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.

项目摘要 准确的染色体隔离至关重要地取决于动态附件 染色体动脉和纺锤微管。近年来带来了巨大的 鉴定动力学的分子成分的进展，但是 这些蛋白质如何与微管相互作用和使染色体运动滞后 在后面。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和 重新构建动物学 - 微动相互作用的生理方面的创新测定 体外。我们有分子工具，设备和专业知识，可用于定量和 严格的方式关于有丝分裂染色体的一些最根本的问题 隔离：（1）动力学如何将其初始微管壁结合到 微管末端附件，（2）它们随后如何挂在微管上， 与小管蛋白组装/拆卸一起移动，（3）以及这些移动链接如何持续 在武力下。在AIM 1中，我们将使用纯化的NDC80蛋白质复合物重新创建这些相互作用 并在战略上选择辅助蛋白质。我们最近重组了微管末端 NDC80的转换，并在加号的指示驱动蛋白CENP-E的辅助下进行转换。不同的NDC80变体 将用于揭示基本机制，其他动力学组件将 添加以揭示其相对影响。我们使用纯化的组件的发现将非常重要 与从有丝分裂提取物分离的天然动力学配合物的活性相比 人类细胞（AIM 2）。我们发现与动力学支架相关的复合物 蛋白质CENP-T可以在散布的微管末端移动。这项基本成就 为我们的功能测定和随后的识别奠定基础 人类细胞中微管末端耦合的关键动力学成分的表征。在 AIM 3我们将使用高级激光镊子技术来批判性地比较纯化的能力 蛋白质和复合物在拉力下移动，模仿姐妹之间的张力 动力学。这些研究的结果将有助于我们建立对 在人动物学上的机械分子耦合，定义了钥匙动物的特定作用 蛋白质NDC80，SKA1等，并揭示动力学之间的功能差异 配合物与不同的支架组装在一起。关于功能行为知之甚少 人类动物学蛋白，我们的研究无疑将提供有关蛋白质的新见解。 动脉色氨酸 - 微管相互作用的基本原理，并促进细胞中的新发现 分区场。