HURP is a Ran-importin beta-regulated protein that stabilizes kinetochore microtubules in the vicinity of chromosomes

Abstract

Background: Formation of a bipolar mitotic spindle in somatic cells requires the cooperation of two assembly pathways, one based on kinetochore capture by centrosomal microtubules, the other on RanGTP-mediated microtubule organization in the vicinity of chromosomes. How RanGTP regulates kinetochore-microtubule (K-fiber) formation is not presently understood.

Results: Here we identify the mitotic spindle protein HURP as a novel target of RanGTP. We show that HURP is a direct cargo of importin beta and that in interphase cells, it shuttles between cytoplasm and nucleus. During mitosis, HURP localizes predominantly to kinetochore microtubules in the vicinity of chromosomes. Overexpression of importin beta or RanT24N (resulting in low RanGTP) negatively regulates its spindle localization, whereas overexpression of RanQ69L (mimicking high RanGTP) enhances HURP association with the spindle. Thus, RanGTP levels control HURP localization to the mitotic spindle in vivo, a conclusion supported by the analysis of tsBN2 cells (mutant in RCC1). Upon depletion of HURP, K-fiber stabilization is impaired and chromosome congression is delayed. Nevertheless, cells eventually align their chromosomes, progress into anaphase, and exit mitosis. HURP is able to bundle microtubules and, in vitro, this function is abolished upon complex formation with importin beta and regulated by Ran. These data indicate that HURP stabilizes K-fibers by virtue of its ability to bind and bundle microtubules.

Conclusions: Our study identifies HURP as a novel component of the Ran-importin beta-regulated spindle assembly pathway, supporting the conclusion that K-fiber formation and stabilization involves both the centrosome-dependent microtubule search and capture mechanism and the RanGTP pathway.