Kinetochore Structure and Function

动粒结构和功能

基本信息

批准号：
7999986
负责人：
Timothy Yen
金额：
$ 8万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-07 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7999986
关键词：
Address Anaphase Antibodies Antineoplastic Agents Binding Binding Proteins Binding Sites CENP-E protein Cells Centromere Chromosome Segregation Chromosomes Complex Congenital Abnormality Data Defect Development Event Exhibits Failure Funding Grant Health Hela Cells Human In Vitro Infertility Kinetochores Light Malignant Neoplasms Mechanics Mediating Metaphase Microtubules Mitosis Mitotic Mitotic Checkpoint Modeling Molecular Molecular Structure Monitor Paper Phosphoric Monoester Hydrolases Phosphorylation Phosphorylation Site Phosphotransferases Positioning Attribute Property Protein Kinase Proteins Publishing Recruitment Activity Reporting Role Signal Transduction Specific qualifier value Staining method Stains Structure Testing Time Work anaphase-promoting complex aurora B kinase cancer cell in vivo inhibitor/antagonist inner centromere protein novel tool

项目摘要

DESCRIPTION (provided by applicant): The broad objective of our lab is to understand the key mechanical and regulatory events that specify accurate chromosome segregation in human cells. We have focused our efforts on characterizing the molecular functions of the kinetochore, a macromolecular structure that mediates the attachment between chromosomes and the spindle. Work supported over the lifetime of this grant has contributed towards addressing outstanding questions such as how kinetochores capture and maintain stable connections with microtubules, how proper attachments generate kinetochore tension and how improper attachments are detected and delays cells from prematurely exiting mitosis. These studies are of direct importance to understanding human health issues such as birth defects, infertility, and cancer. Our studies have also directly stimulated the development of new anti-cancer drugs that may increase the selectivity towards rapidly dividing cancer cells. Our most recent studies have focused on structure and function analysis of CENP-E to determine how it contributes to kinetochore:microtubule attachments and how these activities are monitored by the mitotic checkpoint protein, hBUBRI kinase. Our discovery of the Mitotic Checkpoint Complex (MCC) as the major inhibitor of the Anaphase Promoting Complex (APC) in Hela cells led to a new two-step model for how APC is inhibited by the checkpoint. Our studies of CENP-I provided in vivo evidence that supported the existence of kinetochore-dependent and -independent mechanisms that are required to inhibit the APC. We are addressing the mechanism of action of hBUBRI kinase by identifying residues that are phosphorylated in mitosis. We generated phospho-hBUBRI antibodies that differentially stain kinetochores lacking attachments or tension. Unattached kinetochores and propose to use them to examine how hBUBRI is regulated by microtubule attachments. We recently reported that CENP-F is a new microtubule binding protein that is essential for proper kinetochore attachments and also contributes towards the mitotic checkpoint. Finally, we report a new component of the inner centromere that we call CENP-J. Its characterization has revealed a role in error correction that can be attributed to its ability to recruit the microtubule depolymerase, MCAK to the inner centromere. Depletion of CENP-J cause cells to accumulate aberrant kinetochore attachments that fail to generate tension and thus delay at metaphase. Failure to correct these defective attachments by the time cells overcome the mitotic delay results in lagging chromosomes during the ensuing anaphase. We want to extend our studies to continue to address the major issues about how checkpoint proteins monitor kinetochore attachments, how the "wait anaphase" signal is generated and how errors in attachment are corrected.

描述（由申请人提供）：我们实验室的主要目标是了解人类细胞中精确染色体分离的关键机械和调控事件。我们的工作重点是表征着丝粒的分子功能，着丝粒是一种介导染色体和纺锤体之间附着的大分子结构。在这笔赠款的整个生命周期中支持的工作有助于解决悬而未决的问题，例如动粒如何捕获并维持与微管的稳定连接，正确的附着如何产生动粒张力以及如何检测不正确的附着并延迟细胞过早退出有丝分裂。这些研究对于了解出生缺陷、不孕症和癌症等人类健康问题具有直接重要意义。我们的研究还直接刺激了新型抗癌药物的开发，这些药物可能会增加对快速分裂的癌细胞的选择性。我们最近的研究重点是 CENP-E 的结构和功能分析，以确定它如何对动粒：微管附着做出贡献，以及如何通过有丝分裂检查点蛋白 hBUBRI 激酶监测这些活动。我们发现有丝分裂检查点复合物 (MCC) 作为 Hela 细胞中后期促进复合物 (APC) 的主要抑制剂，从而建立了一个新的两步模型来研究检查点如何抑制 APC。我们对 CENP-I 的研究提供了体内证据，支持抑制 APC 所需的着丝粒依赖和独立机制的存在。我们正在通过鉴定有丝分裂中磷酸化的残基来研究 hBUBRI 激酶的作用机制。我们生成了磷酸化 hBUBRI 抗体，可以对缺乏附着或张力的动粒进行差异染色。未附着的动粒并建议使用它们来检查 hBUBRI 如何受微管附着的调节。我们最近报道，CENP-F 是一种新的微管结合蛋白，对于正确的着丝粒附着至关重要，并且也有助于有丝分裂检查点。最后，我们报告了内部着丝粒的一个新组成部分，我们称之为 CENP-J。它的表征揭示了其在纠错中的作用，这可归因于其将微管解聚酶 MCAK 招募到内部着丝粒的能力。 CENP-J 的耗尽会导致细胞积累异常的着丝粒附着物，这些附着物无法产生张力，从而延迟中期。当细胞克服有丝分裂延迟时未能纠正这些有缺陷的附着会导致染色体在随后的后期滞后。我们希望扩展我们的研究，继续解决有关检查点蛋白如何监测动粒附着、如何生成“等待后期”信号以及如何纠正附着错误的主要问题。