Because paxN and paxC appeared to exert opposing effects on membrane extension, we tested the mutants in combination with one another, or with full-length paxillin, in order to determine whether the N- or C-termini had dominant effects in pax+ cells, as well as whether the two separate halves of the protein could exert complementary effects in the same cell. The experiments described so far were performed with cells cultured on microengineered square ECM islands, so we next tested the effects of paxillin mutation on cells cultured on standard 2D culture substrates to rule out the possibility that the effects we observed were an artifact of this model system. Time-lapse microscopy confirmed that pax+ cells formed both lateral lamellipodia and CDRs in response to PDGF stimulation at early times. These cells typically underwent a single round of CDR formation, which was completed within 10 min, with most dorsal ruffles being completely internalized by 15 min, after which extensive lateral lamellipodia BU 4061T formation continued through 30 min and Nilotinib beyond. Phase-lucent vesicles formed beneath the sites of CDR internalization, then translocated toward the nucleus and decreased in size by 30 min. From the results of the square cell assays, we expected that paxN would rescue directional migration into the scrape wound, whereas paxC would not. Single-cell tracking confirmed this hypothesis, but also revealed that expression of either truncation mutant alone resulted in reduced migration speed. Culturing cells on microfabricated ECM substrates allowed us to control cytoskeletal polarity and FA position, which enabled us to predict where lamellipodia were likely to form when cells were stimulated by a soluble motility factor. Using this system, we were able to detect uncoupling of membrane extension from spatial cues and to analyze the role of paxillin subdomains in this motile response. Normal mouse and human fibroblasts plated on single cell-sized, square FN islands formed large FAs primarily in corner regions and preferentially extended lamellipodia from adjacent sites in response to PDGF. In contrast, paxillin-deficient cells formed more and smaller FAs as well as lamellipodia along the cell periphery, with little spatial preference. These results indicate that paxillin is involved in both promoting membrane extension near FAs, as well as suppressing lamellipodia formation at distant sites. In addition to showing that paxillin is critical for spatially coupling regions of cell distortion and sites of FA assembly to sites where new lamellipodia will form, we found that the N- and Ctermini of paxillin play opposing, but complementary, roles in this process. The N-terminus is critical for suppressing lamellipodia formation and maintaining directional persistence, while the C-terminus actively promotes lamellipodia formation. An unexpected finding was that paxillin mutation also affects the formation of dorsal CDRs, as well as lateral membrane extensions.