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：
7889631
负责人：
STEFAN BEKIRANOV
金额：
$ 32.15万
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/7889631
关键词：
Anaphase Aneuploidy Animal Cap Base Sequence Biochemical Biochemistry Biological Cell Cycle Cell membrane Cells Centromere 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）：每个染色体在有丝分裂过程中正确分离的每个染色体，其动力学必须双极固定纺锤体微管。染色体对生物的失败是细胞非整倍性的主要原因，癌症和先天缺陷的驱动力。之所以会达到双极附件，是因为姊妹动物学体之间会产生张力，这既可以稳定微管的附件并关闭主轴检查点信号。理解细胞如何变成非整倍体的关键是了解染色体如何感知姐妹动物之间的张力并使用它来调节微管附着和主轴检查点信号。位于内侧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，以调节Aurora B激酶，该蛋白是染色体乘客复合物的成员。我们已经将CPC纯化为同质性，并开发了一种在体外研究其激活的系统。这些实验正在揭示正反馈和负反馈循环以及关键突变体，以剖析这些途径在体内的作用。为了表征突变体，我们正在采用爪蟾胚胎的动物帽，使我们能够轻松敲低和替换蛋白质并在正常二倍体组织中解剖表型。体外生物化学，爪蟾提取物和动物帽表型的解剖的结合为在脊椎动物系统中生物化学和细胞生物学方法之间无缝移动提供了独特的机会。我们假设CPC的一个作用是生成可溶性磷酸活性的梯度，该梯度提供空间信息以对细胞的3D空间进行有丝分裂事件的形式。我们还将检验这一重要的假设，并确定Aurora B在生成RhoA中心带中的作用，该rhoA确定了细胞动力学沟的位置。最后，我们将执行内侧丝粒染色质的纯化，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析是癌症基因突变的主要来源。在有丝分裂期间，每个染色体在其动力学之间组装一个内侧丝粒，这是确保准确的染色体分离的关键信号中心。该建议中的实验系统地剖析了内侧丝粒区域，重点是调节染色体乘客复合物，其中包括Aurora B激酶。该实验采用了爪蟾提取物的力量来剖析纯化蛋白质的复杂试剂的功能和重构。我们还通过采用异武器胚胎中细胞周期表型的表型表征来扩展异武系统。生化，细胞生物学和体内技术的这种结合提供了独特的实验能力，可以剖析这一重要问题。