Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3′-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

Here, we report a direct interaction between the beta1 integrin cytoplasmic tail and Rab25, a GTPase that has been linked to tumor aggressiveness and metastasis. Rab25 promotes a mode of migration on 3D matrices that is characterized by the extension of long pseudopodia, and the association of the GTPase with alpha5beta1 promotes localization of vesicles that deliver integrin to the plasma membrane at pseudopodial tips as well as the retention of a pool of cycling alpha5beta1 at the cell front. Furthermore, Rab25-driven tumor-cell invasion into a 3D extracellular matrix environment is strongly dependent on ligation of fibronectin by alpha5beta1 integrin and the capacity of Rab25 to interact with beta1 integrin. These data indicate that Rab25 contributes to tumor progression by directing the localization of integrin-recycling vesicles and thereby enhancing the ability of tumor cells to invade the extracellular matrix.

Summary Integrin adhesion receptors are structurally dynamic proteins that adopt a number of functionally relevant conformations. We have produced a conformation-dependent anti-α5 monoclonal antibody (SNAKA51) that converts α5β1 into a ligand-competent form and promotes fibronectin binding. In adherent fibroblasts, SNAKA51 preferentially bound to integrins in fibrillar adhesions. Clustering of integrins expressing this activation epitope induced directional translocation of α5β1, mimicking fibrillar adhesion formation. Priming of α5β1 by SNAKA51 increased the accumulation of detergent-resistant fibronectin in the extracellular matrix, thus identifying an integrin conformation that promotes matrix assembly. The SNAKA51 epitope was mapped to the calf-1/calf-2 domains. We propose that the action of the antibody causes the legs of the integrin to change conformation and thereby primes the integrin to bind ligand. These findings identify SNAKA51 as the first anti-integrin antibody to selectively recognize a subset of adhesion contacts, and they identify an integrin conformation associated with integrin translocation and fibronectin matrix formation.

Environmental arsenic exposure, through drinking contaminated water, is a significant risk factor for developing vascular diseases and is associated with liver portal hypertension, vascular shunting, and portal fibrosis through unknown mechanisms. We found that the addition of low doses of arsenite to the drinking water of mice resulted in marked pathologic remodeling in liver sinusoidal endothelial cells (SECs), including SEC defenestration, capillarization, increased junctional PECAM-1 expression, protein nitration, and decreased liver clearance of modified albumin. Furthermore, the pathologic changes observed after in vivo exposure were recapitulated in isolated mouse SECs exposed to arsenic in culture. To investigate the role of NADPH oxidase-generated ROS in this remodeling, we examined the effect of arsenite in the drinking water of mice deficient for the p47 subunit of the NADPH oxidase and found that knockout mice were protected from arsenite-induced capillarization and protein nitration. Furthermore, ex vivo arsenic exposure increased SEC superoxide generation, and this effect was inhibited by addition of a Nox2 inhibitor and quenched by the cellpermeant superoxide scavenger. In addition, inhibiting either oxidant generation or Rac1-GTPase blocked ex vivo arsenic-stimulated SEC differentiation and dysfunction. Our data indicate that a Nox2-based oxidase is required for SEC capillarization and that it may play a central role in vessel remodeling following environmentally relevant arsenic exposures.