Architecture-function analysis of the kinetochore motor

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形成其功能和调节机制。动粒是一种大分子马达，它驱动染色体运动，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量的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，这与肿瘤的发生、发育缺陷以及与年龄相关的不孕症有关。因此，定义 动粒体力产生的生物物理机制对于从机制上理解动粒蛋白质中与疾病相关的突变是必要的。尽管在过去的十年里，我们在理解动粒的蛋白质组成方面取得了巨大的进步，但对其作为力生成器的功能的机械理解仍然是难以捉摸的。进一步发展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“建筑-功能”方法来建立动核建筑与其功能之间的机械联系。目的1：建立一种新的荧光显微镜方法来重建纳米尺度的动粒结构。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：确定作用力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‘架构-功能’分析，在这个分析中，我们将研究动核架构的变化对其功能的影响。这项工作将确定由动粒产生力量的生物物理原理。目标3：定义Kinetochore的最小体系结构规范。我们将使用体外实验和人工动粒蛋白组装来确定关键动粒蛋白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建立人造动粒的框架。

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.