Thus, the two halves of paxillin may work together to efficiently drive directional migration by mediating complex cycles of these types of molecular associations. These findings indicate that paxillin may play distinct roles in different subcellular contexts, such as regulating the formation of different kinds of motile processes. These data also suggest cytoplasmic functions for paxillin in controlling CDR extension and membrane trafficking, as well as lamellipodia formation, which may correspond to different modes of migration in vivo. Interestingly, CDRs formed by mesenchymal cells in 2D have been compared to invasive protrusions or invadopodia formed by epithelial cells. Cells in tissue culture have basal membranes that are in contact with ECM and free dorsal surfaces, whereas mesenchymal cells are usually embedded within 3D ECMs in tissues. We found that dorsal protrusion formation in MEFs correlated with the ability of these cells to invade 3D Matrigel plugs. Paxillin-mediated signaling may therefore be critical for determining Tubulin Acetylation Inducer whether a cell migrates along a planar basement membrane or through a 3D interstitial matrix, as occurs, for example, during epithelial-mesenchymal transitions in cancer metastasis. Moreover, switching between Rho- and Rac-mediated modes of migration is a common feature of 3D matrix invasion. Thus, paxillin may be involved in tailoring a cell��s motile response to physical cues in different microenvironments. Importantly, mutation and misregulation of paxillin correlate with metastatic potential in some human breast and lung cancers, suggesting that it may be involved in regulating ECM invasion as well. In any case, the different effects of paxillin deficiency in 2D versus 3D migration underscore the importance of the physical microenvironment on cell behavior, and the central role that paxillin normally plays in this process. In conclusion, detailed examination of fibroblast cells on patterned and unpatterned substrates revealed that they respond to PDGF with an initial round of membrane extension in all directions, followed by progressive spatial fine-tuning that is sensitive to physical cues. Thus, lamellipodia formation becomes preferentially localized to CUDC-907 regions of greatest cell distortion after 15 min of PDGF stimulation in these artificially polarized cells. We found that paxillin can enhance or suppress membrane extension depending on its subcellular context. The presence of paxillin within FAs appears to spatially constrain where Rac is activated inside the cell, and thereby preferentially stimulates motile process formation to adjacent regions. Loss of paxillin results in deregulated spatial pruning of membrane extensions. The N-terminus appears to suppress lateral membrane extension, and the C-terminus enhances lamellipodia formation, but both halves are required for efficient directional migration in 2D. Furthermore, overexpression of the N- or C-terminus alone can tip the balance between ����dorsal���� and ����lateral���� motile process formation in response to PDGF.