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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 Xenopus 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^mut) stimulated spindle assembly, localized moderately on the spindle, and formed narrow spindles. In contrast, GX-MBD2^mutonly 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^mutslid antiparallel MTs faster than GX-WT and GX-MBD1^mut. However, GX-WT and GX-MBD1^mutstatically cross-linked the majority of parallel MTs, whereas GX-MBD2^mutequally 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. 

Microscopic structure tuned by depletant concentration dictates mesoscale dynamics in extensile kinesin-driven microtubule bundles. 

Tight regulation of microtubule (MT) dynamics is necessary for proper spindle assembly and chromosome segregation. The MT destabilizing Kinesin-8, Kif18B, controls astral MT dynamics and spindle positioning. Kif18B interacts with importin α/β as well as with the plus-tip tracking protein EB1, but how these associations modulate Kif18B is not known. We mapped the key binding sites on Kif18B, made residue-specific mutations, and assessed their impact on Kif18B function. Blocking EB1 interaction disrupted Kif18B MT plus-end accumulation and inhibited its ability to control MT length on monopolar spindles in cells. Blocking importin α/β interaction disrupted Kif18B localization without affecting aster size. In vitro, importin α/β increased Kif18B MT association by increasing the on-rate and decreasing the off-rate from MTs, which stimulated MT destabilization. In contrast, EB1 promoted MT destabilization without increasing lattice binding in vitro, which suggests that EB1 and importin α/β have distinct roles in the regulation of Kif18B-mediated MT destabilization. We propose that importin α/β spatially modulate Kif18B association with MTs to facilitate its MT destabilization activity. Our results suggest that Ran regulation is important not only to control molecular motor function near chromatin but also to provide a spatial control mechanism to modulate MT binding of nuclear localization signal–containing spindle assembly factors.