Uncoupling of Membrane Ruffling and Pinocytosis during Ras Signal Transduction (1997)

Activation of Ras stimulates cell surface membrane ruffling and pinocytosis. Although seen as coupled events, our study demonstrates that membrane ruffling and pinocytosis are regulated by distinct Ras signal transduction pathways. Ras controls membrane ruffling via the small GTPase Rac. In BHK-21 cells, expression of the constitutively active Rac1(G12V) mutant, via a Sindbis virus vector, resulted in a dramatic stimulation of membrane ruffling without affecting the uptake of horseradish peroxidase. Expression of Ha-Ras(G12V), an activated Ras mutant, stimulated both membrane ruffling and horseradish peroxidase uptake. The Ha-Ras(G12V)-stimulated pinocytosis but not membrane ruffling was abolished by either wortmannin or co-expression with a dominant negative mutant of Rab5, Rab5(S34N). Expression of the activated Rab5(Q79L) mutant mimics the stimulatory effect of Ha-Ras(G12V) on pinocytosis but not membrane ruffling. Our data indicate that Ha-Ras(G12V) separately activates Rab5-dependent pinocytosis and Rac1-dependent membrane ruffling. Activation of Ras stimulates cell surface membrane ruffling and pinocytosis. Although seen as coupled events, our study demonstrates that membrane ruffling and pinocytosis are regulated by distinct Ras signal transduction pathways. Ras controls membrane ruffling via the small GTPase Rac. In BHK-21 cells, expression of the constitutively active Rac1(G12V) mutant, via a Sindbis virus vector, resulted in a dramatic stimulation of membrane ruffling without affecting the uptake of horseradish peroxidase. Expression of Ha-Ras(G12V), an activated Ras mutant, stimulated both membrane ruffling and horseradish peroxidase uptake. The Ha-Ras(G12V)-stimulated pinocytosis but not membrane ruffling was abolished by either wortmannin or co-expression with a dominant negative mutant of Rab5, Rab5(S34N). Expression of the activated Rab5(Q79L) mutant mimics the stimulatory effect of Ha-Ras(G12V) on pinocytosis but not membrane ruffling. Our data indicate that Ha-Ras(G12V) separately activates Rab5-dependent pinocytosis and Rac1-dependent membrane ruffling. Ras protein is the prototype of a large family of 20–35-kDa monomeric GTPases that serve as molecular switches in regulating diverse cell functions (1Bokoch G.M. Der C.J. FASEB J. 1993; 7: 750-759Crossref PubMed Scopus (184) Google Scholar, 2Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1815) Google Scholar) including cell proliferation and differentiation (3Barbacid M. Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3747) Google Scholar, 4Bollag G. McCormick F. Nature. 1991; 351: 576-579Crossref PubMed Scopus (264) Google Scholar, 5Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1116) Google Scholar). The best characterized Ras-activated pathway involves a cascade of protein kinases including Raf, MEK (mitogen-activated protein kinase kinase), and mitogen-activated protein kinase (6Dent P. Haser W. Haystead T.A.J. Vincent L.A. Roberts T.M. Sturgill T.W. Science. 1992; 257: 1404-1407Crossref PubMed Scopus (495) Google Scholar, 7Howe L.R. Leevers S.J. Gomez N. Nakielny S. Cohen P. Marshall C.J. Cell. 1992; 71: 335-342Abstract Full Text PDF PubMed Scopus (627) Google Scholar, 8Macdonald s.G. Crews C.M. Wu L. Driller J. Clark R. Erickson R.L. McCormick F. Mol. Cell. Biol. 1993; 13: 6615-6620Crossref PubMed Scopus (202) Google Scholar) resulting in the expression of a specific set of target genes (9Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar). This signaling pathway is triggered by a direct interaction between Ras-GTP and Raf (10Moodie S.A. Willumsen B.M. Weber M.J. Wolfman A. Science. 1993; 260: 1658-1661Crossref PubMed Scopus (771) Google Scholar, 11Aelst L.V. Barr M. Marcus S. Polverino A. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6213-6217Crossref PubMed Scopus (502) Google Scholar, 12Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1653) Google Scholar, 13Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (579) Google Scholar, 14Zhang X.-F. