Kinetochore Structure and Function

描述（由申请人提供）：我们实验室的广泛目标是了解在人类细胞中指定准确染色体分离的关键机械和监管事件。我们的工作重点是描述动粒的分子功能，动粒是一种介导染色体和纺锤体之间连接的大分子结构。在该补助金的生命周期内支持的工作有助于解决悬而未决的问题，例如动粒如何捕获和保持与微管的稳定连接，适当的附件如何产生动粒张力以及如何检测到不适当的附件并延迟细胞过早退出有丝分裂。这些研究对了解人类健康问题，如出生缺陷，不孕不育和癌症具有直接重要性。我们的研究还直接刺激了新抗癌药物的开发，这些药物可能会增加对快速分裂的癌细胞的选择性。我们最近的研究集中在CENP-E的结构和功能分析，以确定它如何有助于动粒：微管附着以及这些活动如何由有丝分裂检查点蛋白hBUBRI激酶监测。我们发现有丝分裂检查点复合物（MCC）是Hela细胞中后期促进复合物（APC）的主要抑制剂，这为APC如何被检查点抑制提供了一个新的两步模型。我们对CENP-1的研究提供了体内证据，支持存在抑制APC所需的激动素依赖性和非依赖性机制。我们通过鉴定在有丝分裂中磷酸化的残基来研究hBUBRI激酶的作用机制。我们产生了磷酸化hBUBRI抗体，其差异染色缺乏附件或张力的动粒。并建议使用它们来研究hBUBRI是如何通过微管附着来调节的。我们最近报道，CENP-F是一种新的微管结合蛋白，对于正确的动粒附着是必不可少的，也有助于有丝分裂检查点。最后，我们报告了一个新的内部着丝粒，我们称之为CENP-J的组成部分。它的表征揭示了纠错的作用，可以归因于它的能力，招募微管解聚酶，MCAK的内部着丝粒。CENP-J的消耗导致细胞积累异常的动粒附着，其不能产生张力，从而在中期延迟。当细胞克服有丝分裂延迟时，如果不能纠正这些有缺陷的附着，就会导致随后的后期染色体落后。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，如何产生“等待后期”信号以及如何纠正附着错误的主要问题。

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.