Background: Actin-based motility is critical for diverse cellular processes, including development, wound healing, the immune response, and tumor invasion and metastasis. A common property of all motile cells is their ability to polarize [unreadable] they show distinct asymmetry, with a front containing F-actin protrusive structures and a back containing acto-myosin contractile structures. While most cells possess the ability to generate the individual actin formations underlying motility, there is significant variability in the ability of mammalian cells to coordinate the asymmetric polarity of protrusion and contraction required for directional motility. Objective/Hypotheses: This project will explore two possible fundamental principles behind the variability in cell polarization and motility: 1) non-motile cells express all of the necessary components for motility, but that they are not capable of generating an initial level of activation needed to drive the system to polarity, or 2) non-motile cells are missing the requisite feedback circuits needed for separation of the "front" and the "back". The following Specific Aims will be used to test these hypotheses by attempting to convert non-motile and semi-motile cells into highly motile cells. 1) Overexpressing endogenous proteins or sets of proteins involved in actin regulation, (e.g. co- expression of high levels of antagonistic activators of F-actin and acto-myosin). These proteins will be choosen based on results of RT-PCR experiments used to identify endogenous proteins whose expression correlates with cell motility (e.g. up regulated in highly motile cells relative to semi- and non-motile cells). 2) Introducing synthetic signaling circuits specifically engineered to generate local positive feedback loops for F-actin formation, and long-range cross-inhibition between the signaling pathways responsible for F-actin and acto-myosin. These novel synthetic proteins will be designed to be regulated and localized by specific activators of F-actin and acto-myosin structures using methods developed in the Lim Lab. Relevance: Despite the results of many studies suggesting a link between the malignancy of tumors and the deregulation of actin polymerization, little is understood about how this deregulation occurs. Thus, understanding how non-motile cells can acquire the ability to generate and amplify actin based polarity will provide insight not only into how natural cells normally achieve movement, but also into how non-motile cancer cells gain the ability to metastasize. Such knowledge will be useful for future attempts at controlling metastatic tumors and developing novel therapeutics based on controlling cell motility.