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Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis

2001; Springer Nature; Volume: 20; Issue: 13 Linguagem: Inglês

10.1093/emboj/20.13.3333

1460-2075

Yusuke Ohba,

Immunodeficiency and Autoimmune Disorders

Article2 July 2001free access Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis Yusuke Ohba Yusuke Ohba Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan Search for more papers by this author Koichi Ikuta Koichi Ikuta Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501 Japan Search for more papers by this author Atsuo Ogura Atsuo Ogura Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, 162-8640 Japan Search for more papers by this author Junichiro Matsuda Junichiro Matsuda Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, 162-8640 Japan Search for more papers by this author Naoki Mochizuki Naoki Mochizuki Departmtent of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan Search for more papers by this author Kazuo Nagashima Kazuo Nagashima Laboratory of Molecular and Cellular Pathology, Department of Neuroscience, Graduate School of Medicine, Hokkaido University, Sapporo, 060-8638 Japan Search for more papers by this author Kazuo Kurokawa Kazuo Kurokawa Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan Search for more papers by this author Bruce J. Mayer Bruce J. Mayer Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, 06030 USA Search for more papers by this author Kazushige Maki Kazushige Maki Department of Immune Regulation, Tokyo Medical and Dental University, Tokyo, 113-8519 Japan Search for more papers by this author Jun-ichi Miyazaki Jun-ichi Miyazaki Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Suita, Osaka, 565-0871 Japan Search for more papers by this author Michiyuki Matsuda Corresponding Author Michiyuki Matsuda Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan Search for more papers by this author Yusuke Ohba Yusuke Ohba Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan Search for more papers by this author Koichi Ikuta Koichi Ikuta Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501 Japan Search for more papers by this author Atsuo Ogura Atsuo Ogura Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, 162-8640 Japan Search for more papers by this author Junichiro Matsuda Junichiro Matsuda Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, 162-8640 Japan Search for more papers by this author Naoki Mochizuki Naoki Mochizuki Departmtent of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan Search for more papers by this author Kazuo Nagashima Kazuo Nagashima Laboratory of Molecular and Cellular Pathology, Department of Neuroscience, Graduate School of Medicine, Hokkaido University, Sapporo, 060-8638 Japan Search for more papers by this author Kazuo Kurokawa Kazuo Kurokawa Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan Search for more papers by this author Bruce J. Mayer Bruce J. Mayer Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, 06030 USA Search for more papers by this author Kazushige Maki Kazushige Maki Department of Immune Regulation, Tokyo Medical and Dental University, Tokyo, 113-8519 Japan Search for more papers by this author Jun-ichi Miyazaki Jun-ichi Miyazaki Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Suita, Osaka, 565-0871 Japan Search for more papers by this author Michiyuki Matsuda Corresponding Author Michiyuki Matsuda Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan Search for more papers by this author Author Information Yusuke Ohba1, Koichi Ikuta2, Atsuo Ogura3, Junichiro Matsuda3, Naoki Mochizuki4, Kazuo Nagashima5, Kazuo Kurokawa1, Bruce J. Mayer6, Kazushige Maki7, Jun-ichi Miyazaki8 and Michiyuki Matsuda 1 1Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, 565-0871 Japan 2Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501 Japan 3Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, 162-8640 Japan 4Departmtent of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan 5Laboratory of Molecular and Cellular Pathology, Department of Neuroscience, Graduate School of Medicine, Hokkaido University, Sapporo, 060-8638 Japan 6Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, 06030 USA 7Department of Immune Regulation, Tokyo Medical and Dental University, Tokyo, 113-8519 Japan 8Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Suita, Osaka, 