Structural and Functional Analysis of Mitotic Spindle Forces

这项建议的具体目标是了解纺锤体微管(MTS)在有丝分裂过程中染色体运动中的作用。重点将放在两个方面：1)着丝点在早中期将染色体附着在纺锤体上的能量需求和作用；2)用高分辨率紫外线微束照射纺锤体组件来分析纺锤体的作用力。我们认为染色体的运动是由两类纺锤体MTS控制的，每一类纺锤体MTS都通过中期在两个纺锤体极之间形成一个稳定的连续体。染色体和着丝粒纤维之间的连续体(KMT连续体)被认为是：1)负责将染色体移动到中期板，2)能够在早中期到中期的转变中减少纺锤体的长度，3)间接地与中期纺锤体形状的形成有关，4)直接负责B期染色体到极点的分离。第二连续体由与染色体无关的染色体组成(nKMT连续体)，在正常纺锤体形成过程中以MT曲率的形式被加载到第二连续体中。这一建议的实验重点是确定在前中期到中期转变期间将压缩(能量)加载到形体纺锤体所需的能量需求。使用ATP类似物奎纳克林进行功能分析，以确定动粒和MT相关蛋白在预先将这种能量加载到纺锤体中的作用。第二个重点将是紫外线微束研究，以确定微管以及第二个弹性成分在保持和表达这种能量方面的作用。这些研究将能够分析导致染色体向中期平板聚集和后期染色体运动的纺锤力。利用光学和电子光学对有丝分裂的扰动进行分析。在适当的情况下，还将进行连续切片电子显微镜的三维重建，以确定治疗对纺锤体形态和功能的影响。这个问题的意义在于核分裂的意义：没有它就没有真核生物。尽管经过了100多年的研究，在过去的十年里得到了很大的加强，但它仍然是细胞生物学中尚未解决的基本问题之一。

The specific goal of this proposal is to understand the role of spindle microtubules (MTs) in chromosome motion during mitosis. Emphasis will be placed in two areas: 1) the energy requirements and role of the kinetochore in attaching chromosomes to the spindle during prometaphase and 2) analysis of spindle forces using high resolution UV microbeam irradiation of spindle components. We propose that chromosome movement is controlled by two classes of spindle MTs, each of which forms a stable continuum between the two spindle poles by metaphase. The continuum associated between the chromosomes and the kinetochore fibers (the kMT continuum) is postulated to be: 1) responsible for moving chromosomes to the metaphase plate, 2) able to reduce spindle length during the prometaphase-to-metaphase transition, 3) indirectly related to the creation of the shape of the spindle at metaphase and, 4) directly responsible for chromosome-to-pole separation during anaphase B. The second continuum is composed of those MTs not associated with chromosomes (nKMT continuum) and hold compression which is loaded into it in the form of MT curvature during normal spindle formation. The experimental focus of this proposal is to determine the energy requirements necessary to load compression (energy) into the form spindle during the prometaphase-to-metaphase transition. Functional assays, using the ATP analog quinacrine will be done to establish the role of the kinetochore and MT associated proteins in pre-loading this energy into the spindle. The second emphasis will be a UV microbeam study to determine the role of microtubules and perhaps a second, elastic component in holding and expressing this energy. These studies will allow analysis of spindle forces responsible for the congression of chromosomes to the metaphase plate and the movement of chromosomes during the events of anaphase. Using light and electron optics analysis of perturbations of mitosis will be analyzed. Where appropriate, three dimensional reconstruction of serial section electron micrographs will also be done to determine the effects of treatments on spindle morphology and function. The significance of the problem is the significance of nuclear division: there is no eukaryotic life without it. In spite of over 100 years of research, much intensified during the past decade, it remains one of the unsolved fundamental problems in cell biology.