Our long-term goals are to understand cell motility, which is critical for immunological defense and wound healing, but unwanted during metastatic cancers. Despite the wealth of knowledge describing motility and the role of F-actin cytoskeletal networks, the origin of forces for generating cell protrusions is poorly understood. Long-standing models have yet to be resolved. We have developed a new microscopic approach, laser-deflection particle-tracking microrheology (LDPTM), that can address this problem by fully characterizing subcellular mechanical properties in living cells. In this noninvasive approach, mechanical properties of the cytoskeleton are derived from the residual Brownian motion of individual particles embedded in the network. In reconstituted networks, agreement between LDPTM and traditional rheological approaches is excellent. LDPTM quickly provides a thorough physical characterization of cytoskeletal networks and provides new parameters that directly test models for protrusive forces. In addition, LDPTM is fast enough to resolve 1s "spikes" in stiffness during phagocytosis, which involves many of the same proteins as cell motility. We will continue to use LDPTM to study mammalian cells, but will focus our efforts on the motile amoeba, Dictyostelium discoideum. Because of its well-developed biochemistry and genetics and its fast behavioral response, Dictyostelium offers unique advantages for understanding both cell structure, phagocytosis, and cell motility. Our specific aims are: I. For insights related to motility, use abrupt mechanical changes to help resolve the order of early molecular events during phagocytosis. Use null-mutants and fluorescent localization of GFP-tagged proteins. II. To test models of cell protrusion, monitor mechanical changes within crawling cells caused by wound-healing or chemotaxis. Examine motile animal cells, Listeria-infected cells and motile Dictyostelium. III. To develop a quantitative mechanical theory of F-actin networks, use Dictyostelium to compare in vitro and in vivo measurements. Initially focus on crosslinking proteins. These studies should provide a paradigm for considering cytoskeletal mechanics and its remodeling in many other systems of cell biology.