CYTOSKELETAL DYNAMICS DURING CELL SHAPE CHANGES

描述(改编自申请人的摘要)：更改单元格 形状是许多基本细胞过程的重要组成部分 包括运动、分裂和分化。医学上重要的 形状变化包括血小板在血液凝结过程中所经历的变化 中性粒细胞和巨噬细胞在吞噬过程中表现出来的。这个 这些形状变化的分子基础在于重组 细胞的细胞骨架蛋白，主要是结构蛋白肌动蛋白和 微管蛋白。这两种主要细胞骨架成分的组织状态 进而受一系列肌动蛋白结合和相关微管的调节 蛋白质(MAP)。解释细胞形状变化的分子基础是 这取决于对 ABPs和MAP的体内功能。一种优秀的模型实验系统 用于检测ABPs和MAP功能的是海胆体腔细胞。 这些原始血细胞可以被认为是 脊椎动物的血小板和巨噬细胞 体腔液凝结，吞噬能力强，可活动。 体腔细胞可以被诱导经历同步的、肌动蛋白介导的形状 从片状变为丝状，反之亦然。 这项建议描述了一系列的形态和生化 旨在鉴定体腔细胞ABPs和MAP的实验 阐明它们在体内形状变化过程中的功能。ABPS和 MAP将从肌动蛋白和微管蛋白结合的斑点覆盖层中识别 细胞提取物，来自紫杉醇稳定的微管制剂和 DNA酶亲和层析。光和光的免疫定位 电子显微镜水平将用来检查分布 体腔细胞中的ABPS和MAP，特别强调丝径到 片状脂胞形状改变。这允许检查 肌动蛋白细胞骨架的解体与MAP马达的功能 蛋白质在细胞器官的分布中。作为次要目标， 钙对体腔细胞形状变化的调节也将被研究。 利用钙离子载体处理和钙离子载体的实验 依赖的荧光染料。这些实验的结果应该是 提供对ABP和地图的身份和功能的新见解 体腔细胞的形状改变，也应该有助于导致更好的 对其在医学上有意义的形状变化的理解 血小板和巨噬细胞。

DESCRIPTION (Adapted from the applicant's abstract): Alterations of cell shape are an important component of many fundamental cell processes including movement, division, and differentiation. Medically important shape changes include those undergone by platelets during blood clotting and those exhibited by neutrophils and macrophages during phagocytosis. The molecular basis for these shape changes lies in the reorganization of cell's cytoskeletal proteins, chiefly the structural proteins actin and tubulin. The organization state of these two major cytoskeletal elements is in turn regulated by a host of actin binding and microtubule associated proteins (MAP). Explaining the molecular basis of cell shape changes is dependent on the understanding of the in vivo function of ABPs and MAPs. One excellent model experimental system for examining the functions of ABPs and MAPs is the sea urchin coelomocyte. These primitive blood cells can be considered as a cross between the vertebrate platelet and macrophage in that they change shape during coelomic fluid coagulation, are highly phagocytic and can be motile. Coelomocytes can be induced to undergo synchronous, actin mediated shape changes from a lamellipodial to a filopodial form as well as the reverse. This proposal describes a series of morphological and biochemical experiments aimed at the identification of coelomocyte ABPs and MAPs and the elucidation of their in vivo functions during shape changes. ABPs and MAPs will be identified from actin and tubulin binding overlays of blots of cell extracts, from taxol stabilized microtubule preparations and from DNase affinity chromatography. Immunolocalization at the light and electron microscopic levels will be used to examine the distribution of ABPs and MAPs in coelomocytes with particular emphasis on the filopodial to lamellipodial shape change. This allows for the examination of the dismantling of the actin cytoskeleton and the function of MAP motor proteins in the distribution of cellular organelles.As a secondary aim, the calcium regulation of coelomocyte shape changes will also be investigated using experiments employing calcium ionophore treatments and calcium dependent fluorescent dyes. The results of these experiments should provide new insights into the identity and functions of ABPs and MAPs in coelomocyte shape changes and should also help lead to a better understanding of the medically significant shape changes exhibited by platelets and macrophages.