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-313Crossref PubMed Scopus (684) Google Scholar). In addition to Raf, Ras-GTP also directly interacts with other effector molecules and activates multiple signal transduction pathways. In mammalian cells, the putative Ras effectors include Ras-GAP (GTPase-activating protein) (15Polakis P. McCormick F. J. Biol. Chem. 1993; 268: 9157-9160Abstract Full Text PDF PubMed Google Scholar), Ral-GDS (GDP dissociation stimulator) (16Hofer F. Fields S. Schneider C. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11089-11093Crossref PubMed Scopus (245) Google Scholar, 17Spaargaren M. Bischoff J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12609-12613Crossref PubMed Scopus (247) Google Scholar), and phosphatidylinositol-3-OH kinase (PI 3-kinase) 1The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; α-MEM, α-minimum essential medium; HRP, horseradish peroxidase; PBS, phosphate-buffered saline. 1The abbreviations used are: PI 3-kinase, phosphatidylinositol 3-kinase; α-MEM, α-minimum essential medium; HRP, horseradish peroxidase; PBS, phosphate-buffered saline. (18Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1703) Google Scholar). It has been suggested that multiple Ras functions contribute to mammalian cell proliferation (19White M.A. Nicolette C. Minden A. Polverino A. Aelst L.V. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (626) Google Scholar). Parallel to the activation of nuclear gene expression, Ras-GTP also triggers profound changes in the cytoplasm. The Ras signaling cascade involving the small GTPase Rac1 plays an important role in regulating the actin cytoskeleton and cell surface membrane ruffles (20Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3039) Google Scholar). Ras activation also stimulates pinocytosis (21Bar-Sagi D. Feramisco J.R. Science. 1986; 233: 1061-1068Crossref PubMed Scopus (530) Google Scholar) and transport to endosomes and lysosomes. This process involves the small GTPase, Rab5 (22Bucci C. Parton R.G. Mather I.M. Stunnenberg H. Simons K. Hoflack B. Zerial M. Cell. 1992; 70: 715-728Abstract Full Text PDF PubMed Scopus (1109) Google Scholar, 23Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar). Because cell surface ruffling and pinocytosis both involve organized movements of the plasma membrane, and because Ras stimulates both processes, the two processes have long been thought to be linked (21Bar-Sagi D. Feramisco J.R. Science. 1986; 233: 1061-1068Crossref PubMed Scopus (530) Google Scholar). We have found that membrane ruffling and pinocytosis are regulated by distinct Ras signal transduction pathways. Whereas Ras-stimulated ruffling depends on Rac1 and is independent of PI 3-kinase and Rab5, Ras-stimulated pinocytosis depends on Rab5 and probably PI 3-kinase and is independent of Rac1. BHK-21 cells were grown as described previously (23Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar). Wortmannin (Sigma) in dimethyl sulfoxide at 1 mg/ml (stock solution) was freshly diluted with α-MEM and added to previously rinsed cell monolayers. The wortmannin treatment (15 min, 37 °C) was followed by horseradish peroxidase (HRP) uptake. Dimethyl sulfoxide (<0.1% after dilution) had no effect on pinocytosis or membrane ruffling (data not shown). cDNAs of Ha-Ras, Rac1, and Rab5 were subcloned into the unique XbaI restriction site of the Sindbis vector Toto1000:3′2J (23Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar, 24Hahn C.S. Hahn Y.S. Braciale T.J. Rice C.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2679-2683Crossref PubMed Scopus (205) Google Scholar). The plasmid was then linearized by XhoI digestion and used as a template for in vitro transcription with SP6 RNA polymerase. The resulting RNA transcripts were used for transfection of confluent BHK-21 cell monolayers using a Lipofectin-mediated procedure (Life Technologies, Inc.). Cells were maintained at 37 °C, and the media containing released viruses were harvested 40 h after transfection. Virus titers were generally between 108 and 109 plaque-forming units per ml. Virus stocks were aliquoted and kept frozen at −80 °C before use. Cell lysates (5 μl) were analyzed by SDS-polyacrylamide gel electrophoresis (12% acrylamide), and the proteins were transferred to an Immobilon-P membrane (Millipore) using a Bio-Rad semi-dry transfer apparatus. The membrane was probed with an