Mechanisms of Spindle Assembly

描述（由申请人提供）：遗传物质与子细胞的忠实分离对于生物体的生存至关重要。细胞必须复制其每条染色体的DNA，并将一个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，纺锤体由微管和相关蛋白的动态阵列组成。有丝分裂最复杂的方面之一是染色体向中期板的聚集，这涉及作用于动粒、染色体臂和纺锤体本身的多种力。已经提出了几个模型的分子起源，这些力量，以及何时何地，他们对纺锤体的作用，但我们没有一个清楚的了解，这些力量是如何整合在纺锤体，以实现正确和及时的染色体对齐。在本提案中，我们将：1）通过敲低我们假设对染色体聚集的不同元件有贡献的分子，然后进行高分辨率固定细胞分析，确定由聚集中关键分子的扰动引起的染色体定位中的时间和空间缺陷，其中我们识别所有染色体的位置。纺锤体中的动粒以及测量有丝分裂进程的定时缺陷，（二）开发一种新的动粒跟踪算法和相关的数据分析，以测试以下假设：在国会期间，动粒运动将具有依赖于特定分子机制，以及3）我们将询问如何通过应用目标2中开发的跟踪算法沿着目标1中概述的分子扰动来定量分析前后期染色体运动，从而在空间、分子和时间上控制染色体聚集。我们将进行一系列的分子扰动，这些分子扰动应该会破坏一个或多个聚集途径，然后研究染色体运动是如何改变的。总之，这些研究将提供显着的洞察因素，支配染色体的行为，以及如何染色体的行为是空间和时间协调。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，这对生物体的生存是必不可少的。纺锤体由微管和相关蛋白质组成，用于将染色体连接到纺锤体并确保其准确分离。鉴于有丝分裂纺锤体是许多化疗药物的目标，专门破坏纺锤体MT动力学，阐明纺锤体微管动力学调节的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.