Dissection of the Inner Centromere Regulatory Network

描述(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，其着丝点必须两极连接纺锤体微管。染色体生物定向失败是细胞非整倍体的主要原因，非整倍体是癌症和出生缺陷的驱动力。之所以实现双极连接，是因为姐妹动点之间产生了张力，这既稳定了微管连接，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着丝点之间的张力，并利用这一点来调节微管附着和纺锤体检查点信号。定位于着丝粒内侧的蛋白质是这些过程的中心，这些蛋白质形成一个网络来调节极光B激酶，它是染色体乘客复合体的成员。我们已经将CPC纯化为均一种，并开发了一套系统来研究其体外活性。这些实验正在揭示正反馈环和负反馈环以及关键突变，以剖析这些途径在体内的作用。为了确定突变体的特征，我们使用了非洲爪哇胚胎的动物帽，这使得我们可以很容易地敲除和替换蛋白质，并分析正常二倍体组织中的表型。体外生物化学、非洲爪哇提取物和现在对动物帽子表型的解剖相结合，为在脊椎动物系统中在生化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的作用之一是产生可溶磷活动的梯度，为有丝分裂事件的细胞3D空间模式提供空间信息。我们还将检验这一重要假说，并确定极光B在产生决定细胞动态沟位置的RhoA中央带中的作用。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。在有丝分裂过程中，每条染色体在其着丝点之间组装一个内部着丝粒，这是确保染色体准确分离的关键信号中心。该方案中的实验系统地解剖了内部着丝粒区域，重点是染色体乘客复合体的调节，其中包括Aurora B激酶。这些实验利用非洲爪哇提取物的力量，从纯化的蛋白质中剖析复杂试剂的功能和重组。我们还利用非洲爪哇胚胎中细胞周期表型的表型特征来扩展非洲爪哇的系统。生化、细胞生物学和活体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.