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关键的机械和调控事件，这些事件规定了人类细胞中准确的染色体分离。我们集中精力研究动粒的分子功能，这是一种大分子结构，调节染色体和纺锤体之间的连接。在这笔赠款的有效期内所支持的工作有助于解决悬而未决的问题，例如动细胞如何捕捉和维持与微管的稳定连接、适当的连接如何产生动核张力以及如何检测到不适当的连接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，以确定它如何参与动粒：微管附着，以及这些活动是如何被有丝分裂检查点蛋白hBUBRI激酶监控的。我们在Hela细胞中发现有丝分裂检查点复合体(MCC)作为后期促进复合体(APC)的主要抑制物，这导致了一个新的两步模型来研究检查点是如何抑制APC的。我们对CENP-I的研究提供了体内证据，支持存在抑制APC所需的动粒依赖和非独立机制。我们正在通过鉴定在有丝分裂中被磷酸化的残基来研究hBUBRI激酶的作用机制。我们产生了磷酸化的hBUBRI抗体，这种抗体可以对缺乏附着或张力的动心进行不同的染色。并建议用它们来研究hBUBRI是如何受微管附着调控的。我们最近报道了CENP-F是一种新的微管结合蛋白，它是正常的动粒附着所必需的，也有助于有丝分裂检查点的形成。最后，我们报道了一种新的内着丝粒成分，我们称之为CENP-J。它的特性揭示了它在纠错中的作用，这可以归因于它将微管解聚酶MCAK招募到内着丝粒的能力。CENP-J的耗尽导致细胞积累异常的动粒附着，这些附着不能产生张力，因此在中期延迟。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，就会在随后的后期导致落后的染色体。我们希望扩大我们的研究范围，以继续解决检查点蛋白如何监控动粒附着、如何产生“等待后期”信号以及如何纠正附着中的错误等主要问题。

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.