Mechanisms of "End On" Microtubule Attachment by the Kinetochore

描述（由申请人提供）：着丝点是蛋白质机器，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适当的微管附着。着丝点结合并调节微管的正端来完成这些细胞过程。本研究通过对关键成分的结构-功能分析，结合体外和体内实验，剖析了端-端连接的机制。我们将剖析微管结合活性在Ndc80复合物的Ndc80/Hec1亚基上的作用，Ndc80亚基被认为是一个重要的微管界面。我们还将剖析一个新参与者在着丝点调控中的作用。Cep57是我们最近证明的端上附着所必需的一种蛋白质。Cep57在这个过程中扮演的角色与Ndc80不同，即使Ndc80不受影响，Cep57也是附着所必需的。Cep57直接结合微管和许多着丝点动力学调节因子，将其置于结合微管的蛋白质和调节端上附着微管的蛋白质之间的关键界面。最后，我们将对外着丝点板进行结构分析，以了解为什么着丝点中有如此多的蛋白质直接与微管接触。我们的数据表明，着丝点具有巨大的潜力，作为一个以前未开发的抗有丝分裂化疗靶点，可能对人类疾病的治疗产生深远的影响。

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.