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Abstract：
Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use live-cell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin β signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin β and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin β in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysis-resistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.
摘要：
哺乳动物卵母细胞减数分裂I（MI）期间的同源染色体分离是通过染色体MI纺锤体进行的。尽管在人类卵母细胞中的研究确定RanGTP酶是MI纺锤体功能的关键调节剂，小鼠卵母细胞的实验质疑这一概念的普遍性。这里,我们使用荧光探针和Förster共振能量转移（FRET）生物传感器的活细胞成像来监测小鼠卵母细胞中Ran扰动引起的Ran和importinβ信号传导的变化，同时检查MI纺锤体动力学。我们表明，与以前研究中使用的RanT24N不同，aRanT24N,T42A双突变体抑制RanGEF而不干扰货物与导入蛋白β的结合，并破坏染色体分离中的MI纺锤体功能。Ran和importinβ在微管组织中心（MTOC）和MI主轴组装的聚结中的作用进一步得到了化学抑制剂importazole的使用，其抗GTP水解RanQ69L突变体部分挽救了其作用。这些结果表明RanGTP对于人和小鼠的MI纺锤体组装和功能是必需的。
