Ran-GTP is Non-Essential to Activate NuMA for Spindle Pole Focusing, But Dynamically Polarizes HURP to Control Mitotic Spindle Length

Abstract

During mitosis, a bipolar spindle is assembled around chromosomes to efficiently capture chromosomes. Previous work proposed that a chromosome-derived Ran-GTP gradient promotes spindle assembly around chromosomes by liberating spindle assembly factors (SAFs) from inhibitory importins. However, Ran’s dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic degradation assays to dissect Ran’s mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle pole focusing or for targeting TPX2. In contrast, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions. We demonstrated that Ran-GTP and importin-β coordinately promote HURP’s dynamic microtubule binding-dissociation cycle near chromosomes, which results in stable kinetochore-fiber formation. Intriguingly, this pathway acts to establish proper spindle length preferentially during prometaphase, rather than metaphase. Together, we propose that the Ran pathway is required to activate SAFs specifically near chromosomes, but not generally during human mitotic spindle assembly. Ran-dependent spindle assembly is likely coupled with parallel pathways to activate SAFs, including NuMA, for spindle pole focusing away from chromosomes.