Chromosome Movement in Prometaphase

前中期染色体运动

• 批准号: 6984654
• 负责人: GARY J. GORBSKY
• 金额: $ 35.68万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6984654
• 关键词: Xenopus; Xenopus oocyte; cell component structure /function; cell cycle; cell cycle proteins; centromere; chromosome movement; dynein ATPase; enzyme activity; laboratory rabbit; microtubules; mitotic spindle apparatus; protein kinase; protein structure

DESCRIPTION (provided by applicant): We seek to understand, at the cell biological and molecular levels, how chromosomes move, how they are guided to the metaphase plate, and how microtubule interactions with the kinetochore and other mitotic organelles regulate the mitotic spindle checkpoint. Chromosome movements during prometaphase are driven primarily by dynamic interactions of the mitotic spindle microtubules with the kinetochores. At the same time, kinetochores lacking stable bipolar attachment to microtubules serve to signal the spindle checkpoint that blocks anaphase onset until metaphase alignment is achieved. This checkpoint is silenced when the all sister kinetochores establish stable bipolar microtubule attachments. Thus, chromosome attachment and movement on the mitotic spindle are intimately intertwined with the regulation of the spindle checkpoint. Previously, this lab provided evidence that translocation of the kinetochores along microtubules is the prime mediator of chromosome movement in mitosis. We later discovered that individual kinetochores within a mitotic cell were biochemically distinct and developed the model of kinetochores as catalytic sources for spindle checkpoint signaling. We have now assembled a unique set of tools to dissect the molecular roles of several of the most important regulators of chromosome movement and the spindle checkpoint in vertebrate cells. We will determine if and how microtubule attachment and mechanical tension regulate kinetochore protein dynamics and signaling. We focus on a set of key regulators of microtubule-kinetochore interactions: the Ndc80 protein complex, Polo-like kinase1, the Aurora B kinase, the dynein/dynactin complex and an associated protein complex, the ZW10/Rod complex. We will analyze the functions of these proteins in living Xenopus and mammalian cells. We will recapitulate kinetochore regulation in lysed cell systems, in fractions from mitotic cells, and with purified proteins in vitro. We will collaborate to conduct complementary studies in Xenopus egg extracts. Analyzing the roles of several dynamic protein complexes is a significant challenge. However, only by approaching kinetochore and microtubule protein dynamics in concert with a variety of in vivo and in vitro approaches can we begin to understand the regulation of mitosis in living cells.

描述（由申请人提供）：我们试图在细胞生物学和分子水平上理解染色体如何移动，它们如何被引导到中期板，以及微管与着丝点和其他有丝分裂细胞器的相互作用如何调节有丝分裂纺锤体检查点。前期染色体运动主要由有丝分裂纺锤体微管与着丝点的动态相互作用驱动。与此同时，与微管缺乏稳定的双极连接的着丝点作为纺锤体检查点的信号，阻止后期开始，直到中期对齐实现。当所有姐妹着丝点建立稳定的双极微管附着时，这个检查点沉默。因此，染色体的附着和有丝分裂纺锤体上的运动与纺锤体检查点的调节密切地交织在一起。先前，本实验室提供的证据表明，着丝点沿微管易位是有丝分裂中染色体运动的主要媒介。我们后来发现有丝分裂细胞内的单个着丝点在生化上是不同的，并建立了着丝点作为纺锤体检查点信号传导的催化源的模型。我们现在已经组装了一套独特的工具，来解剖脊椎动物细胞中几个最重要的染色体运动调节因子和纺锤体检查点的分子作用。我们将确定微管附着和机械张力是否以及如何调节着丝点蛋白动力学和信号传导。我们专注于微管-着丝点相互作用的一组关键调节因子：Ndc80蛋白复合物、polo样激酶1、Aurora B激酶、动力蛋白/动力蛋白复合物和相关蛋白复合物ZW10/Rod复合物。我们将分析这些蛋白在爪蟾和哺乳动物细胞中的功能。我们将在裂解的细胞系统、有丝分裂细胞的部分和体外纯化的蛋白质中概括着丝点调控。我们将合作开展爪蟾卵提取物的补充研究。分析几种动态蛋白复合物的作用是一项重大挑战。然而，只有通过将着丝点和微管蛋白动力学与各种体内和体外方法相结合，我们才能开始了解活细胞中有丝分裂的调节。