Molecular dissection of end-on KT attachment to MTs

描述（由申请人提供）：在细胞分裂过程中，细胞将相同的染色体拷贝移动到两个子细胞中。染色体不正确的分离是细胞所不能容忍的，并可能导致细胞死亡，以及出生缺陷和癌症。为了分离染色体，细胞构建有丝分裂纺锤体，这是一个足球形状的结构，由称为微管的大蛋白质细丝组成，微管由微管蛋白亚基组成。微管是“动态的”，这意味着它们会经历生长和收缩，从末端获得和失去亚基的时期。微管末端附着在被称为着丝点的复制染色体上的大型蛋白质复合物上，这种附着允许染色体随着微管的生长和收缩而运动。为了使着丝粒附着能够将微管动力学转化为染色体运动，着丝粒必须“端对端”附着，即微管的末端埋入着丝粒结构中。端上附着体必须足够强大，足以移动整个染色体，但又必须足够动态，即使在附着位点添加和丢失数千个微管蛋白亚基时，这种附着体也能保持不变。虽然我们知道着丝点的哪些蛋白质成分需要与微管结合，但端对端连接是如何实现的仍然是一个谜。本提案概述了使用单分子分析来确定端端微管与着丝点结合的机制和蛋白质需求的实验。

DESCRIPTION (provided by applicant): During cell division, cells move identical copies of chromosomes into two daughter cells. Incorrect segregation of chromosomes is not tolerated by cells, and can lead to cell death, as well as birth defects and cancer. To segregate chromosomes, the cell constructs the mitotic spindle, a football-shaped structure comprised of large protein filaments called microtubules, which are made up of tubulin subunits. Microtubules are "dynamic", meaning they go through periods of growing and shrinking, gaining and losing subunits from their ends. Microtubule ends attach to large protein complexes on the duplicated chromosomes called kinetochores, and this attachment allows movement of chromosomes along with microtubule growing and shrinking. In order for kinetochore attachment to be able to translate microtubule dynamics into chromosome movement, the kinetochore must be attached "end-on", in such a way that the very end of the microtubule is buried in the kinetochore structure. An end-on attachment must be strong enough to move an entire chromosome, but dynamic enough that the attachment can be retained even as thousands of tubulin subunits are added and lost from the site of attachment. While we understand which protein components of the kinetochore are required to bind to microtubules, how end-on attachment is achieved is still mysterious. This proposal outlines experiments using single molecule assays to determine the mechanism and protein requirements for end-on microtubule binding to kinetochores. PUBLIC HEALTH RELEVANCE: The research we have proposed has high relevance to cancer progression and tumorigenesis, as many cancers posses chromosome segregation defects, and many kinetochore proteins are overexpressed in cancer cells. Understanding how segregation occurs normally with help us to understand what goes wrong in cancer cells, and will reveal more targets for cancer therapeutics.