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Vol. 16, Issue 9, 4294-4303, September 2005
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Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, Japan
Submitted December 14, 2004;
Revised June 13, 2005;
Accepted June 21, 2005
Monitoring Editor: Anne Ridley
We examined the spatio-temporal activity of RhoA in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In HeLa cells migrating at a low cell density, RhoA was activated both at the contractile tail and at the leading edge. However, RhoA was activated only at the leading edge in MDCK cells migrating as a monolayer sheet. In growth factor-stimulated Cos1 and NIH3T3 cells, the activity of RhoA was greatly decreased at the plasma membrane, but remained high at the membrane ruffles in nascent lamellipodia. These observations are in agreement with the proposed role played by RhoA in stress fiber formation, but they also implicated RhoA in the regulation of membrane ruffling, the induction of which is a typical phenotype of activated Rac. In agreement with this view, dominant negative RhoA was found to inhibit membrane ruffling induced by active Rac. Furthermore, we found that Cdc42 activity was also required for high RhoA activity in membrane ruffles. Finally, we found that mDia1, but not ROCK, was stably associated with membrane ruffles. In conclusion, these results suggested that RhoA cooperates with Rac1 and Cdc42 to induce membrane ruffles via the recruitment of mDia.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Michiyuki Matsuda (matsudam{at}biken.osaka-u.ac.jp).
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