Structural and Functional Analysis of Mitotic Spindle Forces

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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.