565-0871 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:3333-3341https://doi.org/10.1093/emboj/20.13.3333 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G−/− homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF-hC3G. Expression of Cre abolished the expression of C3G in MEF-hC3G and inhibited cell adhesion-induced activation of Rap1. The Cre-expressing MEF-hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R-Ras. Expression of Epac and CalDAG-GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G-deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G-dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse. Introduction Ras-family G proteins function as molecular switches in growth, differentiation, survival and adhesion of eukaryotic cells (Bos, 1997; Campbell et al., 1998). They cycle between GDP-bound inactive and GTP-bound active forms. This cycling is mediated by guanine nucleotide exchange factor (GEF), the activator, and GTPase-activating proteins (GAPs), the inactivator (Bourne et al., 1990; Downward, 1992). A variety of signals, external or internal, control the on/off status of the Ras-family G proteins via the activation or inactivation of GEFs and GAPs (Overbeck et al., 1995; Bos, 1997). C3G was identified as one of the two major proteins bound to the Src homology 3 (SH3) domain of the Crk oncogene product (Knudsen et al., 1994; Tanaka et al., 1994). The C-terminus of C3G consists of the CDC25 homology domain, which is a catalytic domain of the Ras-family GEF. C3G contains three Crk SH3-binding sequences and one p130Cas SH3-binding sequence in the central region (Kirsch et al., 1998). The N-terminal region of C3G negatively regulates its GEF activity (Ichiba et al., 1999). Many types of stimulation induce binding of the Crk–C3G complex to phosphotyrosine-containing proteins, including receptor-type tyrosine kinases, p130Cas and paxillin (reviewed by Kiyokawa et al., 1997). Following translocation from the cytosol to the cell membrane, C3G becomes phosphorylated on Tyr504, and the negative regulation by the N-terminal region is repressed to increase the GEF activity (Ichiba et al., 1999). C3G promotes the guanine nucleotide exchange reaction of Rap1 and Rap2, and, to a lesser extent, it also stimulates R-Ras and TC21 (Gotoh et al., 1995, 1997; Ohba et al., 2000a,b). In contrast to Ras, the function of which has been studied extensively, the physiological role of the substrates of C3G remains elusive. A function of Rap1 is to antagonize Ras (Kitayama et al., 1989; Yatani et al., 1991; Sakoda et al., 1992; Boussiotis et al., 1997; Okada et al., 1998), probably by inhibiting the Ras-dependent activation of mitogen-activated protein kinase (MAPK) (Cook et al., 1993; Hu et al., 1997). However, in some cell types, Rap1 activates the MAPK cascade, as does Ras (Yoshida et al., 1992; Vossler et al., 1997; Altschuler and Ribeiro-Neto, 1998; York et al., 1998). Moreover, it is reported that growth factor-induced activation of Rap1 does not correlate with the repression of Ras-dependent MAPK activation (Zwartkruis et al., 1998). Recently, it has been shown that Rap1 contributes integrin-mediated cell adhesion (Posern et al., 1998; Tsukamoto et al., 1999; Katagiri et al., 2000; Reedquist et al., 2000), although the mechanism underlying this phenomenon is yet to be analyzed. Rap2, the amino acid sequence of which shows 60% identity with Rap1, is regulated by the same set of GEFs and GAPs as is Rap1 (Ohba et al., 2000a). Rap2 binds to a set of effectors very similar to those of Rap1 (Janoueix-Lerosey et al., 1998; Nancy et al., 1999), and it inhibits Ras-dependent MAPK activation, as does Rap1 (Ohba et al., 2000a). A unique feature of Rap2 is its low sensitivity to GAPs, which permits more than half of Rap2 to remain in a GTP-bound active state in adherent cells. R-Ras and TC21 are the two members of the R-Ras subfamily and are regulated by the same GEFs and GAPs (Ohba et al., 2000b). R-Ras and TC21 activate MAPK, as does the classical Ras (Cox et al., 1994; Graham et al., 1999; Movilla et al., 1999; Rosario et al., 1999); however, they may have unique functions, such as inhibition of apoptosis (Suzuki et al., 1997) and stimulation of cell adhesion (Zhang et al., 1996). The C3G–Rap1 signaling cascade has been studied genetically in Drosophila