Length control of the metaphase spindle
Length control of the metaphase spindle
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DOI：
10.1016/j.cub.2005.09.054
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发表时间：
2005-11-22
期刊：
影响因子：
9.2
通讯作者：
Vale, RD
中科院分区：
文献类型：
--
作者：
Goshima, G;Wollman, R;Vale, RD
背景资料：在给定的细胞类型中，中期纺锤体的极间距离是合理恒定的;在脊椎动物雌性卵母细胞的情况下，这种稳态长度可以保持相当长的时间，在此期间微管保持高度动态。虽然一些分子扰动已被证明会影响纺锤体长度，但对决定中期纺锤体长度的因素还没有达成全面的理解。使用果蝇S2细胞系，我们耗尽或过度表达蛋白质，这些蛋白质要么在纺锤体微管之间产生滑动力，（驱动蛋白-5，驱动蛋白-14，动力蛋白），促进微管聚合（EB 1，Mast/Orbit [CLASP]，Minispurity [Dis 1/XMAP 215/TOG]）或解聚（驱动蛋白-8，13），或介导姐妹染色单体凝聚（Rad 21），以探索这些力量如何影响纺锤体长度。使用高通量自动显微镜和> 4000个纺锤体的半自动图像分析，我们发现微管聚合因子的RNAi或驱动蛋白-8的过表达后纺锤体尺寸减小。而更长的纺锤体是由Rad 21、驱动蛋白-8或驱动蛋白-13的敲低产生的。相反，与之前的报告不同，双极主轴长度对电机产生的滑动力的增加相对不敏感。然而，在驱动蛋白-5滑动马达的临界浓度下，观察到纺锤体结构中的超灵敏的单极到双极转变。这些观察结果可以解释的定量模型，提出了微管解聚率和微管sliding forces.Conclusions之间的耦合：通过整合广泛的RNAi与高通量的图像处理方法和数学建模，我们得出的结论是中期纺锤体长度是敏感的微管动力学和姐妹染色单体凝聚力的改变，但对微管滑动力的改变稳健。
Background: The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved.Results: Using the Drosophila S2 cell line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/ XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of > 4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8. whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-1 3. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-tobipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces.Conclusions: By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.