Coupling kinetochore microtubule dynamics to chromosome motion

项目摘要 准确的染色体分离关键取决于染色体之间的动态连接。 染色体动粒和纺锤体微管。近年来， 在确定动粒的分子组成方面取得了进展，但 这些蛋白质如何与微管相互作用并使染色体运动是滞后的 后面我们建议通过使用还原主义的多尺度方法来解决这一缺陷， 创新的检测方法，可重建激动素-微管相互作用的生理学方面 体外我们拥有分子工具、设备和专业知识， 一些关于有丝分裂染色体的最基本的问题 分离：（1）着丝粒如何将其最初的微管壁结合转化为 微管末端附着，（2）它们如何随后挂在微管末端， 与微管蛋白组装/拆卸一起移动，（3）以及这些移动的联系如何持续 在武力下。在目标1中，我们将使用纯化的Ndc 80蛋白复合物重建这些相互作用 和策略性选择的辅助蛋白质。我们最近重组了微管末端 通过Ndc 80的转化，由正末端定向驱动蛋白CENP-E辅助。不同Ndc 80变体 将被用来揭示潜在的机制，额外的动粒组件将 以显示其相对影响。我们对纯化成分的发现将是至关重要的 与分离自有丝分裂细胞提取物的天然动粒复合物的活性相比， 人细胞（Aim 2）。我们发现与动粒支架相关的复合物 CENP-T蛋白可以在微管末端移动。这一重要成就 为我们的功能测定和随后的鉴定奠定了基础， 在人细胞中用于微管末端偶联的关键动粒组分的表征。在 目的3：我们将使用先进的激光镊子技术， 蛋白质和复合物在拉力下移动，模仿姐妹之间的张力 动粒这些研究的结果将有助于我们建立一个综合的观点， 机械分子偶联在人体动粒，定义关键动粒的具体作用 蛋白Ndc 80，Ska 1等，并揭示了动粒之间的功能差异 用不同的支架组装复合物。关于其功能行为知之甚少， 人类动粒蛋白，我们的研究无疑将提供新的见解， 运动舞蹈微管相互作用的基本原理，并促进细胞中的新发现 除法场

PROJECT SUMMARY Accurate chromosome segregation crucially depends on the dynamic attachments between chromosomal kinetochores and spindle microtubules. Recent years brought tremendous progress in identifying molecular components of the kinetochores, but mechanistic studies of how these proteins interact with microtubules and enable chromosome motions are lagging behind. We propose to address this deficiency by using reductionist multiscale approaches and innovative assays that reconstitute physiological aspects of kinetochore-microtubule interactions in vitro. We have molecular tools, equipment and expertise to address in a quantitative and rigorous manner some of the most fundamental questions about mitotic chromosome segregation: (1) how the kinetochores convert their initial microtubule-wall binding into microtubule-end attachment, (2) how they subsequently hang onto the microtubule ends and move in conjunction with tubulin assembly/disassembly, (3) and how these mobile links persist under force. In Aim 1 we will recreate these interactions using purified Ndc80 protein complex and strategically chosen assisting proteins. We recently reconstituted microtubule end conversion by Ndc80, assisted by a plus-end directed kinesin CENP-E. Different Ndc80 variants will be used to uncover the underlying mechanism, and additional kinetochore components will be added to reveal their relative impact. Our findings with purified components will be critically compared with the activity of native kinetochore complexes isolated from extracts of mitotic human cells (Aim 2). We have found that complexes associated with the kinetochore scaffold protein CENP-T can move at the dissembling microtubule ends. This essential achievement lays the groundwork for our functional assays, and subsequent identification and characterization of key kinetochore components for microtubule end coupling in human cells. In Aim 3 we will use advanced laser tweezers techniques to critically compare the ability of purified proteins and complexes to move under pulling force, mimicking tension between sister kinetochores. The results from these studies will help us to construct an integrative view of the mechano-molecular coupling at human kinetochore, define the specific roles of key kinetochore proteins Ndc80, Ska1 and others, and reveal functional difference between kinetochore complexes assembled with different scaffolds. Little is known about functional behavior of human kinetochore proteins, and our research will undoubtedly provide novel insights into the fundamentals of kinetochore-microtubule interactions, and promote new discoveries in the cell division field.