Abstract
The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly1,2. Here we examine the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1 (ref. 3). We find that Crm1 localizes to kinetochores, and that Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. We further show that Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres were under increased tension. In leptomycin B-treated cells, centromeres frequently associated with continuous microtubule bundles that spanned the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects were observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, our findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells.
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Acknowledgements
We thank E. D. Salmon and C. L. Rieder for insightful discussions. We thank E. D. Salmon, A. Khodjakov, I. Mattaj, and B. B. Quimby for critical comments on this manuscript. We thank D. Görlich for anti-Crm1 antibodies and Crm1 cDNA clones, and M. Yoshida for leptomycin B. We thank T. Anan and F. Ayaydin for help with the U2OSH2B–GFP and HeLaH2B–GFP cell lines. We thank the members of the Lippincott-Schwartz group for their assistance and for the use of the spinning disk confocal microscope.
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Arnaoutov, A., Azuma, Y., Ribbeck, K. et al. Crm1 is a mitotic effector of Ran-GTP in somatic cells. Nat Cell Biol 7, 626–632 (2005). https://doi.org/10.1038/ncb1263
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DOI: https://doi.org/10.1038/ncb1263
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