Abstract
The chromosome-derived Ran-GTP gradient is believed to promote spindle assembly by releasing spindle assembly factors (SAFs) such as NuMA and HURP from inhibitory importins near chromosomes. The Ran-GTP gradient plays critical roles in meiosis, but how the Ran-based network spatiotemporally defines SAF localization and function in mitosis remains incompletely understood. Here, we systematically depleted RCC1 (Ran-GEF), RanGAP1, and importin-β using auxin-inducible degron (AID) technology in somatic human cells. We demonstrate that the Ran-Importin network does not substantially affect NuMA localization and functions at spindle poles. In contrast, the Ran-based network polarizes both HURP and importin-β on K-fibers near chromosomes, where HURP, but not importin-β, stabilizes microtubules. In addition, acute RCC1 degradation during metaphase reveals that HURP’s K-fiber localization is dynamically maintained by Ran-GTP even after spindle assembly. Together, we propose that the Ran-Importin network locally promotes microtubule-binding and dissociation cycle of HURP, but not NuMA, to dynamically organize stable K-fibers near chromosomes in mitotic human cells.
Summary Using auxin-inducible degron technology, we systematically analyzed the mechanisms of Ran-based polarization of spindle assembly factors on human mitotic spindles. We find that the Ran-based network dynamically polarizes and maintains HURP, but not NuMA, by promoting local microtubule binding-dissociation cycle.
Highlights
Ran-GTP is dispensable for NuMA localization and function at spindle poles in mitotic human cells.
Ran-Importin network is indispensable for HURP and importin-β to accumulate at K-fibers near chromosomes.
HURP, but not importin-β, is required to stabilize K-fibers.
HURP is dynamically maintained on K-fibers even after spindle assembly.