MOLECULAR GENETIC ANALYSIS OF THE CYTOSKELETON

细胞骨架是一种复杂的动态结构， 在决定细胞形状、运动性和形态发生中的作用。 这些过程对于正常发育、伤口愈合和 免疫反应。 此外，对监管的理解 运动性可用于预防肿瘤细胞转移。 我们 正在使用分子遗传技术来确定体内功能 以及两种细胞骨架蛋白的相互作用。 网骨藻与哺乳动物的运动细胞非常相似， 行为和蛋白质的补充，组织 细胞骨架 网骨藻ABP-120和ABP-240均交联肌动蛋白 细丝成正交阵列，都与人类非肌肉 细丝蛋白 人类细丝蛋白最近被证明是重要的， 癌细胞运动性 结构、功能和免疫学证据 表明ABP-240是丝状蛋白的网骨藻等效物。 网骨藻ABP-120有一个肌动蛋白结合位点， 但该蛋白质具有不同的结构构象。 是 推测网骨藻ABP-120和ABP-240将发挥相关作用， 在细胞骨架组织中的不同功能。 该项目将 完成了网囊藻ABP-240基因的克隆和序列测定 为了确定其结构和一级序列的相似性， 网囊藻ABP-120和细丝蛋白。 为了评估体内 ABP-240的功能，我们将创建突变细胞系， ABP-240基因在预测功能位点存在突变。 的 ABP-120和ABP-240具有重叠功能的可能性， 通过制造缺少这两种基因的突变体来解决。 为了最小化 由于缺乏其中一种或两种基因， 在这些蛋白质中，将采用一种新技术， 在细胞中有条件地表达任一蛋白质。 将对突变细胞系进行广泛分析，以确定其在 这两种蛋白质的体内功能。 生长速度和行为 将评估细胞的一般活力。 ABP-120和ABP-240特异性单克隆抗体组合 与荧光鬼笔环肽将用于确定相对3- 肌动蛋白丝的空间定位和两个交联 野生型和突变型细胞中的蛋白质。 细胞的计算机分析 运动性将用于营养细胞和趋化定向细胞 来确定伪足的伸展和运动 都被突变改变了 细胞运动和形状控制的大多数模型都集中在组织上 肌动蛋白细胞骨架。 细胞内的肌动蛋白丝 主要以正交网络的形式。 所述实验 将直接测试这些模型，通过改变负责 F-肌动蛋白交联和研究这些扰动如何影响 细胞骨架组织和运动性。

The cytoskeleton is a complex and dynamic structure that plays a crucial role in the determination of cell shape, motility and morphogenesis. These processes are critical for normal development, wound healing and the immune response. In addition, an understanding of the regulation of motility can be applied to the prevention of tumor cell metastasis. We are using molecular genetic techniques to determine the in vivo function and interactions of two cytoskeletal proteins. Dictyostelium amoebae are remarkably like mammalian motile cells both in behavior and in the complement of proteins that organize the cytoskeleton. Dictyostelium ABP-120 and ABP-240 both cross-link actin filaments into orthogonal arrays and are both related to human non-muscle filamin. Human filamin has recently been shown to be important for cancer cell motility. Structural, functional and immunological evidence indicates that ABP-240 is the Dictyostelium equivalent of filamin. Dictyostelium ABP-120 has an actin binding site that is related to filamin but the protein has a different structural conformation. It is presumed that Dictyostelium ABP-120 and ABP-240 would perform related but distinct functions in cytoskeletal organization. This project will accomplish the cloning and sequencing of the Dictyostelium ABP-240 gene in order to define its structural and primary sequence similarity to both Dictyostelium ABP-120 and filamin. In order to assess the in vivo function of ABP-240, we will create mutant cell lines either lacking the ABP-240 gene possessing mutations in predicted functional sites. The possibility that ABP-120 and ABP-240 have overlapping functions will be addressed by making mutants that lack both genes. In order to minimize the cancer of compensatory mutations caused by the lack of either or both of these proteins, a new technique will be employed which will allow either protein to be conditionally expressed in cells. The mutant cell lines will be extensively analyzed to determine the in vivo function of these two proteins. The rate of growth and behavior during development will assess the general viability of the cells. Monoclonal antibodies specific for ABP-120 and ABP-240, in combination with fluorescent phalloidin will be used to determine the relative 3- dimensional localization of actin filaments and the two cross-linking proteins in wild-type and mutant cells. Computerized analysis of cell motility will be used on vegetative and chemotactically oriented cells to determine what behavior aspects of pseudopod extension and movement are altered by the mutations. Most models of cell movement and shape control focus on the organization of the actin cytoskeleton. The actin filaments in cells are found primarily in the form of orthogonal networks. The described experiments will directly test these models by altering the proteins responsible for F-actin cross-linking and investigating how these perturbations affect cytoskeletal organization and motility.