Abstract
During spindle assembly, microtubules are spatially organized by the chromosome-derived Ran-GTP gradient. Previous work demonstrated that Ran-GTP releases spindle assembly factors such as HURP from inhibitory importins to assemble microtubules near chromosomes. However, the significance and mechanisms of Ran-mediated spindle assembly remains poorly understood, especially in somatic cells. Here, we systematically depleted RCC1 (Ran-GEF), RanGAP1, and importin-β in human cells using auxin-inducible degron technology. We demonstrate that depletion of RCC1, but not RanGAP1, causes short metaphase spindles that lack HURP on kinetochore-fibers (k-fibers). Surprisingly, we find that importin-β co-localizes with HURP to k-fibers, where it acts as an active, not inhibitory, regulator for HURP. HURP and importin-β are mutually dependent for their k-fiber localization and coordinately regulated by Ran-GTP. In addition, importin-β mutants lacking Ran-GTP binding fail to accumulate on k-fibers. Together, we propose a model in which, in the presence of microtubules, importin-β still interacts with HURP following Ran-GTP binding and further promotes HURP’s microtubule association to stabilize k-fibers.