Architecture-function analysis of the kinetochore motor

着丝粒马达的结构功能分析

• 批准号: 8480061
• 负责人: Ajit Joglekar
• 金额: $ 28.06万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8480061
• 关键词: 3-Dimensional; Architecture; Back; Binding; Biochemical; Biophysics; Cell division; Cell physiology; Cells; Chromosomal Instability; Chromosomes; Complex; Coupled; DNA; Data; Defect; Development; Disease; Ensure; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Foundations; Generations; Genome; Goals; In Vitro; Infertility; Kinetochores; Knowledge; Lead; Life; Link; Location; Maps; Measures; Mediating; Methodology; Methods; Microscopy; Microtubule Depolymerization; Microtubule Polymerization; Microtubules; Modeling; Molecular; Motor; Mutation; Pattern; Positioning Attribute; Postdoctoral Fellow; Printing; Property; Proteins; Publishing; Regulation; Relative (related person); Resolution; Role; Shapes; Techniques; Testing; Work; Yeasts; age related; base; cell motility; chemotherapy; chromosome movement; daughter cell; design; feeding; gene therapy; grasp; in vitro Assay; in vivo; insight; nanoscale; novel; polymerization; protein distribution; protein structure; public health relevance; reconstitution; reconstruction; research study; segregation; sensor; stoichiometry; tumorigenesis

DESCRIPTION (provided by applicant): Our goal is to understand how the nanoscale arrangement of kinetochore proteins shapes its functional and regulatory mechanisms. The kinetochore is a macromolecular motor that drives chromosome movement and ensures their accurate segregation during cell division. Kinetochore force generation required for chromosome movement is critical for inheritance of a complete genome by both daughter cells. Kinetochore misregulation leads to chromosomal instability, which has been linked to tumorigenesis, developmental defects, as well as age- related infertility. Therefore, definition of the biophysical mechanism of kinetochore force generation is necessary to develop a mechanistic understanding of disease relevant mutations in kinetochore proteins. Although the last decade has witnessed tremendous progress in our understanding the protein composition of the kinetochore, a mechanistic understanding of its function as a force generator remains elusive. The primary obstacle in further progress is a lack of understanding of the molecular architecture of the kinetochore. Therefore, we propose a novel 'architecture-function' approach to establish mechanistic link between kinetochore architecture and its function. Aim 1: Develop a new fluorescence microscopy method to reconstruct the nanoscale kinetochore architecture. We have developed a new technique to determine nanoscale distribution of proteins in live cells. Our preliminary reconstruction of kinetochore architecture suggests an integrative model of how the kinetochore generates microtubule polymerization and depolymerization coupled force. Our technique will be useful for determining the architecture of other cellular machines. Aim 2: Determine how the location of force generating molecules defines their function. We will subject our new model to an 'architecture-function' analysis, wherein we will study the impact of changes in kinetochore architecture on its function. This work will define the biophysical principles of force generation by the kinetochore. Aim 3: Define the minimal architectural specification for the kinetochore. We will use in vitro experiments and artificial kinetochore protein assemblies to determine the necessary and sufficient architectural features of a key kinetochore protein, Ndc80, for reconstituting its distribution and function observed in vivo. This work will establish a framework for building artificial kinetochores in cells.

描述（由申请人提供）：我们的目标是了解动力学蛋白的纳米级排列如何塑造其功能和调节机制。动力学是一种大分子电动机，可驱动染色体运动并确保其在细胞分裂期间的准确分离。染色体运动所需的动力学力产生对于两个子细胞对完整基因组的遗传至关重要。动力学的误导会导致染色体不稳定性，这与肿瘤发生，发育缺陷以及与年龄相关的不育有关。因此，定义 动力学力产生的生物物理机制对于在动元蛋白中对疾病相关突变的机械理解是必要的。尽管过去十年在我们理解动力学的蛋白质组成方面取得了巨大的进步，但对其作为力发生器功能的机械理解仍然难以捉摸。进一步进步的主要障碍是缺乏对动力学的分子结构的了解。因此，我们提出了一种新颖的“体系结构功能”方法，以建立动力学体系结构及其功能之间的机械联系。 AIM 1：开发一种新的荧光显微镜方法来重建纳米级运动学结构。我们已经开发了一种新技术来确定活细胞中蛋白质的纳米级分布。我们对动脉结构的初步重建提出了一个集成模型，该模型是如何生成微管聚合和解聚耦合力。我们的技术将有助于确定其他蜂窝机的架构。目标2：确定产生分子的力的位置如何定义其功能。我们将对新模型进行“体系结构功能”分析，其中我们将研究动物学体系结构对其功能的变化的影响。这项工作将定义动力学生成的生物物理原理。 AIM 3：定义动力学的最低架构规范。我们将使用体外实验和人工动力学蛋白质组件来确定关键动物学蛋白NDC80的必要和足够的结构特征，以重新建立其在体内观察到的分布和功能。这 工作将建立一个在细胞中构建人工动物学的框架。