Architecture-function analysis of the kinetochore motor

着丝粒马达的结构功能分析

基本信息

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
项目类别：
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

项目摘要

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的产生对于两个子细胞遗传完整基因组至关重要。动粒失调会导致染色体不稳定，这与肿瘤发生、发育缺陷以及年龄相关的不孕症有关。因此，定义着丝粒力产生的生物物理机制对于发展对着丝粒蛋白疾病相关突变的机制理解是必要的。尽管过去十年我们在理解动粒蛋白质组成方面取得了巨大进展，但对其作为力发生器的功能的机械理解仍然难以捉摸。进一步进展的主要障碍是缺乏对动粒分子结构的了解。因此，我们提出了一种新颖的“结构-功能”方法来建立动粒结构与其功能之间的机械联系。目标 1：开发一种新的荧光显微镜方法来重建纳米级动粒结构。我们开发了一种新技术来确定活细胞中蛋白质的纳米级分布。我们对动粒结构的初步重建提出了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蜂窝机器的架构。目标 2：确定产生力的分子的位置如何定义其功能。我们将对我们的新模型进行“结构-功能”分析，其中我们将研究着丝粒结构的变化对其功能的影响。这项工作将定义动粒产生力的生物物理原理。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着丝粒蛋白组装来确定关键着丝粒蛋白 Ndc80 的必要和充分的结构特征，以重建其在体内观察到的分布和功能。这这项工作将建立一个在细胞中构建人工动粒的框架。