Dissection of the Inner Centromere Regulatory Network

内着丝粒调节网络的解剖

基本信息

批准号：
8241085
负责人：
STEFAN BEKIRANOV
金额：
$ 31.82万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8241085
关键词：
Anaphase Aneuploidy Animal Cap Base Sequence Biochemical Biochemistry Biological Cell Cycle Cell membrane Cells Centromere ChIP-seq Chromatin Chromosome Segregation Chromosome Territory Chromosomes Complex Congenital Abnormality Cytokinesis DNA Sequence Data Deposition Diploidy Dissection Electron Microscope Embryo Ensure Event Failure Feedback Gene Mutation Grant In Vitro Kinetochores Location Malignant Neoplasms Measures Metaphase Micrococcal Nuclease Microtubules Mitosis Mitotic Mitotic Chromosome Mitotic spindle Pathway interactions Pattern Phenotype Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Plus End of the Microtubule Process Prometaphase Proteins Reading Reagent Regulation Research Role Signal Transduction Sister Sister Chromatid Source Structure System Techniques Testing Tissues To specify Work Xenopus aurora B kinase aurora kinase cohesion driving force in vivo inner centromere protein insight member morphogens mutant novel protein complex public health relevance reconstitution research study self organization

项目摘要

DESCRIPTION (provided by applicant): For each chromosome to properly segregate during mitosis, its kinetochores must bipolarly attach spindle microtubules. The failure of chromosomes to biorient is a major cause of cellular aneuploidy, a driving force in cancer and birth defects. Bipolar attachment is achieved because tension is produced between sister kinetochores, which both stabilizes microtubule attachments and turns off spindle checkpoint signals. A key to understanding how cells become aneuploid is to understand how chromosomes sense tension between sister kinetochores and use this to regulate microtubule attachment and spindle checkpoint signals. Proteins that localize to the inner centromere are central to these processes and these proteins form a network to regulate the Aurora B kinase which is a member of the chromosome passenger complex. We have purified the CPC to homogeneity and developed a system to study its activation in vitro. These experiments are uncovering both positive and negative feedback loops as well as the key mutants to dissect the role of these pathways in vivo. To characterize mutants we are employing the animal caps of Xenopus embryos which allow us to easily knockdown and replace proteins and dissect phenotypes in normal diploid tissue. The combination of in vitro biochemistry, Xenopus extracts and now dissection of phenotypes in animal caps provides a unique opportunity to move seamlessly between biochemical and cell biological approaches in a vertebrate system. We hypothesize that one role of the CPC is to generate gradients of soluble phosphoactivity that provide spatial information to pattern the 3D space of the cell for mitotic events. We will also test this important hypothesis as well as determine the role of Aurora B in generating a central band of RhoA that determines the location of the cytokinetic furrow. Finally we will perform purification of inner centromere chromatin to systematically identify proteins that localize to this chromosome territory as well as the DNA sequences that they are assembled upon. PUBLIC HEALTH RELEVANCE: The missegregation of chromosomes during mitosis is a major source of genetic mutations in cancer. During mitosis every chromosome assembles an inner centromere between its kinetochores, which is a key signaling center to ensure accurate chromosome segregation. The experiments in this proposal systematically dissect the inner centromere region with an emphasis on the regulation of the Chromosome Passenger Complex, which includes the Aurora B kinase. The experiments employ the power of Xenopus extracts to dissect function and reconstitution of complex reagents from purified proteins. We also expand the Xenopus system by employing phenotypic characterization of cell cycle phenotypes in Xenopus embryos. This combination of biochemical, cell biological and in vivo techniques provides unique experimental power to dissect this important problem.

描述（由申请人提供）：为了使每条染色体在有丝分裂期间正确分离，其动粒必须双极地附着纺锤体微管。染色体生物定向失败是细胞非整倍性的主要原因，也是癌症和出生缺陷的驱动力。双极附着的实现是因为姐妹着丝粒之间产生了张力，这既稳定了微管附着又关闭了纺锤体检查点信号。了解细胞如何成为非整倍体的关键是了解染色体如何感知姐妹动粒之间的张力，并利用它来调节微管附着和纺锤体检查点信号。定位于内部着丝粒的蛋白质是这些过程的核心，这些蛋白质形成一个网络来调节极光 B 激酶，极光 B 激酶是染色体乘客复合体的成员。我们已将 CPC 纯化至均质，并开发了一个系统来研究其体外激活。这些实验揭示了正反馈环和负反馈环以及关键突变体，以剖析这些途径在体内的作用。为了表征突变体，我们使用爪蟾胚胎的动物帽，这使我们能够轻松敲低和替换蛋白质并解剖正常二倍体组织中的表型。体外生物化学、非洲爪蟾提取物和现在对动物帽表型的解剖相结合，为脊椎动物系统中生化和细胞生物学方法之间的无缝转换提供了独特的机会。我们假设 CPC 的作用之一是产生可溶性磷酸活性的梯度，从而提供空间信息来为有丝分裂事件的细胞 3D 空间进行模式化。我们还将测试这一重要的假设，并确定 Aurora B 在生成 RhoA 中央带（决定细胞分裂沟位置）中的作用。最后，我们将对内部着丝粒染色质进行纯化，以系统地识别定位于该染色体区域的蛋白质以及它们组装的 DNA 序列。公共卫生相关性：有丝分裂过程中染色体的错误分离是癌症基因突变的主要来源。在有丝分裂期间，每条染色体在其着丝粒之间组装一个内部着丝粒，这是确保准确染色体分离的关键信号中心。该提案中的实验系统地剖析了内部着丝粒区域，重点是染色体乘客复合体（包括 Aurora B 激酶）的调节。这些实验利用非洲爪蟾提取物的力量来剖析纯化蛋白质的功能和重构复杂试剂。我们还通过利用非洲爪蟾胚胎细胞周期表型的表型特征来扩展非洲爪蟾系统。生物化学、细胞生物学和体内技术的结合为剖析这一重要问题提供了独特的实验能力。