Arnaoutov Alexei, Dasso Mary
Laboratory of Gene Regulation and Development, NICHD, NIH, Building 18, Room 106, 20892, Bethesda, MD, USA
Dev Cell. 2003 Jul;5(1):99-111. doi: 10.1016/s1534-5807(03)00194-1.
The Ran GTPase is required for nuclear assembly, nuclear transport, spindle assembly, and mitotic regulation. While the first three processes are relatively well understood, details of Ran's role in mitotic progression remain obscure. We have found that elevated levels of Ran's exchange factor (RCC1) abrogate the spindle assembly checkpoint in Xenopus egg extracts, restore APC/C activity, and disrupt the kinetochore localization of checkpoint regulators, including Mad2, CENP-E, Bub1, and Bub3. Depletion of Ran's GTPase activating protein (RanGAP1) and its accessory factor (RanBP1) similarly abrogates checkpoint arrest. By contrast, the addition of RanGAP1 and RanBP1 to extracts with exogenous RCC1 restores the spindle checkpoint. Together, these observations suggest that the spindle checkpoint is directly responsive to Ran-GTP levels. Finally, we observe a clear wave of RCC1 association to mitotic chromosomes at the metaphase-anaphase transition in normal cycling extracts, suggesting that this mechanism has an important role in unperturbed cell cycles.
Ran GTP酶对于细胞核组装、核运输、纺锤体组装和有丝分裂调控是必需的。虽然前三个过程相对较为人所熟知，但Ran在有丝分裂进程中的作用细节仍不清楚。我们发现，Ran交换因子（RCC1）水平升高会消除非洲爪蟾卵提取物中的纺锤体组装检查点，恢复后期促进复合物/细胞周期体（APC/C）的活性，并破坏检查点调节因子（包括Mad2、着丝粒蛋白E、Bub1和Bub3）的动粒定位。Ran的GTP酶激活蛋白（RanGAP1）及其辅助因子（RanBP1）的缺失同样会消除检查点阻滞。相比之下，向含有外源性RCC1的提取物中添加RanGAP1和RanBP1可恢复纺锤体检查点。这些观察结果共同表明，纺锤体检查点直接对Ran-GTP水平作出反应。最后，我们在正常循环提取物的中期-后期转换时观察到RCC1与有丝分裂染色体有明显的结合波，表明这种机制在未受干扰的细胞周期中具有重要作用。
