin-14s differentially control microtubule sliding and cross-linking and provide insight into how molecular motors can mediate the dynamic organization of microtubules in the spindle. Competing Interest Statement The authors have declared no competing interest. Footnotes The new version includes additional analyses of the spindle assembly data (Figure 5) and the Microtubule Cross-linking experiments (Figures 6 and 7). Abbreviations - GX - GFP-XCTK2 - HSET - human SET protein homolog - K-14 - Kinesin-14 - MBD - microtubule binding domain - MT - microtubule - Ncd - Drosophila non-claret disjunctional protein - NLS - nuclear localization signal - XCTK2 - Xenopus carboxy-terminal kinesin 2