Kinetochore Structure and Function

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的染色体分离的关键机械和调控事件。我们的工作重点是描述着丝点的分子功能，着丝点是一种大分子结构，介导染色体与纺锤体之间的连接。该基金支持的工作有助于解决悬而未决的问题，例如着丝点如何捕获并保持与微管的稳定连接，适当的附着如何产生着丝点张力，以及如何检测到不适当的附着并延迟细胞过早脱离有丝分裂。这些研究对于理解诸如出生缺陷、不孕症和癌症等人类健康问题具有直接的重要性。我们的研究也直接刺激了新的抗癌药物的开发，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的结构和功能分析上，以确定它如何参与着丝点：微管附着，以及这些活性如何被有丝分裂检查点蛋白hBUBRI激酶监测。我们在Hela细胞中发现有丝分裂检查点复合体（MCC）是后期促进复合体（APC）的主要抑制剂，这为检查点如何抑制APC提供了一个新的两步模型。我们对CENP-I的研究提供了体内证据，支持着丝酶依赖和独立机制的存在，这些机制是抑制APC所必需的。我们正在通过鉴定有丝分裂中磷酸化的残基来解决hBUBRI激酶的作用机制。我们产生了磷酸hbubri抗体，对缺乏附着或张力的着丝点进行差异染色。未附着的动着点，并建议使用它们来研究hBUBRI是如何被微管附着调节的。我们最近报道了CENP-F是一种新的微管结合蛋白，对于着丝点的正常附着和有丝分裂检查点也有重要作用。最后，我们报道了内着丝粒的一个新成分，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合酶（MCAK）招募到内部着丝粒的能力。CENP-J的缺失导致细胞积累异常的着丝点附着物，不能产生张力，从而导致中期延迟。当细胞克服有丝分裂延迟时，未能纠正这些有缺陷的附着物，导致在随后的后期染色体滞后。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着丝点附着，“等待后期”信号如何产生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.