Mechanisms to move and steer chromosomes

项目概要/摘要 我们将阐明有丝分裂纺锤体上染色体运动的动力机制， 控制这些运动方向的机制。真菌使用环形Dam 1复合体， 与多个“臂状”Ndc 80复合物一起工作以移动染色体。这个环可以被推向极点， 当解聚微管在正端弯曲时产生的“动力冲程”为微管的运动提供动力， 染色体然而，目前还不清楚大多数真核生物，包括人类，如何动力染色体运动 因为他们缺少Dam 1环复合体我们将纯化的人Ndc 80和Ska复合物在 通过EM断层扫描来观察微管，以阐明人运动舞蹈微管附着的结构。 这些新的结构将Ska定位在微管上，也表明Ndc 80复合物在微管上寡聚化。 微管形成一种我们称之为“滑足”的结构。这个新结构表明 后生动物动粒如何被解聚微管的曲率推动的机制 就像酵母。我们已经开发了两种体内试验，使我们能够测量滑动脚的形成 并测量需要斯卡的染色体运动。此外，我们将采用单分子 测定以测量滑动脚在体外产生力的需求。使用这些新的检测方法，我们 将确定驱动人类染色体在有丝分裂纺锤体上运动的机制。 令人惊讶的是，在大多数染色体上，两个姐妹动粒中只有一个有滑动脚。这是令人兴奋 因为染色体运动需要一个姐妹主动解聚~20个微管， 拉染色体，而它的姐妹必须被动地附着在生长的微管上。我们将确定 在两个姐妹动粒上产生滑动足形成的不对称性的调节途径。我们 假设这些通路不仅调节滑足的形成， 在微管与其姐妹动粒结合的同时，微管发生解聚。我们 将建立在这些发现，以确定机制，直接染色体运动，无论是向或 远离有丝分裂纺锤体的两极 了解这些位于染色单体分离中心的基本机制， 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， 抗微管蛋白化学治疗剂的功效。

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.