Title: Integrated mechanochemical control of directional cell migration by paxillin
Description: In this dissertation, I explore the mechanism by which physical cues from the cellular microenvironment influence directional cell migration, specifically by spatially orienting motile processes. Previous work in the Ingber lab showed cells cultured on square-shaped, single cell-sized extracellular matrix (ECM) islands preferentially extend lamellipodial membrane extensions from corner regions (Parker et al., 2002), and that this requires a spatiotemporal balance of the activities of the small GTPases Rac and Rho (Brock et al., 2003; Brock et al., 2005). Corner regions of square cells are enriched in focal adhesions (FAs), sites of attachment between the ECM and the cytoskeleton. In this work, I show that physical cues from the ECM direct migration at the subcellular level through the FA scaffold protein paxillin. Loss of paxillin resulted in uncoupling of spatial cues and lamellipodia formation in square cells, and this loss of spatial organization corresponds to a directional motility defect, as demonstrated by single-cell tracking in scrape wound assays. In addition, Rac and Rho GTPase activites were spatiotemporally misregulated in paxillin knockout cells. Studies using paxillin truncation mutants revealed that paxillin both negatively and positively regulates lamellipodia formation through its N- and C-termini, respectively, but both halves of paxillin are required for efficient cell migration. Mutations of the paxillin LD4 motif, which mediates binding of Pkl/Git Arf6GAP proteins (Turner et al., 2001), abolished corner lamellipodia formation and alter Pkl association.

Depletion of Pkl by RNAi also resulted in a loss of spatial restriction of lamellipodia formation. In addition, constitutively active Arf6 rescued side lamellipodia suppression, but not corner lamellipodia promotion, in pax-/- cells. Endosomal vesicle formation was also found to be spatially correlated with lamellipodia in a paxillin and Arf6 dependent manner, suggesting a functional link between FAs and membrane trafficking in motile cells. Thus, paxillin may integrate the spatiotemporal regulation of lamellipodia formation with physical cues from the ECM by recruitment of membrane-associated signaling complexes via its N-terminus, and targeting these macromolecular assemblies to FAs through its C-terminus, to regulate the balance of GTPase activation in different subcellular microdomains. Finally, I observed that pax-/- cells showed an increased propensity to form circular dorsal ruffles in response to PDGF, and that this correlated with an enhanced capacity of the cells to invade Matrigel in a 3D migration assay. These results suggest that paxillin may be critical for control of directional motility in both 2D and 3D microenvironments, and may play multiple roles during normal tissue development as well as in the progression of metastatic cancer.