Current Biology
Volume 31, Issue 1, 11 January 2021, Pages 115-127.e3
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Article
Ran-GTP Is Non-essential to Activate NuMA for Mitotic Spindle-Pole Focusing but Dynamically Polarizes HURP Near Chromosomes

https://doi.org/10.1016/j.cub.2020.09.091Get rights and content
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Highlights

  • Using AID technology, we developed mitotic depletion assays for the Ran pathway

  • The Ran pathway is non-essential to activate NuMA for spindle-pole focusing

  • Ran-GTP is not required to target TPX2 but is required to localize HURP and HSET

  • The Ran pathway maintains HURP’s polarized spindle localization during metaphase

Summary

Spindle assembly is spatially regulated by a chromosome-derived Ran- GTP gradient. Previous work proposed that Ran-GTP activates spindle assembly factors (SAFs) around chromosomes by dissociating inhibitory importins from SAFs. However, it is unclear whether the Ran-GTP gradient equivalently activates SAFs that localize at distinct spindle regions. In addition, 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 depletion 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. On the other hand, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions to set proper spindle length during prometaphase. We demonstrated that Ran-GTP and importin-β coordinately promote HURP’s dynamic microtubule binding-dissociation cycle, which maintains HURP near chromosomes during metaphase. Together, we propose that the Ran pathway acts on spindle assembly independently of its interphase functions in mitotic human cells but does not equivalently regulate all Ran-regulated SAFs. Ran-dependent spindle assembly is likely coupled with additional parallel pathways that activate SAFs distantly located from the chromosomes.

Keywords

mitotic spindle
Ran-GTP
NuMA
HURP
auxin-inducible degradation

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