Arnaoutov Alexei, Azuma Yoshiaki, Ribbeck Katharina, Joseph Jomon, Boyarchuk Yekaterina, Karpova Tatiana, McNally James, Dasso Mary
Laboratory of Gene Regulation and Development, NICHD, NIH, Building 18, Room 106, Bethesda, MD 20892-5431, USA.
Nat Cell Biol. 2005 Jun;7(6):626-32. doi: 10.1038/ncb1263.
The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. 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.
Ran GTP酶控制多种细胞过程，包括核运输、有丝分裂检查点、纺锤体组装和有丝分裂后核膜重新组装。在这里，我们研究了Crm1的有丝分裂功能，Crm1是一种与Ran-GTP结合的核输出受体，负责富含亮氨酸的货物（带有核输出序列）和Snurportin-1的输出（参考文献3）。我们发现Crm1定位于动粒，并且Crm1三元复合物的组装对于Ran-GTP依赖性地将Ran GTP酶激活蛋白1（Ran-GAP1）和Ran结合蛋白2（Ran-BP2）募集到动粒至关重要。我们进一步表明，雷帕霉素B对Crm1的抑制会破坏有丝分裂进程和染色体分离。对雷帕霉素B处理的细胞内纺锤体的分析表明，它们的着丝粒处于增加的张力之下。在雷帕霉素B处理的细胞中，着丝粒经常与跨越着丝粒的连续微管束相关联，这表明它们的动粒没有维持与单个动粒纤维的离散端对端附着。在温度敏感的Ran途径突变体（tsBN2细胞）中也观察到了类似的纺锤体缺陷。综上所述，我们的研究结果表明，Crm1和Ran-GTP对于Ran-BP2/Ran-GAP1募集到动粒、动粒纤维的定义以及后期染色体分离至关重要。因此，Crm1是体细胞中有丝分裂纺锤体组装和功能的关键Ran-GTP效应物。
