Structural and Functional Analysis of Mitotic Spindle Forces

本提案的具体目标是了解 纺锤体微管（MT）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）能源需求， 动粒在染色体与纺锤体结合过程中的作用 前中期和2）使用高分辨率UV分析纺锤体力 主轴部件的微束辐照。我们认为染色体 运动由两类主轴MT控制， 在分裂中期形成一个稳定的纺锤体两极之间的连续体。 染色体和动粒之间的连续体 纤维（kMT连续体）被假定为：1）负责 将染色体移至中期板，2）能够减少纺锤体 在前中期到中期过渡期间的长度，3）间接 与中期纺锤体形状的形成有关，4） 在分裂后期直接导致染色体与极分离 B。 第二个连续体是由那些与 染色体（nKMT连续体）并保持加载到 在正常纺锤体形成过程中，它以MT曲率的形式存在。 的 该建议的实验重点是确定能量 将压缩（能量）加载到模板中所需的要求 纺锤体在前中期到中期的转变。 功能 使用ATP类似物奎纳克林进行测定，以确定 动粒和MT相关蛋白在预加载这一过程中的作用 能量进入纺锤体。 第二个重点将是紫外线微束 研究确定微管的作用，也许是第二， 保持和表达这种能量的弹性成分。 这些研究 将允许主轴力的分析负责国会的 染色体向分裂中期板的运动 在后期的事件中。 利用光和电子光学分析 将分析有丝分裂的扰动。 适当时，三 连续切片电子显微照片的三维重建将 并测定了处理对纺锤体的影响 形态和功能。 问题的重要性在于 核分裂的意义：没有核分裂就没有真核生命 它。尽管有100多年的研究，在20世纪60年代， 在过去的十年里，它仍然是细胞中未解决的基本问题之一， 生物学

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.