Molecular dissection of end-on KT attachment to MTs

描述（由申请人提供）：在细胞分裂期间，细胞将相同的染色体拷贝移入两个子细胞。细胞不能容忍染色体的不正确分离，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，细胞构建了有丝分裂纺锤体，这是一种由称为微管的大蛋白丝组成的足球状结构，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动粒的复制染色体上的大蛋白复合物上，这种附着允许染色体随着微管的生长和收缩而沿着运动。为了使动粒附着能够将微管动力学转化为染色体运动，动粒必须以微管的末端被掩埋在动粒结构中的方式“端部上”附着。末端连接必须足够强，以移动整个染色体，但足够动态，即使数千个微管蛋白亚基被添加并从连接位点丢失，连接也可以保留。虽然我们了解了着丝粒的哪些蛋白质成分需要与微管结合，但如何实现末端连接仍然是个谜。该提案概述了使用单分子测定来确定微管与着丝粒末端结合的机制和蛋白质要求的实验。 公共卫生相关性：我们提出的研究与癌症进展和肿瘤发生高度相关，因为许多癌症都存在染色体分离缺陷，并且许多动粒蛋白在癌细胞中过表达。了解分离是如何正常发生的，有助于我们了解癌细胞中发生了什么问题，并将揭示癌症治疗的更多靶点。

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.