Mechanisms of Spindle Assembly

描述(由申请人提供)：将遗传物质忠实地分离到子代细胞对生物体的生存至关重要。细胞必须复制其每条染色体的DNA，并将一份拷贝分配给两个子细胞。染色体分离是由有丝分裂纺锤体介导的，纺锤体由一系列动态的微管和相关蛋白质组成。有丝分裂最复杂的方面之一是染色体向中期板的聚集，这涉及到作用于着丝粒、染色体臂和纺锤体本身的多种力。对于这些力的分子起源以及它们何时何地作用于纺锤体，已经提出了几个模型，但我们还不清楚这些力是如何整合到纺锤体内，以实现适当和及时的染色体比对。在目前的方案中，我们将：1)通过击倒我们假设与染色体聚集的不同元素有关的分子，然后执行高分辨率固定细胞分析，确定所有染色体/着丝点在纺锤体中的位置，并测量有丝分裂进展的时间缺陷，从而确定由于会议中关键分子的扰动而引起的染色体定位的时间和空间缺陷；2)开发新的动粒跟踪算法和相关的数据分析，以检验在会议期间的动粒运动将具有依赖于特定分子机制的可测量特征的假设；以及3)我们将询问染色体聚集在空间上是如何的，通过应用目标2中开发的跟踪算法以及目标1中概述的分子扰动来进行分子和时间控制，以定量分析染色体前期和后期的运动。我们将进行一系列分子扰动，这些扰动应该扰乱一个或多个聚集路径，然后询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洞察力。公共卫生相关性：有丝分裂纺锤体将遗传物质忠实地分离到子细胞，这对有机体的生存至关重要。纺锤体由微管和相关蛋白质组成，用来将染色体连接到纺锤体上，并确保它们的准确分离。鉴于有丝分裂纺锤体是许多特异性干扰纺锤体MT动力学的化疗药物的靶标，阐明纺锤体微管动力学的调控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.