ARF1 mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts.
journal contributionposted on 15.12.2016, 16:21 by J. C. Norman, D. Jones, S. T. Barry, M. R. Holt, S. Cockcroft, D. R. Critchley
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 GTPgammaS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPgammaS 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 Delta17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPgammaS- 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 Delta17ARF1 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.
The work in D.R. Critchley’s laboratory was supported by the Medical Research Council, UK, and that in S. Cockcroft’s laboratory by the Wellcome Trust.
CitationJournal of Cell Biology, 1998, 143 (7), pp. 1981-1995
Author affiliation/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/MBSP Non-Medical Departments/Molecular & Cell Biology
VersionVoR (Version of Record)
Published inJournal of Cell Biology
PublisherRockefeller University Press
3T3 CellsADP-Ribosylation Factor 1ADP-Ribosylation FactorsActin CytoskeletonAnimalsBacterial ProteinsBiological TransportCell Membrane PermeabilityCulture Media, Serum-FreeCytoskeletal ProteinsGTP-Binding ProteinsGuanosine 5'-O-(3-Thiotriphosphate)Intercellular JunctionsMiceMicroinjectionsPaxillinPhosphoproteinsRecombinant Fusion ProteinsSequence DeletionStreptolysinsrho GTP-Binding Proteins