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粒蛋白的纳米级排列如何塑造其功能和调节机制。动粒是一种大分子马达，驱动染色体运动，并确保它们在细胞分裂过程中准确分离。染色体运动所需的动粒力产生对于两个子细胞的完整基因组的遗传是至关重要的。动粒失调导致染色体不稳定，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 动粒力产生的生物物理机制对于发展对动粒蛋白中疾病相关突变的机制理解是必要的。尽管过去十年我们在了解动粒的蛋白质组成方面取得了巨大进展，但对其作为力发生器功能的机械理解仍然难以捉摸。进一步研究的主要障碍是对动粒的分子结构缺乏了解。因此，我们提出了一种新的“架构功能”的方法来建立机械的联系动粒架构和它的功能。目的1：发展一种新的荧光显微镜方法来重建纳米级动粒结构。我们已经开发出一种新的技术来确定纳米级分布的蛋白质在活细胞中。我们对动粒结构的初步重建提出了一个关于动粒如何产生微管聚合和解聚耦合力的综合模型。我们的技术将有助于确定其他细胞机器的架构。目标2：确定力产生分子的位置如何定义它们的功能。我们将对我们的新模型进行“结构-功能”分析，其中我们将研究动粒结构变化对其功能的影响。这项工作将定义由动粒产生力的生物物理学原理。目标3：定义动粒的最小架构规范。我们将使用体外实验和人工动粒蛋白组装来确定一个关键的动粒蛋白，Ndc 80，重建其分布和功能在体内观察到的必要和足够的建筑特征。这 这项工作将建立一个在细胞中构建人工动粒的框架。