Ha-Ras specific monoclonal antibody (AB1, from Oncogene Science), and the immunoblot was developed using the ECL reagents from Amersham Corp. Confluent BHK-21 monolayers were mock-infected or infected with the vector or recombinant viruses as described (23Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar). At 4 h post-infection or otherwise indicated, cells were washed twice with serum-free α-MEM, and HRP uptake was initiated by adding HRP (5mg/ml) in α-MEM (1ml) containing 0.2% bovine serum albumin. Cell lysates were assayed for HRP activity as described (23Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar). At 4 h post-infection, early endosomes were marked by a 10-min HRP uptake. Cells were rinsed four times with ice-cold PBS, fixed in 2% glutaraldehyde (in PBS) for 30 min, and washed four more times with PBS. HRP reaction was developed for 10 min by incubating with 50 mm Tris-HCl, pH 7.4, containing 2 mg/ml diaminobenzidine (Sigma) and 0.01% H2O2 (Sigma). Cells were then processed for Poly/Bed (Polysciences, Inc.) embedding, semi-thick section (∼200 nm) preparation, and electron microscopy. At 4 h post-infection, cells were fixed with 2% paraformaldehyde (in PBS) for 30 min, peameabilized, and quenched with PBS containing 0.05% Triton X-100, 0.1 nNH4Cl, and 0.2% gelatin, followed by staining with rhodamine-phalloidin (25Nobes C.D. Hawkins P. Stephens L. Hall A. J. Cell Sci. 1995; 108: 225-233Crossref PubMed Google Scholar). The coverslips were mounted in 70% glycerol (in PBS), and the actin localization and cell edge membrane ruffles were visualized by a Zeiss axiovert microscope and a Bio-Rad confocal scanning imaging system. Pinocytosis is regulated by serum growth factors but the molecular mechanisms and biological significance are not well understood. Many growth factors also stimulate cell surface membrane ruffling via rearrangement of actin filaments (20Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3039) Google Scholar). It has long been thought that membrane ruffling contributes to increased pinocytosis (reviewed in Ref. 26Swanson J.A. Watts C. Trends Cell Biol. 1995; 5: 424-428Abstract Full Text PDF PubMed Scopus (656) Google Scholar). One example is oncogenic Ras-stimulated pinocytosis and membrane ruffling (21Bar-Sagi D. Feramisco J.R. Science. 1986; 233: 1061-1068Crossref PubMed Scopus (530) Google Scholar). To understand the molecular basis of the two processes, we used a combination of reagents that specifically alter pinocytosis and membrane ruffling and found, to our surprise, that pinocytosis and membrane ruffling are independent cellular processes regulated by distinct Ras signal transduction pathways. To determine the effect of Ras activation on pinocytosis, we transiently expressed a constitutively active Ras mutant, Ha-Ras(G12V), in cultured BHK-21 cells (23Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar, 24Hahn C.S. Hahn Y.S. Braciale T.J. Rice C.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2679-2683Crossref PubMed Scopus (205) Google Scholar) and monitored pinocytosis of HRP (23Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar). Fig. 1 A shows immunoblot analysis of Ha-Ras(G12V) expression at different times after virus infection. At 4 h after infection, abundant Ha-Ras(G12V) expression was detected. Thereafter, the protein accumulated throughout virus infection (Fig. 1 A). Endogenous Ras protein was not detected. HRP accumulation increased dramatically in cells expressing Ha-Ras(G12V) (Fig. 1 B). At 4 h after infection, cells expressing Ha-Ras(G12V) showed a 3-fold stimulation of HRP uptake compared with the control cells (Fig. 1 B). Ha-Ras(G12V) also increased the rate of HRP uptake (Fig. 1 C). By electron microscopy, HRP was identified in intracellular endosomes (Fig.2), which were significantly enlarged in cells expressing Ha-Ras(G12V) (Fig. 2), suggesting increased endosome activity.Figure 2Electron microscopy of early endosomal structures. Expression of Ha-Ras(G12V) or Rab5(Q79L) leads to the formation of enlarged endosomal structures (B andC, respectively) that are not seen in control cells (A). Arrows indicate HRP-marked endosomes.N denotes nucleus. Magnification 1:12,800.Bar = 1 cm = 730 nm.View Large Image Figure ViewerDownload (PPT) Ha-Ras(G12V) interacts with multiple effector molecules via its effector domain (residues 32–40), (10Moodie S.A. Willumsen B.M. Weber M.J. Wolfman A. Science. 