Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly.
Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly.
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DOI：
10.1091/mbc.e13-05-0232
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发表时间：
2013-10
影响因子：
3.3
通讯作者：
Vogel J
中科院分区：
文献类型：
--
作者：
Nazarova E;O'Toole E;Kaitna S;Francois P;Winey M;Vogel J
主轴结构与动力学行为的相关性表明，一对反平行的微管足以形成双极性主轴，而极性微管在主轴装配过程中保持极位移的速度。形成的极间微管的数量部分通过γ-微管蛋白磷酸化控制。在纺锤体组装过程中，微管可以附着在着丝点上或成对形成反平行对或极性间微管，它们跨越两个纺锤体极点，有助于有丝分裂极点分离和染色体分离。极性间微管规范中的事件了解甚少。利用三维电子断层扫描和活细胞主轴动力学行为分析，研究了主轴装配过程。出乎意料的是，我们发现γ-微管蛋白中一个进化上保守的Cdk1位点（S360）的磷酸化状态与极性微管的数量和组织有关。模拟S360磷酸化（S360D）导致双极纺锤体具有正常数量的微管，但缺乏极性间微管。抑制S360磷酸化（S360A）导致纺锤体具有极性间微管和高角度、反平行的微管对。后者也在长度<1 μm的野生型纺锤体中检测到，这表明高角度微管配对代表了极性间微管形成的中间步骤。主轴结构与动力学行为的相关性表明，微管配对足以分离主轴极点，而极间微管在主轴装配早期保持极位移速度。我们的研究结果表明，在主轴装配过程中形成的极间微管的数量部分是通过主轴极的活动来控制的。
Correlation of spindle architecture with dynamic behavior shows that pairs of antiparallel microtubules are sufficient to form a bipolar spindle, whereas interpolar microtubules maintain the speed of pole displacement during spindle assembly. The number of interpolar microtubules formed is controlled in part through γ-tubulin phosphorylation. During spindle assembly, microtubules may attach to kinetochores or pair to form antiparallel pairs or interpolar microtubules, which span the two spindle poles and contribute to mitotic pole separation and chromosome segregation. Events in the specification of the interpolar microtubules are poorly understood. Using three-dimensional electron tomography and analysis of spindle dynamical behavior in living cells, we investigated the process of spindle assembly. Unexpectedly, we found that the phosphorylation state of an evolutionarily conserved Cdk1 site (S360) in γ-tubulin is correlated with the number and organization of interpolar microtubules. Mimicking S360 phosphorylation (S360D) results in bipolar spindles with a normal number of microtubules but lacking interpolar microtubules. Inhibiting S360 phosphorylation (S360A) results in spindles with interpolar microtubules and high-angle, antiparallel microtubule pairs. The latter are also detected in wild-type spindles <1 μm in length, suggesting that high-angle microtubule pairing represents an intermediate step in interpolar microtubule formation. Correlation of spindle architecture with dynamical behavior suggests that microtubule pairing is sufficient to separate the spindle poles, whereas interpolar microtubules maintain the velocity of pole displacement during early spindle assembly. Our findings suggest that the number of interpolar microtubules formed during spindle assembly is controlled in part through activities at the spindle poles.