Following the gradient sensing phase, cells reorganize their internal components into a symmetry-broken configuration, resulting in an elongated cell with a clearly identifiable back, front, and side. This polarized state is coupled to motility through the formation of membrane extensions, pseudopodia, which occur mostly at the front of the cell. Using quantitative measurements on cells that are vertically restricted, we have determined that these pseudopodia are closely correlated with membrane areas ("patches") of increased concentration of activated Ras, Ras-GTP. What remains unclear, however, is the role Ras-GTP plays in restricting the pseudopodia to the front of the cell. Furthermore, the mechanisms for the transient nature of the patches and pseudopodia are unclear. Finally, there is a vigorous and ongoing debate whether new pseudopodia split off existing ones via a specialized fip-splitting mechanism or whether new pseudopodia are created in a stochastic fashion, largely independent of the location of the existing pseudopod. The goal of this project will be to examine the mechanisms of cell polarity using microfluidics technology in combination with modeling efforts. The quantitative results from the experiments will be used to construct mathematical models. Conversely, through specific predictions, these models will guide the experiments. Again, we think that such two-way communication between experiments and modeling is essential in making progress in understanding the role of polarity in chemotaxis.