1993; 260: 1658-1661Crossref PubMed Scopus (771) Google Scholar, 11Aelst L.V. Barr M. Marcus S. Polverino A. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6213-6217Crossref PubMed Scopus (502) Google Scholar, 12Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1653) Google Scholar, 13Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (579) Google Scholar, 14Zhang X.-F. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-313Crossref PubMed Scopus (684) Google Scholar, 15Polakis P. McCormick F. J. Biol. Chem. 1993; 268: 9157-9160Abstract Full Text PDF PubMed Google Scholar, 16Hofer F. Fields S. Schneider C. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11089-11093Crossref PubMed Scopus (245) Google Scholar, 17Spaargaren M. Bischoff J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12609-12613Crossref PubMed Scopus (247) Google Scholar, 18Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1703) Google Scholar, 19White M.A. Nicolette C. Minden A. Polverino A. Aelst L.V. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (626) Google Scholar, 27Bischoff F.R. Ponsting H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10830-10834Crossref PubMed Scopus (220) Google Scholar, 28Kikuchi A. Demo S.D. Ye Z.-H. Chen Y.-W. Williams L.T. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (244) Google Scholar). To identify the Ras pathway(s) leading to the stimulation of pinocytosis, we expressed an effector domain mutant of Ha-Ras(G12V), Ha-Ras(G12V,D38A), and tested the effect on HRP uptake. Ras(G12V,D38A) completely abolished the ability of Ha-Ras(G12V) to stimulate HRP uptake (Fig.3 A). Since the D38A mutation can abolish the interaction between Ha-Ras(G12V) and its effector molecules including PI 3-kinase (18Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1703) Google Scholar), this result demonstrated the existence of a responsible Ras signal transduction pathway. Expression of the constitutively active Raf and RalB mutants (the C-terminal 344 residues of human c-Raf (29Stanton V.P. Nichols D.W. Laudano A.P. Cooper G.M. Mol. Cell. Biol. 1989; 9: 639-647Crossref PubMed Scopus (189) Google Scholar) and RalB(G23V), respectively), putative Ras effectors, failed to mimic the stimulatory effect of Ha-Ras(G12V) on HRP uptake (data not shown). Wortmannin, a potent PI 3-kinase inhibitor (30Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1041) Google Scholar, 31Thelen M. Wymann M.P. Langen H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4960-4964Crossref PubMed Scopus (202) Google Scholar), completely blocked the stimulation of HRP uptake by Ha-Ras(G12V) (Fig. 3 B). Wortmannin (100 nm) also inhibited HRP uptake in control cells (Fig. 3 B) (32Jones A.T. Clague M.J. Biochem. J. 1995; 311: 31-34Crossref PubMed Scopus (108) Google Scholar,33Li G. D'Souza-Schorey C. Barbieri M.A. Roberts R.L. Klippel A. Williams L.T. Stahl P.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211Crossref PubMed Scopus (245) Google Scholar). Early endosome fusion immediately follows the internalization step at the plasma membrane and appears to be rate-limiting (22Bucci C. Parton R.G. Mather I.M. Stunnenberg H. Simons K. Hoflack B. Zerial M. Cell. 1992; 70: 715-728Abstract Full Text PDF PubMed Scopus (1109) Google Scholar, 23Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar). Wortmannin inhibits a function required for the activation of Rab5 (33Li G. D'Souza-Schorey C. Barbieri M.A. Roberts R.L. Klippel A. Williams L.T. Stahl P.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211Crossref PubMed Scopus (245) Google Scholar), a GTPase essential for early endosome fusion. Expression of the constitutively active Rab5 mutant, Rab5(Q79L), leads to increased early endosome fusion and increased pinocytosis while expression of the dominant negative Rab5(S34N) mutant results in decreased early endosome fusion and decreased pinocytosis (23Li G. Stahl P.D. J. Biol. Chem. 1993; 268: 24475-24480Abstract Full Text PDF PubMed Google Scholar, 34Stenmark H. Parton R.G. Steele-Mortimer O. Lutcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (761) Google Scholar). The positive correlation between early endosome fusion activity and cellular pinocytic activity led us to suggest that Ha-Ras(G12V) sequentially activates a wortmannin-sensitive function (possibly PI 3-kinase) and Rab5, leading to an increase in early endosome fusion and pinocytosis. We found that Rab5(Q79L) mimicked the stimulatory effect of Ha-Ras(G12V) on HRP uptake, and this stimulation could be blocked neither by wortmannin (33Li G. D'Souza-Schorey C. Barbieri M.A. Roberts R.L. Klippel A. Williams L.T. Stahl P.