Coupling kinetochore microtubule dynamics to chromosome motion

将动粒微管动力学与染色体运动耦合

基本信息

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
项目类别：
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

项目摘要

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.

项目概要准确的染色体分离关键取决于染色体之间的动态附着染色体动粒和纺锤体微管。近年来带来了巨大的在识别动粒分子成分方面取得了进展，但其机制研究这些蛋白质如何与微管相互作用并促进染色体运动是滞后的在后面。我们建议通过使用还原论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方面的创新测定体外。我们拥有分子工具、设备和专业知识，可以定量和分析严格地回答有关有丝分裂染色体的一些最基本的问题分离：（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) 它们随后如何挂在微管末端，以及与微管蛋白组装/拆卸一起移动，(3) 以及这些移动链接如何持续存在在力量的作用下。在目标 1 中，我们将使用纯化的 Ndc80 蛋白复合物重新创建这些相互作用和战略性选择的辅助蛋白质。我们最近重建了微管末端在正端定向驱动蛋白 CENP-E 的协助下，由 Ndc80 进行转化。不同的 Ndc80 变体将用于揭示潜在机制，并且额外的着丝粒组件将添加以揭示它们的相对影响。我们对纯化成分的研究结果将至关重要与从有丝分裂提取物中分离的天然着丝粒复合物的活性进行比较人类细胞（目标 2）。我们发现与着丝粒支架相关的复合物蛋白质CENP-T可以在解体微管末端移动。这一重要成就为我们的功能测定以及随后的鉴定和鉴定奠定了基础人体细胞微管末端耦合关键着丝粒成分的表征。在目标 3 我们将使用先进的激光镊子技术来严格比较纯化的能力蛋白质和复合物在拉力下移动，模仿姐妹之间的紧张关系动粒。这些研究的结果将帮助我们构建一个综合的观点人体动粒的力-分子耦合，定义关键动粒的具体作用蛋白质 Ndc80、Ska1 等，并揭示着丝粒之间的功能差异用不同的支架组装的复合物。对功能行为知之甚少人类着丝粒蛋白，我们的研究无疑将为人类着丝粒蛋白提供新的见解动粒-微管相互作用的基础知识，并促进细胞的新发现分部领域。