ARF1 mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts

Focal adhesion assembly and actin stress fiber formation were studied in se rum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O, Permea bilization in the presence of GTP gamma S stimulated rho-dependent formatio n of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTP gamma S at 8 min a fter permeabilization still induced paxillin recruitment to focal adhesion- like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rh o-independent, but could be evoked using constitutively active Q71L ADP-rib osylation factor (ARF1), and blocked by NH2-terminally truncated Delta 17AR F1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTP gamma S-induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regu late paxillin recruitment to focal adhesions was confirmed by microinjectio n of Q71LARF1 and Delta 17ARF1 into intact cells. Interestingly, these expe riments showed that V14RhoA-induced assembly of actin stress fibers was pot entiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesio ns at the ends of prominent actin stress fibers.