Dissection of the Inner Centromere Regulatory Network

描述（由申请人提供）：为了使每条染色体在有丝分裂过程中正确分离，它的着丝点必须两极连接纺锤体微管。染色体双定向失败是细胞非整倍体的主要原因，是癌症和出生缺陷的驱动力。双极性附着的实现是因为姐妹着丝点之间产生张力，这既稳定了微管附着，又关闭了纺锤体检查点信号。理解细胞如何变成非整倍体的关键是理解染色体如何感知姐妹着丝点之间的张力，并利用它来调节微管附着和纺锤体检查点信号。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染色体乘客复合体的成员Aurora B激酶。我们将CPC纯化至均匀性，并建立了体外活性研究体系。这些实验揭示了正反馈回路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。为了描述突变体，我们使用了非洲爪蟾胚胎的动物帽，这使我们能够轻松地敲除和替换蛋白质，并解剖正常二倍体组织的表型。体外生物化学，爪蟾提取物和现在解剖动物帽的表型的结合提供了一个独特的机会，在脊椎动物系统的生物化学和细胞生物学方法之间无缝移动。我们假设CPC的一个作用是产生可溶性磷活性的梯度，为有丝分裂事件的细胞三维空间提供空间信息。我们还将测试这一重要假设，并确定极光B在产生RhoA中心带中的作用，RhoA中心带决定了细胞动力学沟的位置。最后，我们将进行内部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。

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.