Amoeboid Motility--A Cellular and Genetic Approach

变形细胞运动性是许多真核细胞的一种特性，在生理过程中起着关键作用， 如炎症、伤口愈合、神经元靶向和转移性侵袭。这项建议的目的是 利用线虫简单、特化的精子研究细胞爬行的分子机制， 猪蛔虫实验系统。这些细胞显示出与常规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， 精子的器官是基于沿着前缘沿着聚集的主要精子蛋白（MSP）细丝， 然后在片足的底部分解这些独特的细丝没有结构极性 分子马达蛋白不是精子运动所必需的。MSP细胞骨架动力学的耦合 提出了一种用于运动的“推拉”机构，其中用于前缘突出的力和 细胞体收缩产生于片足的相对两端，并与组装体相连 细胞骨架的状态。推拉模型将通过表征 运动装置和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丝中MSP亚基的方向，并定义 这种相互作用促进了细丝内在地成束成更大的阵列。根据这一信息，MSP 将构建突变体来研究长丝聚合和集束对产生 运动的力量。生物化学和分子生物学方法将用于分析膜和胞质 蛋白质所需的成核MSP聚合的前沿，并探讨pH值和 磷酸化在调节这一过程。细胞体收缩的力是由 MSP细胞骨架的去溶胀将通过确定诱导MSP细丝收缩的条件来测试 凝胶体外和检查组织的细胞骨架在基地的片足。长期 本项目的目的是确定精子运动的机制，以便比较肌动蛋白和MSP， 的系统可以用来理解变形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