MOLECULAR GENETIC ANALYSIS OF THE CYTOSKELETON

细胞骨架是一种复杂而动态的结构，在细胞内起着至关重要的作用。 在确定细胞形状、运动性和形态发生方面的作用。 这些过程对正常发育、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的转移。我们 正在使用分子遗传学技术来确定体内的功能 以及两种细胞骨架蛋白之间的相互作用。 网生鞭毛虫与哺乳动物的运动细胞非常相似，在 行为和组成组织 细胞骨架。网柄网柄菌ABP-120和ABP-240均为交联型肌动蛋白 长丝排列成正交阵，两者都与人体非肌肉相关 丝状物。人类细丝蛋白最近被证明对 癌细胞的运动性。结构、功能和免疫学证据 表明ABP-240是丝状菌的等价物。 DictyostelialaBP-120有一个肌动蛋白结合位点，与 丝蛋白，但蛋白质有不同的结构构象。它是 推测DictyostelialABP-120和ABP-240将执行相关的但 在细胞骨架组织中的不同功能。这个项目将 完成毛盘基菌ABP-240基因的克隆和测序 为了确定其结构和一级序列的相似性 DictyostelialABP-120和丝状菌。为了评估体内的 ABP-240的功能，我们将创造突变的细胞系，要么缺乏 预测功能位点有突变的ABP-240基因。这个 ABP-120和ABP-240功能重叠的可能性将是 通过制造同时缺乏这两种基因的突变体来解决。为了尽可能地减少 由于缺乏其中一种或两种都缺乏而导致的代偿性突变的癌症 在这些蛋白质中，将采用一种新技术，它将使 任何一种蛋白质在细胞中有条件地表达。 将对突变的细胞系进行广泛的分析，以确定 这两种蛋白质的活体功能。增长的速度和行为 在发育过程中将评估细胞的一般活性。 联合应用抗ABP-120和ABP-240的单抗 用荧光法将鬼臼糖苷用于测定相对的3- 肌动蛋白细丝的空间定位及其两个交联物 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化学定向细胞 确定伪足伸展和移动的哪些行为方面 都会被突变所改变。 大多数细胞运动和形状控制模型都集中在组织上 肌动蛋白细胞骨架。在细胞中发现了肌动蛋白细丝 主要是以正交网络的形式。所描述的实验 将通过改变负责的蛋白质来直接测试这些模型 F-肌动蛋白的交联和研究这些扰动如何影响 细胞骨架的组织和运动。

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.