melanogaster. Overexpression of wild-type C3G (DC3G) does not cause any detectable abnormality in the developing eye; however, expression of membrane-targeted active C3G leads to an adult rough-eye phenotype, as does the expression of active Ras or active Rap1 (Hariharan et al., 1991; Ishimaru et al., 1999). This rough-eye phenotype due to the membrane-targeted C3G is suppressed by reduction of the gene dose of Ras1, ksr, rolled (MAPK) or Rap1, indicating that the effect of DC3G overactivation is mediated by the RAS–MAPK pathway and RAP1. Overexpression of Rapgap, the inactivator, also induces a rough-eye phenotype, which is exacerbated by reduction of the Rap1 gene dosage (Chen et al., 1997). Removal of maternal Rap1 inhibits ventral furrow closure and head involution of the embryo (Hariharan et al., 1991), and it also perturbs the migration of pole cells and mesodermal cells (Asha et al., 1999). Rap1 is required for imaginal disc development, but not for adult survival (Asha et al., 1999). Thus, the C3G–Rap1 pathway in Drosophila has at least two roles: as a regulator of morphogenesis in the adult stage and as a mediator of cell proliferation and cell fate specification in the developmental stage. Here, we demonstrate that the C3G gene is essential for mouse embryogenesis and that C3G-dependent Rap1 activation promotes cell adhesion and cell spreading, but represses cell migration. Results Structure of the mouse C3G gene We sequenced the mouse C3G genome to determine the exon–intron structure (Figure 1A). We could not obtain a genomic clone that contained the initiation codon of mouse C3G. Therefore, an exon, which corresponded to cDNA from nucleotide 281 to 394 of human C3G cDNA, was temporarily designated as exon 2. The 23rd exon contained the stop codon. Figure 1.Generation of C3G knockout mice. (A) Schematic structure of the mouse C3G gene and C3G protein. Coding exons 2–23 are shown at the top. A targeting vector, p836BL/R, consisted of 3.5 and 6.0 kb homologous DNA fragments, the PGK-neo cassette replacing exons 15 and 16, and the MC1-tk cassette at the 5′ end of the targeting vector. The diagnostic probe (probe B) and the EcoRV sites (vertical arrows) are indicated at the bottom. P, proline-rich Crk-binding regions; CDC25, CDC25 homology region. (B) Southern blot analysis of mouse genomic DNA. EcoRV-digested DNAs from ES cells were separated on a 0.5% agarose gel, transferred to a nylon membrane and hybridized with fluorescein isothiocyanate (FITC)-labeled probe B. The mouse genotype was identified by 9.4 and 6.5 kb fragments that were derived from the wild-type and mutant alleles, respectively. (C) Horizontal sections of uterus of a C3G+/− mouse crossed with a C3G+/− mouse and examined at E7.5. Embryos of C3G−/− mice did not conform to any histological structure, as shown on the right. A control section from a C3G+/− embryo is shown on the left. Download figure Download PowerPoint Generation of C3G knockout mice To produce a targeted disruption of the mouse C3G gene, we constructed a C3G disruption vector, p836BL/R, containing 3.5 and 6.0 kb homologous DNA fragments, the PGK-neo cassette replacing exons 15 and 16, and the MC1-tk cassette being at the 5′ end of the targeting vector (Figure 1A). Embryonic stem (ES) cells were transfected with linearized p836BL/R by electroporation, and clones resistant to both G418 and 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil were isolated. Homologous recombinant clones were detected by Southern blot analysis with probe B, a flanking sequence on the 3′ side (Figure 1B), and the neo probe (data not shown). Of 165 clones, two were targeted for the C3G gene. Both of the targeted clones were injected into C57BL/6/blastocysts, which gave rise to chimeras. Chimeric mice were crossed with C57BL/6 mice, and mice heterozygous for the mutation in the C3G locus were identified among offspring with agouti coat color by Southern blot analysis. Homozygous mutant offspring could not be recovered from crosses between heterozygous mice, suggesting that the mutation causes lethality during embryogenesis. To determine the stage at which mutant embryos died, we collected embryos from staged matings and genotyped them by using a PCR assay (Table I). When we examined implantation sites at embryonic days 7.5 and 8.5 (E7.5 and E8.5), nearly a quarter of the sites contained only degenerating