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211Crossref PubMed Scopus (245) Google Scholar) nor by the dominant negative Ha-Ras(S17N) mutant (Fig.3 C). Co-expression of the activated Rab5(Q79L) and Ha-Ras(G12V) mutants did not lead to a synergistic stimulation of HRP uptake (Fig. 3 C). Furthermore, like cells expressing Rab5(Q79L) (Fig. 2 C), cells expressing Ha-Ras(G12V) exhibited enlarged endosomal structures (Fig. 2 B), indicative of enhanced endosome fusion. Finally, co-expression of the dominant negative Rab5(S34N) mutant completely blocked the stimulatory effect of Ha-Ras(G12V) on HRP uptake (Fig. 3 D). The pioneering work of Hall and colleagues (20Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3039) Google Scholar) has partially defined the Ras signal transduction pathway regulating membrane ruffling. Our study begins to shed light on this important aspect of Ras signal transduction. We show that oncogenic Ras-stimulated pinocytosis is sensitive to wortmannin and is dependent on Rab5 function. Because Ras can directly activate a PI 3-kinase (18Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1703) Google Scholar) and PI 3-kinase activity can stimulate Rab5-dependent early endosome fusion in vitro (33Li G. D'Souza-Schorey C. Barbieri M.A. Roberts R.L. Klippel A. Williams L.T. Stahl P.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211Crossref PubMed Scopus (245) Google Scholar), we suggest that Ras sequentially activates PI 3-kinase and Rab5, leading to a quantitative increase in endosome fusion and pinocytosis. Ridley et al. (20Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3039) Google Scholar) previously characterized a Ras signal transduction pathway that promotes membrane ruffling via activation of the small GTPase Rac1. To further examine the relationship of Ras-stimulated membrane ruffling and pinocytosis, we expressed the constitutively active and dominant negative Rac1 mutants. As shown in Fig. 3 E, expression of Rac1(G12V) did not stimulate HRP uptake while under the same conditions Ha-Ras(G12V) stimulated HRP uptake 3-fold. Fig. 3 F shows the co-expression of constructs used in this study. These data immediately suggested that membrane ruffling and pinocytosis are controlled by distinct Ras signal transduction pathways. We then examined membrane ruffling (Fig. 4). Consistent with previous reports (20Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3039) Google Scholar,25Nobes C.D. Hawkins P. Stephens L. Hall A. J. Cell Sci. 1995; 108: 225-233Crossref PubMed Google Scholar), expression of Rac1(G12V) resulted in a dramatic production of cell surface membrane ruffles (Fig. 4 C). Under the same conditions, expression of Ha-Ras(G12V) also stimulated membrane ruffling (Fig. 4 D). Cells expressing the activated Rab5(Q79L) mutant, a strong stimulator of pinocytosis, did not show membrane ruffling (Fig. 4 E), nor did the vector virus-infected (Fig. 4 B) or mock-infected control cells (Fig. 4 A). In agreement with Nobes et al. (25Nobes C.D. Hawkins P. Stephens L. Hall A. J. Cell Sci. 1995; 108: 225-233Crossref PubMed Google Scholar), we found that wortmannin (100 nm) failed to block Rac(G12V)-induced or Ha-Ras(G12V)-induced membrane ruffling (data not shown), under conditions where Ha-Ras(G12V)-stimulated pinocytosis was completely inhibited (Fig. 3 B). From these data, we conclude that the Ras signal transduction pathway leading to the activation of Rac1 and membrane ruffling does not contribute to the stimulation of pinocytosis. Taken together, as summarized in Fig. 5, it is clear that pinocytosis and membrane ruffling are regulated by distinct sets of molecules during Ras signal transduction.