Mechanisms of "End On" Microtubule Attachment by the Kinetochore

描述（由申请人提供）：动粒是在染色体分离期间协调许多事件的蛋白质机器，包括移动染色体、产生纺锤体检查点信号和纠正不正确的微管附着。着丝粒结合并调节微管的正末端以执行这些细胞过程。该提案剖析了端对附件的机制，使用的结构-功能分析的关键组成部分在体外和体内试验的组合。我们将解剖的Ndc 80/Hec 1亚基的Ndc 80复合物，这是一个重要的微管接口上的微管结合活性的作用。我们还将剖析一个新的球员在着丝点调节的功能。Cep 57是我们最近发现的一种蛋白质，它是末端连接所必需的。Cep 57在该过程中起着与Ndc 80不同的作用，即使Ndc 80不受影响，Cep 57也是附着所必需的。Cep 57直接结合微管和许多动粒动力学的调节剂，将其置于结合微管的蛋白质和调节末端附着微管的蛋白质之间的关键界面。最后，我们将对外动粒板进行结构分析，以了解为什么动粒中有如此多的蛋白质直接接触微管。我们的数据表明，动粒具有巨大的潜力，作为一个以前未开发的目标，抗有丝分裂化疗，可能有深远的影响，对人类疾病的治疗。 公共卫生：有丝分裂期间染色体的错误分离是癌症基因突变的主要来源。在有丝分裂过程中，每条染色体组装两个称为动粒的大型蛋白质机器，它们驱动复制的DNA链分离到两个子细胞。动粒蛋白在癌症中经常发生突变，这种机器已成为化疗药物的重要新靶点。该提案中的实验将阐明重要蛋白质在动粒中的作用，长期目标是了解这种复杂的机器如何分离染色体以及癌症如何改变其蛋白质以更快地进化。

DESCRIPTION (provided by applicant): Kinetochores are proteinaceous machines that coordinate numerous events during chromosome segregation, including moving chromosomes, generating spindle checkpoint signals and correcting improper microtubule attachments. Kinetochores bind and regulate the plus-ends of microtubules to perform these cellular processes. This proposal dissects the mechanisms of end-on attachment using a structure-function analysis of key components in a combination of in vitro and in vivo assays. We will dissect the role of a microtubule binding activity on the Ndc80/Hec1 subunit of the Ndc80 complex, which is proposed to be an important microtubule interface. We will also dissect the function of a new player in kinetochore regulation. Cep57 is a protein that we have shown recently is required for end-on attachment. Cep57 plays a different role in the process than Ndc80 and is required for attachment even when Ndc80 is unaffected. Cep57 directly binds microtubules and a number of regulators of kinetochore dynamics, placing it at a critical interface between the proteins that bind microtubules and the proteins that regulate end-on attached microtubules. Finally, we will perform a structural analysis of the outer kinetochore plate to understand why so many proteins in the kinetochore directly contact the microtubule. Our data suggest that the kinetochore has enormous potential as a previously untapped target for anti-mitotic chemotherapy that could have a profound impact on the treatment of human disease. PUBLIC HEALTH REVELANCE: The missegregation of chromosomes during mitosis is a major source of genetic mutations in cancer. During mitosis, every chromosome assembles two large proteinaceous machines called kinetochores that drive the segregation of the replicated DNA strands to the two daughter cells. Kinetochore proteins are often mutated in cancers, and this machine has become an important new target for chemotherapeutics. The experiments in this proposal will elucidate the roles of important proteins in the kinetochore, with the long-term goal of understanding how this complex machine segregates chromosomes and how cancers change their proteins to evolve more rapidly.