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© The Rockefeller University Press, 0021-9525/1998//1981 $5.00
The Journal of Cell Biology, Volume 143, Number 7, , 1998 1981-1995

ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts

J.C. Norman^*, D. Jones, S.T. Barry^*, M.R. Holt^*, S. Cockcroft, and D.R. Critchley^*

^* Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom; and Department of Physiology, University College London, London WC1E 6JJ, United Kingdom

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPS at 8 min after 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 rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH₂-terminally truncated 17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and 17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.

Key Words: focal adhesions • paxillin • vinculin • rho • ARF1 • permeabilized cells

Abbreviations used in this paper: ARF, ADP-ribosylation factor; ECM, extracellular matrix; LPA, lysophosphatidic acid; MLC, myosin light chain; PBS/ME, phosphate buffered saline/2 mM MgCl₂/3 mM EGTA; PLD, phospholipase D; PA, phosphatidic acid; PIP2, phosphatidyl inositol 4,5 bisphosphate; PI4P, phosphatidyl inositol 4-phosphate; SAM, substrate-attached material; SL-O, streptolysin-O.

S.T. Barry's current address is Receptor Systems, Glaxo-Wellcome, Medicines Research Centre, Stevenage, Herts SG1 7NY, UK.

M.R. Holt's current address is Department of Physiology, University College London, London WC1E 6JJ, UK.

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