SIGNAL TRANSDUCTION AND ACTIN DURING AMOEBOID CHEMOTAXIS

变形虫趋化过程中的信号转导和肌动蛋白

• 批准号: 3294955
• 负责人: JOHN S CONDEELIS
• 金额: $ 23.3万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3294955
• 关键词: Dictyostelium; actin binding protein; actins; affinity chromatography; antisense nucleic acid; biological signal transduction; cellular polarity; chemoattractants; chemotaxis; fluorescence microscopy; gene deletion mutation; gene expression; genetic manipulation; laboratory mouse; microorganism genetics; monoclonal antibody; nucleic acid sequence; online computer; phosphorylation; polymerization; protein isoforms; protein structure function; video recording system

Amoeboid cells of Dictyostelium discoideum are chemotactic to a variety of agents including extracellular cAMP. As in other chemotactic amoeboid cells, such as leukocytes and metastatic tumor cells, stimulation of Dictyostelium amoebae with chemoattractant elicits a burst of actin polymerization. The location and timing of this polymerization response helps determine subsequent cell polarity, and creates part of the force for pseudopod extension. In the previous funding period we identified and purified a protein, aginactin, that may be involved in regulating chemoattractant induced actin polymerization in cells by uncapping the barbed ends of cytoskeleton associated actin filaments. In this renewal we propose to characterize aginactin at the molecular level. Aginactin is a cytosolic isoform of the HSC-70 family of proteins. We will identity which members of the HSC-70 family have aginactin-like capping activity through the use of antibodies that specifically crossreact with the subset of HSC-70s that have capping activity and by protein biochemistry. We will analyze the domain structure of aginactin to identity phosphorylation, actin binding and capping domains in the protein to gain insights into the mechanism of capping and its regulation. The localization of aginactin in situ will be determined relative to F-actin and the free barbed ends of actin filaments particularly immediately before and after chemotactic stimulation that causes uncapping of barbed ends. To determine the function of aginactin in vivo we will prepare gene disruption, replacement and/or antisense constructs that can be used to disrupt or lower the expression of, or overexpress aginactin in vivo. Phenotypes of transformants prepared by these methods will be analyzed using established biochemical assays, fluorescence microscopy and computer assisted video microscopy to detect even subtle differences compared to wild type and control cells. The combined approaches of protein chemistry, bacterial expression and molecular genetics in Dictyostelium will provide a definitive analysis of the structure and function of aginactin.

DICTYOSTELIUM DISCOIDEUM的变形虫细胞对多种 包括细胞外营地在内的特工。如其他趋化性变形虫 细胞，例如白细胞和转移性肿瘤细胞，刺激 用趋化吸引剂引起肌动蛋白爆发的变形虫 聚合。此聚合响应的位置和时机 有助于确定随后的细胞极性，并创建一部分力 用于假脚架扩展。在上一个资金期内，我们确定了 纯化了可能与调节有关 化学吸引剂诱导细胞中的肌动蛋白聚合通过解开 细胞骨架相关的肌动蛋白丝的刺末端。 在这种更新中，我们建议在分子上表征活动蛋白 等级。活动蛋白是HSC-70蛋白质家族的胞质同工型。 我们将识别哪些HSC-70家族的成员具有类似活动蛋白的成员 通过使用特定的抗体来封盖活动 与具有上限活动的HSC-70的子集进行交叉反应 蛋白质生物化学。我们将分析活动蛋白的结构域结构 蛋白质中的身份磷酸化，肌动蛋白结合和上限结构域 洞悉封盖及其调节的机制。这 凝激素原位的定位将相对于F-肌动蛋白确定 肌动蛋白丝的自由刺末端，特别是立即 趋化性刺激前后导致刺刺的刺激 结束。 为了确定脂肪酸蛋白酶在体内的功能，我们将制备基因 可以用来用于的破坏，替代和/或反义结构 破坏或降低体内或过表达活动蛋白的表达。 通过这些方法制备的转化体的表型将进行分析 使用已建立的生化测定，荧光显微镜和计算机 辅助视频显微镜以检测到甚至微妙的差异 野生型和对照细胞。 蛋白质化学，细菌表达和 dictyostelium中的分子遗传学将对 活动蛋白的结构和功能。