Architecture-function analysis of the kinetochore motor

描述（由申请人提供）：我们的目标是了解着丝点蛋白的纳米级排列如何塑造其功能和调节机制。着丝点是驱动染色体运动的大分子马达，在细胞分裂过程中保证染色体的准确分离。染色体运动所需的着丝点力的产生是两个子细胞完整基因组遗传的关键。着丝点失调导致染色体不稳定，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

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.