Mechanisms of "End On" Microtubule Attachment by the Kinetochore

描述(申请人提供)：动点是一种蛋白质机器，在染色体分离过程中协调许多事件，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。动点结合并调节微管的加端来执行这些细胞过程。这项建议通过对体外和体内试验相结合的关键成分的结构-功能分析来剖析末端附着的机制。我们将剖析微管结合活性在Ndc80复合体的Ndc80/Hec1亚单位上的作用，这被认为是一个重要的微管界面。我们还将剖析一个新的参与者在动粒调控中的作用。Cep57是我们最近发现的一种蛋白质，它是末端连接所必需的。Cep57在这一过程中扮演着与Ndc80不同的角色，即使在Ndc80不受影响的情况下，Cep57也是附着所必需的。Cep57直接结合微管和许多动粒动力学调节器，将其置于结合微管的蛋白质和调节末端连接的微管的蛋白质之间的关键界面上。最后，我们将对外部动粒芯板进行结构分析，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，着丝粒具有巨大的潜力，可以作为以前尚未开发的抗有丝分裂化疗的靶点，这可能会对人类疾病的治疗产生深远的影响。 公共卫生评论：有丝分裂过程中染色体的错误分离是癌症基因突变的主要来源。在有丝分裂期间，每条染色体都会组装两个名为动点的大型蛋白质机器，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，这台机器已成为化疗的重要新靶点。这项计划中的实验将阐明重要蛋白质在动粒中的作用，长期目标是了解这台复杂的机器是如何分离染色体的，以及癌症是如何改变它们的蛋白质以更快地进化的。

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.