Mechanisms of Spindle Assembly

描述（由申请人提供）：将遗传物质忠实地分离到子细胞对生物体的生存至关重要。细胞必须复制每条染色体的DNA，并将一个副本分配给两个子细胞。染色体分离是由有丝分裂纺锤体介导的，它是由微管和相关蛋白的动态阵列组成的。有丝分裂中最复杂的一个方面是染色体向中期板聚集，这涉及到许多作用于着丝点、染色体臂和纺锤体本身的力。对于这些力的分子起源，以及它们在何时何地作用于纺锤体，已经提出了几种模型，但我们对这些力如何在纺锤体内整合以实现适当和及时的染色体排列还没有清楚的了解。在本建议中，我们将：1)通过敲除我们假设对染色体聚集的不同要素有贡献的分子，确定由聚集中关键分子的扰动引起的染色体定位的时间和空间缺陷，然后进行高分辨率的固定细胞分析，其中我们确定纺锤体中所有染色体/着丝点的位置，并测量有丝分裂进程时间上的缺陷。2)开发一种新的着丝点跟踪算法和相关的数据分析，以测试着丝点在聚集过程中的运动将具有依赖于特定分子机制的可测量特征的假设。3)我们将询问染色体聚集是如何在空间上进行的。通过应用Aim 2中开发的跟踪算法以及Aim 1中概述的分子扰动来定量分析前期后期染色体运动，从而进行分子和时间控制。我们将执行一系列的分子扰动，应该破坏一个或多个途径的国会，然后问染色体运动是如何改变的。总之，这些研究将为控制染色体行为的因素以及染色体行为如何在空间和时间上协调提供重要的见解。公共卫生相关性：有丝分裂纺锤体向子细胞忠实分离遗传物质对生物体的生存至关重要。纺锤体由微管和相关蛋白组成，用于将染色体连接到纺锤体上并确保其准确分离。鉴于有丝分裂纺锤体是许多化疗药物的靶标，这些化疗药物专门破坏纺锤体MT动力学，阐明纺锤体微管动力学的调节机制对生物医学研究具有重要意义。

DESCRIPTION (provided by applicant): The faithful segregation of genetic material to daughter cells is essential for the survival of an organism. The cell must replicate the DNA of each of its chromosomes and distribute one copy to each of the two daughter cells. Chromosome segregation is mediated by the mitotic spindle, which is composed of a dynamic array of microtubules and associated proteins. One of the most complex aspects of mitosis is the congression of chromosomes to the metaphase plate, which involves a multitude of forces acting on the kinetochore, on the chromosome arms, and on the spindle itself. Several models have been proposed for the molecular origins of these forces as well as for when and where they act on the spindle, but we do not have a clear understanding of how these forces are integrated within the spindle to achieve proper and timely chromosome alignment. In the present proposal we will: 1) Determine the temporal and spatial defects in chromosome positioning caused by perturbation of key molecules in the congression by knocking down molecules that we hypothesize contribute to different elements of chromosome congression and then performing a high-resolution fixed cell analysis in which we identify the locations of all chromosomes/ kinetochores in the spindle as well as measure the defects in the timing of mitotic progression, 2) Develop a new kinetochore tracking algorithm and associated data analysis to test the hypothesis that kinetochore movements during congression will have measurable characteristics that depend upon a specific molecular mechanism, and 3) We will ask how chromosome congression is spatially, molecularly and temporally controlled by applying the tracking algorithms developed in Aim 2 along with the molecular perturbations outlined in Aim 1 to quantitatively analyze pre-anaphase chromosome movement. We will perform a series of molecular perturbations that should disrupt one or more pathways of congression and then ask how chromosome movement is altered. Together these studies will provide significant insight into the factors that govern chromosome behavior as well as how chromosome behavior is spatially and temporally coordinated. PUBLIC HEALTH RELEVANCE: The faithful segregation of genetic material by the mitotic spindle to daughter cells is essential for the survival of an organism. The spindle is composed of microtubules and associated proteins that are utilized to attach the chromosomes to the spindle and to ensure their accurate segregation. Given that the mitotic spindle is a target of numerous chemotherapeutic agents that specifically disrupt spindle MT dynamics, elucidating the mechanisms by which spindle microtubule dynamics are regulated has important implications for biomedical research.