Dissection of the Inner Centromere Regulatory Network

描述（由申请方提供）：为了使每条染色体在有丝分裂期间正确分离，其动粒必须双极附着纺锤体微管。染色体不能双定向是细胞非整倍体的主要原因，是癌症和出生缺陷的驱动力。双极附着的实现是因为姐妹动粒之间产生张力，这既稳定了微管附着又关闭了纺锤体检查点信号。理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中心，并且这些蛋白质形成网络以调节染色体乘客复合物的成员极光B激酶。我们已经将CPC纯化至均一，并开发了一个系统来研究其体外活化。这些实验揭示了正反馈和负反馈回路以及关键突变体，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，这使我们能够容易地敲除和替换蛋白质，并在正常的二倍体组织中解剖表型。体外生物化学、非洲爪蟾提取物和现在动物帽中的表型解剖的组合提供了在脊椎动物系统中在生物化学和细胞生物学方法之间无缝移动的独特机会。我们假设CPC的一个作用是产生可溶性磷酸化活性的梯度，提供空间信息以形成细胞有丝分裂事件的3D空间。我们也将测试这一重要的假设，以及确定的作用，极光B在产生一个中央带的RhoA，确定的位置的细胞动力学沟。最后，我们将进行内着丝粒染色质的纯化，以系统地鉴定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：有丝分裂期间染色体的错误分离是癌症基因突变的主要来源。在有丝分裂过程中，每个染色体在其动粒之间组装一个内部着丝粒，这是确保染色体精确分离的关键信号中心。在这个提议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物的调节，其中包括极光B激酶。实验采用爪蟾提取物的力量来剖析功能和从纯化的蛋白质中重建复合试剂。我们还扩大了非洲爪蟾系统采用细胞周期表型的表型表征非洲爪蟾胚胎。这种生物化学、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力量。

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.