Chromosome and Spindle Dynamics in Yeast

描述（由申请人提供）：本提案的目的是了解有丝分裂过程中染色体和纺锤体的动态和结构特性。动粒是位于真核生物染色体上的动态蛋白质-DNA组装体，其对于将染色体DNA连接到有丝分裂纺锤体的微管是必需的。在许多类型的肿瘤细胞中已经记录了染色体分离保真度的破坏，并且可能是肿瘤进展的起始事件。染色体非整倍性通过增加肿瘤抑制基因中隐性突变的发现率而加速肿瘤的发展。动粒蛋白的鉴定是了解染色体分离机制的关键步骤之一。理解动粒功能和染色体分离的关键将是阐明动粒与动态微管之间的联系机制。随着芽殖酵母活细胞荧光成像技术的出现，酵母基因组计划的完成，以及描述蛋白质-蛋白质相互作用网络的努力，我们准备解决着丝粒捕获微管，实现双极取向，促进染色体动力学和分离的机械过程。 具体的目标是确定的作用动粒组件在管理动粒微管动力学和动粒附着/脱离，定量的动粒蛋白质的动态特性，并确定这些动态组件如何与微管相互作用，产生一个机械健全的联系。我们将确定允许着丝粒侧翼DNA在微管附着后高度延伸的结构要求，以及染色体拉伸是否有助于纺锤体检查点。我们的方法需要染色体工程，染色体和蛋白质动力学的定量荧光显微镜和动粒组件的遗传操作的分析。

DESCRIPTION (provided by applicant): The objective of this proposal is to understand the dynamic and structural properties of chromosomes and the spindle during mitosis. Kinetochores are dynamic protein-DNA assemblies situated along eukaryotic chromosomes that are essential for linking chromosomal DNA to microtubules of the mitotic spindle. A breakdown in the fidelity of chromosome segregation has been documented in many types of tumor cells and is likely to be an initiating event in tumor progression. Chromosomal aneuploidy will accelerate tumor development by increasing the rate at which recessive mutations in tumor suppressor genes are uncovered. The identification of kinetochore proteins is one of many critical steps toward understanding mechanisms of chromosome segregation. The key to understanding kinetochore function and chromosome segregation will be the elucidation of mechanisms that link kinetochores to dynamic microtubules. With the advent of live cell fluorescence imaging techniques in budding yeast, the completion of the yeast genome project, and efforts to describe the network of protein-protein interactions, we are poised to address mechanistic processes by which kinetochores capture microtubules, attain bipolar orientation, and promote chromosome dynamics and segregation. The specific objectives are to determine the role of kinetochore components in governing kinetochore microtubule dynamics and kinetochore attachment/detachment, quantitative the dynamic properties of kinetochore proteins, and determine how these dynamic assemblies interact with microtubules to generate a mechanically sound linkage. We will determine the structural requirements that allow centromere flanking DNA to become highly extended upon microtubule attachment, and whether chromosome stretching contributes to the spindle checkpoint. Our approach entails chromosome engineering, analysis of chromosome and protein dynamics by quantitative fluorescence microscopy and genetic manipulation of kinetochore components.