Determining how the spindle assembly checkpoint monitors chromosome biorientation

描述（由申请人提供）：染色体分离必须仔细调节，因为错误分离具有灾难性后果，如基因组不稳定和癌症。为了正确分离，所有复制的染色体必须在有丝分裂纺锤体上正确定向，每对姐妹动粒从相对的纺锤体极附着到微管上。即使单个染色体未能实现这种“双向”构型，也会触发主要的细胞周期检查点反应，称为纺锤体组装检查点（SAC），其停止细胞周期并防止姐妹染色单体分离。SAC的许多下游效应物已经被很好地表征，但是关于染色体错位如何触发SAC的初始激活的机制细节仍然知之甚少。在这种调节中的关键参与者是控制必需的有丝分裂激酶Aurora B活性的染色体乘客复合物（CPC）。强调必须 CPC与细胞周期调控的关系，已知CPC的成员在癌细胞中上调，目前正被用作药物开发的靶点。然而，尽管如此重要，但对产品总分类各组成部分的职能缺乏透彻的了解。这里提出的研究培训计划描述了三个互补的实验线，以研究CPC控制极光B活性的机制，从而协调染色体结构和细胞周期进程。这些研究将为染色体生物学和细胞周期调控提供重要的见解，这将与基因组维护以及对癌症进展的理解产生广泛的相关性。本文提出的研究培训计划的目标是：1）确定CPC如何影响Aurora B在有丝分裂染色体上的定位和动态周转; 2）阐明染色体双定向影响Aurora B激酶的哪些靶点; 3）揭示与特异性结合配偶体的相互作用是否赋予CPC功能对染色体结构的敏感性。为了实现这些目标，三个补充线的实验将进行使用非洲爪蟾卵提取物系统，它已被广泛用于表征细胞周期调控的生物化学，因为它提供了前所未有的控制细胞周期进程。非洲爪蟾系统的力量将与高分辨率显微镜分析和最近的创新相结合，在有丝分裂染色质的重建和纯化。这些实验将检验目前对CPC功能的假设，并将为将尖端技术应用于重要的 染色体生物学和细胞周期控制领域。 公共卫生相关性：许多癌症和发育缺陷（如唐氏综合症）的根本原因是未能协调细胞分裂与染色体分离。这里提出的研究调查了一组蛋白质的功能，这些蛋白质通过在细胞分裂过程中的关键点监测染色体的正确方向来控制这种协调。该项目将深入了解这些重要蛋白质的功能，这些蛋白质将指导药物开发，并对我们理解染色体生物学和细胞周期调控及其对人类健康的影响产生广泛影响。

DESCRIPTION (provided by applicant): Chromosome segregation must be carefully regulated because missegregation has catastrophic consequences such as genome instability and cancer. In order to segregate properly, all replicated chromosomes must orient correctly on the mitotic spindle with each pair of sister kinetochores making attachments to microtubules from opposing spindle poles. Failure of even a single chromosome to achieve this "bioriented" configuration triggers a major cell cycle checkpoint response, known as the spindle assembly checkpoint (SAC), which halts the cell cycle and prevents separation of sister chromatids. Many of the downstream effectors of the SAC have been well characterized, but the mechanistic details of how misaligned chromosomes trigger the initial activation of the SAC remain poorly understood. A critical player in this regulation is the chromosomal passenger complex (CPC) that controls the activity of the essential mitotic kinase Aurora B. Highlighting the importance of the CPC to cell cycle control, members of CPC are known to be upregulated in cancer cells and are currently being used as targets in drug development. However, despite this importance a thorough understanding of the functions of CPC components is lacking. The research training plan proposed here describes three complementary lines of experimentation to investigate the mechanisms by which the CPC controls the activity of Aurora B and thereby coordinates chromosome structure and cell cycle progression. These studies will provide critical insights into chromosome biology and the regulation of the cell cycle that will have broad relevance to genome maintenance as well as specific impacts on the understanding of cancer progression. The goals of the research training plan proposed here are 1) to determine how the CPC effects the localization and dynamic turnover of Aurora B on mitotic chromosomes 2) to elucidate which targets of Aurora B kinase are effected by chromosome biorientation and 3) to uncover whether the interaction with specific binding partners confers the sensitivity of CPC function to chromosome structure. To meet these goals, three complementary lines of experimentation will be undertaken using the Xenopus egg extract system, which has been widely used to characterize the biochemistry of cell cycle regulation because of the unprecedented control it provides of cell cycle progression. The power of the Xenopus system will be combined with high-resolution microscopic analysis and recent innovations in the reconstitution and purification of mitotic chromatin. These experiments will test current hypotheses for the function of the CPC and will provide valuable training in the application of cutting edge technologies to the important fields of chromosome biology and cell cycle control. PUBLIC HEALTH RELEVANCE: An underlying cause of many cancers and developmental defects such as Down syndrome is a failure to coordinate cell division with the segregation of chromosomes. The research proposed here investigates the function of a group of proteins that control this coordination by monitoring the correct orientation of chromosomes at a critical point during cell division. This project will provide insights into the function of these important protens that will guide drug development and have broad impacts on our understanding of chromosome biology and cell cycle regulation and their effects on human health.