Amoeboid Motility--A Cellular and Genetic Approach

变形虫运动性——细胞和遗传方法

• 批准号: 7892239
• 负责人: THOMAS M ROBERTS
• 金额: $ 12.72万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-03 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7892239
• 关键词: Actins; Ascaris suum; Behavior; Biochemical; Biological; Bundling; Cell membrane; Cells; Communication; Complex; Coupling; Crystallography; Cytoskeletal Modeling; Cytoskeleton; Data; Electron Microscopy; Eukaryotic Cell; Exhibits; Filament; Funding; Gel; Generations; Genetic; Goals; In Vitro; Individual; Inflammation; Laboratories; Link; Locomotion; Membrane; Methods; Modeling; Molecular; Molecular Motors; Molecular Probes; Movement; Nematoda; Neurons; Phosphorylation; Physiological Processes; Play; Process; Production; Property; Protein Subunits; Proteins; Research Personnel; Role; Site; Sperm Motility; Structure; System; Testing; Wound Healing; base; cell motility; cell type; dimer; mutant; neuronal cell body; polymerization; reconstitution; skills; sperm cell; sperm protein

Amoeboid cell motility, a property of many eukaryotic cells, plays a key role in physiological processes such as inflammation, wound healing, neuronal targeting, and metastatic invasion. The purpose of this proposal is to investigate the molecular mechanism of cell crawling using the simple, specialized sperm of the nematode, Ascaris suum, as an experimental system. These cells display the same motile behavior as conventional crawling cells but lack the actin machinery usually associated with cell migration. Instead, the motility apparatus of sperm is based on major sperm protein (MSP) filaments that assemble along the leading edge, and disassemble at the base of the lamellipod. These unique filaments have no structural polarity indicating that molecular motor proteins are not required for sperm motility. The coupling of MSP cytoskeletal dynamics to locomotion suggests a "push-pull" mechanism for movement in which forces for leading edge protrusion and cell body retraction are produced at opposite ends of the lamellipod and linked reciprocally to the assembly status of the cytoskeleton. The push-pull model will be evaluated by characterizing the components of the motility apparatus and integrating this information to define how the cell machinery produces movement. Stuctural studies will be extended to determine the orientation of the MSP subunits in filaments and to define the interactions that promote intrinsic bundling of filaments into larger arrays. Based on this information MSP mutants will be constructed to study the contributions of filament polymerization and bundling to generating the forces for movement. Biochemical and molecular methods will be used to analyze the membrane and cytosolic proteins required to nucleate MSP polymerization at the leading edge and explore the roles of pH and phosphorylation in regulating this process. The hypothesis that the force for cell body retraction is produced by deswelling of the MSP cytoskeleton will be tested by defining conditions that induce shrinkage of MSP filament gels in vitro and examining the organization of the cytoskeleton at the base of the lamellipod. The long term goal of this project is to define the mechanism of sperm locomotion so that comparison of actin- and MSP- based systems can be used to understand the basic principles of amoeboid cell motility

变形虫细胞运动是许多真核细胞的一种特性，在生理过程中起着关键作用，例如 如炎症、伤口愈合、神经元靶向和转移侵袭。该提案的目的是 利用线虫简单、特化的精子研究细胞爬行的分子机制， 猪蛔虫，作为一个实验系统。这些细胞表现出与传统细胞相同的运动行为 爬行细胞，但缺乏通常与细胞迁移相关的肌动蛋白机制。取而代之的是动力 精子的结构基于沿着前缘组装的主要精子蛋白（MSP）细丝， 并在片脚的底部拆卸。这些独特的细丝没有结构极性表明 精子运动不需要分子运动蛋白。 MSP细胞骨架动力学的耦合 运动提出了一种“推拉”运动机制，其中前缘突出和 细胞体收缩在板状足的相对两端产生，并与组件相互关联 细胞骨架的状态。将通过表征推拉模型的组件来评估 运动装置并整合这些信息来定义细胞机器如何产生运动。 结构研究将扩展到确定细丝中 MSP 亚基的方向并定义 促进细丝内在捆绑成更大阵列的相互作用。根据此信息 MSP 将构建突变体来研究丝聚合和捆绑对产生 运动的力量。将使用生化和分子方法来分析膜和细胞质 在前沿 MSP 聚合成核所需的蛋白质，并探索 pH 和 磷酸化调节这一过程。假设细胞体收缩的力是由 通过定义诱导 MSP 丝收缩的条件来测试 MSP 细胞骨架的消肿 体外凝胶并检查片足底部细胞骨架的组织。长期来看 该项目的目标是定义精子运动的机制，以便比较肌动蛋白-和 MSP- 基于系统可用于了解变形虫细胞运动的基本原理