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Vol. 15, Issue 3, 1003-1010, March 2004
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Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
Submitted August 20, 2003;
Revised October 28, 2003;
Accepted November 18, 2003
Monitoring Editor: Anne Ridley
A major function of Rho-family GTPases is to regulate the organization of the actin cytoskeleton; filopodia, lamellipodia, and stress fiber are regarded as typical phenotypes of the activated Cdc42, Rac, and Rho, respectively. Using probes based on fluorescent resonance energy transfer, we report on the spatiotemporal regulation of Rac1 and Cdc42 at lamellipodia and membrane ruffles. In epidermal growth factor (EGF)-stimulated Cos1 and A431 cells, both Rac1 and Cdc42 were activated diffusely at the plasma membrane, followed by lamellipodial protrusion and membrane ruffling. Although Rac1 activity subsided rapidly, Cdc42 activity was sustained at lamellipodia. A critical role of Cdc42 in these EGF-induced morphological changes was demonstrated as follows. First, phorbol 12-myristate 13-acetate, which activated Rac1 but not Cdc42, could not induce full-grown lamellipodia in Cos1 cells. Second, a GTPase-activating protein for Cdc42, KIAA1204/CdGAP, inhibited lamellipodial protrusion and membrane ruffling without interfering with Rac1 activation. Third, expression of the Cdc42-binding domain of N-WASP inhibited the EGF-induced morphological changes. Therefore, Rac1 and Cdc42 seem to synergistically induce lamellipodia and membrane ruffles in EGF-stimulated Cos1 cells and A431 cells.
Abbreviations used: CFP, cyan fluorescent protein; EGF, epidermal growth factor; FRET, fluorescent resonance energy transfer; GAP, GTPase-activating protein; GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; mRFP, monomeric red fluorescent protein; PMA, phorbol 12-myristate 13-acetate; TPEM, two-photon excitation fluorescence microscopy; YFP, yellow fluorescent protein.
Online version of this article contains supplementary videos for some figures. Online version is available at www.molbiolcell.org.
* Corresponding author. E-mail address: matsudam{at}biken.osakau.ac.jp.
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