Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts.
Mitotic spindle assembly around RCC1-coated beads in Xenopus egg extracts.
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DOI：
10.1371/journal.pbio.1001225
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发表时间：
2011-12
期刊：
影响因子：
9.8
通讯作者：
Heald R
中科院分区：
文献类型：
--
作者：
Halpin D;Kalab P;Wang J;Weis K;Heald R
包裹有鸟核苷酸交换因子RCC1和动蛋白马达蛋白的珠子足以诱导非洲爪哇卵细胞质中有丝分裂的纺锤体组装。在细胞分裂期间，染色体上的遗传物质通过一种名为有丝分裂纺锤体的动态微管结构分配给子细胞。在这里，我们建立了一个重组系统，以评估单个染色体蛋白对非洲爪哇卵子提取物中直径10微米的多孔玻璃珠周围有丝分裂纺锤体形成的贡献。我们发现，染色体凝聚调节因子1(RCC1)，即小GTP酶RAN的鸟核苷酸交换因子(Gef)，可以诱导双极纺锤体的形成。值得注意的是，RCC1珠子在纺锤体内从一个极到另一个极振荡，这种行为可以通过添加Kinesin和RCC1来转换为更典型、更稳定的关联。这些结果确定了两种足以模拟染色质介导的纺锤体组装的活性，并为未来完全由纯化的组件重建纺锤体组装奠定了基础。有丝分裂纺锤体是一种两极结构，负责在分裂细胞中分离两组复制的染色体，从而将一组染色体传递给两个子细胞中的每一个。它是由称为微管的动态细丝以及数百个其他组件组成的，这些组件有助于微管的组织和动态以及染色体的运动。为了了解哪些蛋白质对纺锤体的形成和功能是必不可少的，我们希望能够从纯化的成分中构建它。作为实现这一目标的一步，我们将单个蛋白质与惰性玻璃珠(作为染色体的替代品)偶联，以确定什么蛋白质组合可以在来自青蛙卵的复杂细胞质中诱导纺锤体组装。我们发现，一种名为RCC1的单一酶足以激活一条途径，该途径稳定并组织微管形成围绕珠子的双极结构，但随后珠子在纺锤体的两极之间来回振荡。通过将基于微管的马达蛋白与珠子上的RCC1偶联，我们能够将珠子平衡在纺锤体的中心。因此，固定在珠子上的两种蛋白质可以代替一条染色体，并诱导稳定的纺锤体形成。
Beads coated with the guanine nucleotide exchange factor RCC1 and a kinesin motor protein are sufficient to induce mitotic spindle assembly in Xenopus egg cytoplasm. During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components. The mitotic spindle is a bipolar structure that is responsible for separating the two sets of duplicated chromosomes in a dividing cell, thereby delivering one set to each of the two daughter cells. It is built from dynamic filaments called microtubules, as well as hundreds of other components that contribute to the organization and dynamics of the microtubules and to chromosome movement. To understand which proteins are essential for spindle formation and function, we would like to be able to build it from purified components. As a step towards this goal, we coupled individual proteins to inert glass beads (as a substitute for chromosomes), to determine what combination of proteins can induce spindle assembly in a complex cytoplasm derived from frog eggs. We found that a single enzyme called RCC1 is sufficient to activate a pathway that stabilizes and organizes microtubules into a bipolar structure around the bead, but that this bead then oscillated back and forth between the poles of the spindle. By coupling a microtubule-based motor protein together with RCC1 on the bead, we were able to balance the bead in the center of the spindle. Thus, two proteins immobilized on a bead can substitute for a chromosome and induce stable spindle formation.
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