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© The Rockefeller University Press, 0021-9525/1997//927 $5.00
The Journal of Cell Biology, Volume 138, Number 4, , 1997 927-938

Article

Rho- and Rac-dependent Assembly of Focal Adhesion Complexes and Actin Filaments in Permeabilized Fibroblasts: An Essential Role for Ezrin/Radixin/Moesin Proteins



Deborah J.G. Mackay*, Fred Esch§, Heinz Furthmayr||, and Alan Hall*,{ddagger}

* MRC Laboratory for Molecular Cell Biology, {ddagger} Department of Biochemistry, and § Eisai London Research Laboratories, University College London, London WC1E 6BT, United Kingdom; and || Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305

The small GTPases Rho and Rac regulate actin filament assembly and the formation of integrin adhesion complexes to produce stress fibers and lamellipodia, respectively, in mammalian cells. Although numerous candidate effectors that might mediate these responses have been identified using the yeast two-hybrid and affinity purification techniques, their cellular roles remain unclear. We now describe a biological assay that allows components of the Rho and Rac signaling pathways to be identified. Permeabilization of serum-starved Swiss 3T3 cells with digitonin in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP{gamma}S) induces both actin filament and focal adhesion complex assembly through activation of endogenous Rho and Rac. These responses are lost when GTP{gamma}S is added 6 min after permeabilization, but can be reconstituted using concentrated cytosolic extracts. We have achieved a 10,000-fold purification of the activity present in pig brain cytosol and protein sequence analysis shows it to contain moesin. Using recombinant proteins, we show that moesin and its close relatives ezrin and radixin can reconstitute stress fiber assembly, cortical actin polymerization and focal complex formation in response to activation of Rho and Rac.

Abbreviations used in this paper: GAP, GTPase activating protein; GDI, guanine nucleotide dissociation inhibitor; GST, glutathione-S-transferase; GTP{gamma}S, guanosine 5'-O-(3-thiotriphosphate); MLC, myosin light chain; PIP2, phosphatidylinositol-4,5-bisphosphate; PVDF, polyvinylidene difluoride; ROCK, Rho-kinase.

Please address all correspondence to Alan Hall, MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK. Tel.: 44-171-380-7909. Fax: 44-171-380-7909. e-mail: Alan.Hall{at}ucl.ac.uk

We are very grateful to B. Balch for encouragement and advice early in this project, P. Mangeat and S. Tsukita for moesin and ERM antibodies, T. Bridges for recombinant proteins and N. Tapon for GST-RhoGAP.

This work was generously supported by the Wellcome Trust (to D.J.G. Mackay and A. Hall).


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