The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5
The rate of bipolar spindle assembly depends on the microtubule-gliding velocity of the mitotic kinesin Eg5
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DOI：
10.1016/j.cub.2004.09.052
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发表时间：
2004-10-05
期刊：
影响因子：
9.2
通讯作者：
Kapoor, TM
中科院分区：
文献类型：
--
作者：
Kwok, BH;Yang, JG;Kapoor, TM
在早期胚胎周期中，有丝分裂纺锤体组装所需的时间必须与自主细胞周期振荡相匹配，因为这两个过程之间缺乏协调将导致染色体分离错误[1]。广泛保守的BimC驱动蛋白家族成员对于所有真核生物的纺锤体形成至关重要，BimC功能的完全丧失导致单极纺锤体具有未分离的两个纺锤极[2-6]。然而，BimC运动活动在主轴装配过程中的确切作用尚不清楚。为了研究BimC驱动蛋白的运动活性对纺锤体组装的贡献，我们产生并鉴定了Eg5突变体，一种脊椎动物BimC驱动蛋白，其体外微管滑动速度降低。在爪蟾卵提取物中，我们用重组野生型或突变型运动蛋白代替内源性Eg5。通过使用中心体依赖和中心体独立的纺锤体组装分析，我们发现，决定纺锤体大小和形状的机制对于Eg5运动减少6倍是稳定的。然而，当Eg5运动功能受损时，主轴组装过程变慢。Eg5的这种作用与它对中心体分离的贡献无关。我们提供的证据表明，Eg5是脊椎动物驱动纺锤体组装的细胞机制的限速成分。
During early embryonic cycles, the time required for mitotic spindle assembly must match the autonomous cell cycle oscillations because a lack of coordination between these two processes will result in chromosome segregation errors [1]. Members of the widely conserved BimC kinesin family are essential for spindle formation in all eukaryotes, and complete loss of BimC function results in monopolar spindles that have two spindle poles that are not separated [2-6]. However, the precise roles of BimC motor activity in the spindle assembly process are not known. To examine the contribution of BimC kinesin's motor activity to spindle assembly, we generated and characterized mutants of Eg5, a vertebrate BimC kinesin, with reduced in vitro microtubule-gliding velocities. In Xenopus egg extracts, we replaced endogenous Eg5 with recombinant wild-type or mutant motor proteins. By using centrosome-dependent and centrosome-independent spindle assembly assays, we found that mechanisms that determine spindle size and shape were robust to similar to6-fold reductions in Eg5 motility. However, the spindle assembly process was slower when Eg5 motor function was impaired. This role of Eg5 was independent of its contribution to centrosome separation. We provide evidence that Eg5 is a rate-limiting component of the cellular machinery that drives spindle assembly in vertebrates.