Coupling kinetochore microtubule dynamics to chromosome motion

将动粒微管动力学与染色体运动耦合

• 批准号: 9381209
• 负责人: Ekaterina L Grishchuk
• 金额: $ 39.84万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9381209
• 关键词: Abnormal Cell; Achievement; Address; Affect; Affinity; Amaze; Behavior; Binding; Binding Proteins; Biological Assay; CENP-E protein; Cell Proliferation; Cell division; Cells; Chromosome Segregation; Chromosomes; Complex; Coupled; Coupling; Defect; Development; Diffusion; Dissection; Equipment; Fostering; Frequencies; Health; Human; In Vitro; Kinesin; Kinetics; Kinetochores; Lead; Length; Link; Maintenance; Malignant Neoplasms; Mechanics; Microspheres; Microtubules; Mission; Mitotic; Mitotic Chromosome; Molecular; Molecular Abnormality; Molecular Genetics; Molecular Machines; Motion; Physiological; Play; Point Mutation; Pregnancy loss; Property; Proteins; Public Health; Recombinant Proteins; Recombinants; Recruitment Activity; Research; Role; Scaffolding Protein; Sister; Surface; System; Techniques; Testing; Time; Tubulin; United States National Institutes of Health; Variant; centromere protein C; design; experience; improved; innovation; insight; laser tweezer; mutant; novel; novel anticancer drug; particle; protein complex; receptor; reconstitution; scaffold; targeted cancer therapy; tool

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.

项目总结 准确的染色体分离关键取决于染色体之间的动态连接 染色体动点和纺锤体微管。最近几年带来了巨大的 动粒分子组成鉴定的进展，但对其机理的研究 这些蛋白质如何与微管相互作用并使染色体运动变得迟缓 在后面。我们建议使用简化论的多尺度方法来解决这一缺陷 重建动粒-微管相互作用的生理学方面的创新分析 在试管中。我们拥有分子工具、设备和专业知识，可以在定量和 关于有丝分裂染色体的一些最基本的问题 分离：(1)动点如何将它们最初的微管壁结合转化为 微管末端附着，(2)它们随后如何附着在微管末端，以及 移动与微管蛋白组装/拆解相结合，(3)以及这些移动链接如何持续 在武力的作用下。在目标1中，我们将使用纯化的NDC80蛋白复合体重建这些相互作用 和经过战略选择的辅助蛋白质。我们最近重建了微管末端 Ndc80的转化，在CENP-E的正端定向运动的辅助下。不同的Ndc80变种 将被用来揭示潜在的机制，并且附加的动粒组件将 被添加进来，以揭示它们的相对影响。我们对提纯成分的发现将是至关重要的 与从有丝分裂提取物中分离的天然动粒复合体的活性比较 人类细胞(目标2)。我们发现与动粒支架相关的复合体 CENP-T蛋白可以在分解的微管末端移动。这一重要成就 为我们的功能分析以及随后的鉴定和 人类细胞微管末端偶联的关键动粒成分的特征。在……里面 目的3我们将使用先进的激光镊子技术来关键地比较纯化的能力 蛋白质和复合体在拉力下移动，模仿姐妹之间的紧张关系 动感家务。这些研究的结果将有助于我们构建一个综合的观点 人类动粒的机械力-分子耦合，定义了关键的动粒的具体作用 Ndc80、Ska1等蛋白质，揭示了动粒之间的功能差异 用不同的支架组装的复合体。的功能行为知之甚少。 人类动粒蛋白，而我们的研究无疑将提供对 动粒-微管相互作用的基础，并促进细胞中的新发现 师字段。