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

Proper spindle assembly requires the Kinesin-14 (K-14) family of motors to organize microtubules (MT) into the bipolar spindle by cross-linking and sliding antiparallel 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 K-14 XCTK2 tail and found that MBD1 MT affinity was weaker than MBD2. Comparable with full-length GFP-XCTK2 wild-type protein (GX-WT), GFP-XCTK2 containing the MBD1 mutations (GX-MBD1) stimulated spindle assembly, localized moderately on the spindle, and formed narrow spindles. In contrast, GX-MBD2 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-MBD2 slid antiparallel MTs faster than GX-WT and GX-MBD1. However, GX-WT and GX-MBD1 statically cross-linked the majority of parallel MTs, whereas GX-MBD2 equally slid and statically cross-linked parallel MTs without affecting their sliding velocity. These results provide a mechanism by which the two different MBDs in the K-14 tail balance antiparallel 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.