Mechanisms to move and steer chromosomes

项目摘要/摘要 我们将阐明驱动有丝分裂纺锤体上染色体运动的机制，以及 控制这些运动方向的机制。真菌使用环形的Dam1复合体，它 它与多个“手臂状”的Ndc80复合体一起工作，以移动染色体。这个环可以被推向极地 当解聚正端的微管曲线时产生的“功率冲程”为运动提供动力 染色体。然而，目前还不清楚包括人类在内的大多数真核生物是如何推动染色体运动的。 因为他们缺少Dam1环复合体。我们将纯化的人NDC80和Ska复合体可视化显示在 微管的EM断层扫描，以阐明人的动粒-微管附着的结构。 这些新结构使Ska定位在微管上，也表明Ndc80络合物在微管上齐聚 微管形成一种我们称之为“滑足”的结构。这一新结构表明可测试性 解聚微管的曲率如何推动后生动物动粒的机制 就像酵母一样。我们已经开发了两种活体测试方法，使我们能够测量滑动脚的形成 并测量需要Ska的染色体运动。此外，我们还将使用单分子 测定滑足在体外产生力量的需要。使用这些新的分析方法，我们 将确定人类染色体在有丝分裂纺锤体上移动的动力机制。 令人惊讶的是，在大多数染色体上，两个姐妹着丝点中只有一个有滑动的脚。真令人兴奋 因为染色体运动需要一个姐妹积极参与解聚~20个微管以 拉着染色体，而它的姐妹必须被动地附着在生长中的微管上。我们将确定 在两个姐妹运动中枢上产生滑步形成不对称性的调节通路。我们 假设这些通路不仅调节滑动的脚的形成，而且还可以确保一个姐妹 有解聚的微管，而与其姊妹着丝粒结合的微管正在聚合。我们 将建立在这些发现的基础上，以确定引导染色体运动的机制 远离有丝分裂纺锤体上的两极。 了解这些位于染色单体分离中心的基本机制是很重要的 确定癌细胞如何降低有丝分裂的保真度以产生基因组不稳定性并增加 抗微管蛋白化疗药物的疗效。

Project Summary/Abstract We will elucidate the mechanisms that power the movements of chromosomes on the mitotic spindle and the mechanisms that control the direction of these movements. Fungi use the ring shaped Dam1 complex, which works with multiple “arm-like” Ndc80 complexes to move chromosomes. This ring can be pushed poleward by a “power stroke” generated when depolymerizing microtubules curve at the plus end to power the movement of chromosomes. However, it is unclear how most eukaryotes, including humans, power chromosome movement since they lack the Dam1 ring complex. We visualized purified human Ndc80 and Ska complexes on microtubules by EM tomography to elucidate the structure of the human kinetochore-microtubule attachment. These new structures orient Ska on microtubules and also suggest Ndc80 complexes oligomerize on microtubules to form a structure we have named the “sliding foot”. This new structure suggests testable mechanisms for how metazoans kinetochores are pushed by the curvature of a depolymerizing microtubule like yeast. We have developed two in vivo assays that allow us to measure the formation of the sliding feet and to measure the chromosome movements that require Ska. In addition, we will employ single molecule assays to measure the requirement of the sliding foot to generate force in vitro. Using these new assays, we will identify the mechanism that powers the movements of human chromosomes on the mitotic spindle. Surprisingly, on most chromosomes only one of the two sister kinetochores has sliding feet. This is exciting because chromosome movements require one sister to actively engage depolymerizing ~20 microtubules to pull chromosomes, while its sister must passively attach to growing microtubules. We will identify the regulatory pathways that generate the asymmetry of sliding foot formation on the two sister kinetochores. We hypothesize that these pathways not only regulate sliding foot formation but can also ensure that one sister has depolymerizing microtubules while the microtubules bound to its sister kinetochore are polymerizing. We will build on these findings to identify the mechanisms that direct chromosome movements either towards or away from poles on the mitotic spindle. It is important to understand these basic mechanisms that lie at the center of the chromatid segregation to determine how cancer cells lower the fidelity of mitosis to generate genomic instability and to increase the efficacy of anti-tubulin chemotherapeutics.