Amoeboid Motility--A Cellular and Genetic Approach

阿米巴细胞的运动性是许多真核细胞的一种特性，在许多生理过程中起着关键作用 如炎症、伤口愈合、神经元靶向和转移侵袭。这项建议的目的是 利用线虫简单而特化的精子研究细胞爬行的分子机制， 猪蛔虫作为实验系统。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。相反，这种能动性 精子器官的基础是沿精子前沿聚集的主要精子蛋白(MSP)细丝， 并在圆柱体的底部进行拆卸。这些独特的细丝没有结构上的极性指示 精子运动不需要分子马达蛋白。MSP细胞骨架动力学的耦合 提出了一种“推-拉”的运动机制，在这种机制中，前缘突出的力和 细胞体回缩产生在瓣椭圆形的相对两端，并相互连接到组件 细胞骨架的状态。推-拉模型将通过表征 运动装置，并结合这些信息来定义细胞机械如何产生运动。 结构研究将扩展到确定MSP亚基在细丝中的取向并确定 这种相互作用促进了细丝的内在集束，形成更大的阵列。基于这一信息，MSP 将构建突变体来研究细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细胞膜和细胞质 MSP聚合前沿成核所需的蛋白质和探索pH和 在调节这一过程中的磷酸化。假设细胞体回缩的力是由 将通过定义导致MSP细丝收缩的条件来测试MSP细胞骨架的消胀 体外凝胶法和细胞骨架组织学检查。从长远来看 这个项目的目标是确定精子运动的机制，以便比较肌动蛋白和MSP-1。 基础系统可以用来理解阿米巴细胞运动的基本原理

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