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. Structural 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.
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