Most importantly, these studies revealed that in addition to regulating directional migration in 2D, paxillin is a critical mediator of ECM invasion and migration in 3D, and this more complex response correlates with formation of CDRs in 2D cultures. Cells migrating on ECM substrates that vary in the mechanical compliance move in the direction in which they exert the highest traction forces. In square-shaped cells, traction forces are concentrated in corner regions, likely due to positive feedback between geometric constraints and contractilitydependent assembly of FAs. In addition to containing high concentrations of signaling molecules, FAs may be ����permissive zones���� for membrane extension in that actin-driven protrusions are not blocked by CHIR-99021 cortical actin. In support of this hypothesis, myosin-mediated cortical tension has been shown to inhibit branching in endothelial cells, and inhibition of myosin II subjacent to the plasma membrane can induce localized membrane protrusion. At early time-points after PDGF stimulation, cells with and without paxillin formed both dorsal and lateral membrane extensions. This suggests that paxillin is not required for the initial burst of actin-driven ruffling in response to growth factor stimulation, and that this early process may be molecularly distinct from later rounds of lamellipodia formation. Although many paxillin domains have been studied, little is known about the conformation of paxillin in vivo. Vinculin in FAs undergoes a conformational change that BYL719 clinical trial relieves an intramolecular association between the head and tail regions, exposing protein-protein interaction domains that are hidden in the cytosolic form. Like vinculin, paxillin may adopt different conformations upon FA recruitment that expose or sequester various protein-interaction sites, which could explain the complex effects of the truncation mutants on the formation of different protrusive structures. It is possible that the different effects of the paxN and paxC truncation mutants are due to exposure of binding domains that are usually only available in specific subcellular contexts. The paxN and paxC truncation mutants may thus act as ����dominant negatives����, sequestering proteins away from other binding partners, or ����dominant positives���� that can interact with proteins that normally would be unavailable in a given subcellular context. Paxillin binds the ArfGAPs Git1 and Pkl/Git2 via its N-terminal LD4 motif, and these proteins have been implicated in directional motility through both positive and negative mechanisms. Git-1 has been reported to either inhibit membrane extension or promote cell migration depending on its location within the cell, whereas Pkl appears to be involved in control of directional cell migration in fibroblasts. Localization of Pkl to FAs is regulated by tyrosine phosphorylation, and its dephosphorylation is mediated by PTP-PEST, which binds to paxillin via its C-terminal LIM domains.