embryonic tissue within the decidual stroma. In some cases, little DNA for the PCR assay was retrieved from the degenerating tissue and, therefore, these cases were excluded from the data shown in Table I. However, the remaining samples with sufficient amounts of DNA were identified with C3G−/− mice. Histological examination of the implantation sites with these abnormal embryos revealed the absence of typical fetal or placental structures (Figure 1C). These findings indicate that C3G was essential for embryogenesis, and the mutant embryos died shortly after implantation (around day 5). Table 1. Genotyping of embryos arising from C3G heterozygous crosses Gestational age No. of embryos or postnatal mice Total +/+ +/− −/− E7.5 23 7 14 3 E8.5 19 5 13 1 Postnatal 91 36 55 0 Complementation of mouse C3G by the human C3G gene To confirm that the embryonic lethality of C3G homozygous mice resulted from the C3G deficiency, we expressed human C3G to rescue the viability of C3G−/− mice. The human C3G cDNA was placed under the regulation of the chicken β-actin promoter and the cytomegalovirus (CMV) immediate early enhancer (Figure 2A). A 5.7 kb SalI–BamHI fragment was microinjected into the pronuclei of fertilized eggs. Of 36 offspring, seven founders (F0 mice) were identified by PCR screening at the age of 4 weeks. All founders were fertile and were crossed with C3G+/− mice. In F1 mice, we could not observe any effect of the expression of the human C3G gene. C3G+/− mice carrying the human C3G transgene, indicated hereafter as C3G+/−/hu, were then crossed. From two transgenic lines, we obtained mice deficient in mouse C3G and expressing human C3G, C3G−/−/hu (Figure 2B). However, in both mouse lines, the numbers of C3G−/−/hu mice were lower than expected (Table II), indicating that expression of human C3G did not completely rescue the mouse C3G deficiency. We used a CAG promoter, which shows potent activity in a wide range of cells (Niwa et al., 1991), to express the human C3G gene; however, in some tissues and at some developmental stages, it may not provide sufficient C3G. We never obtained C3G-deficient mice without the human C3G transgene, confirming that the C3G gene was essential for embryogenesis and that the human C3G gene complemented the C3G deficiency. Figure 2.Complementation of mouse C3G deficiency by the human C3G transgene. (A) Schematic illustration of an expression plasmid of the human C3G transgene. The human C3G gene, which is sandwiched with two loxP recombination sites, is placed downstream of the chicken β-actin promoter and the CMV immediate early enhancer. SalI and BamHI sites, which were used to isolate the transgene expression unit, are indicated by arrows. (B) Southern blot analysis of transgenic mice crossed with C3G knockout mice. The genotype of mouse C3G and the presence of the human C3G transgene are indicated at the top. Download figure Download PowerPoint Table 2. Genotyping of living mice from C3G+/−/hu crosses Parents No. of mice +/+/hu +/+/− +/−/hu +/−/− −/−/hu +/−/huA × +/−/− 9 6 8 17 3 −/−/huA × +/−/− 0 0 11 16 9 +/−/huB × +/−/− 10 17 10 29 3 −/−/huB × +/−/− 0 0 14 15 2 A and B indicate transgenic mouse lines A and B, respectively. Isolation of embryonic fibroblasts from C3G−/−/hu mice To study the role of C3G further, we obtained mouse embryonic fibroblasts (MEFs) from C3G−/−/hu embryos at E12.5. In these cell lines, which were named MEF-hC3G, the integrated human C3G transgene could be excised by infection with the Cre-expressing retrovirus, MX-pie-Cre, or adenovirus, AxCANCre. Equal amounts of genomic DNA of MEF-hC3G and irrelevant mouse genomic DNA mixed with standard C3G plasmid were digested with XhoI and analyzed by Southern blotting (Figure 3A). MEF-hC3G cells were estimated to carry 20 copies of the human C3G transgene per cell. By Cre expression, the quantity of integrated C3G was decreased to less than one copy per cell. We further confirmed by immunoblotting that removal of the human C3G transgene abolished the expression of C3G protein in MEF-hC3G cells (Figure 3B). We did not detect any truncated C3G protein that might be generated by homologous recombination by use of antibodies raised against three different regions of C3G (Figure 3C). However, contrary to our expectation, the loss of C3G in MEF-hC3G did not decrease the basal level amounts of GTP-bound forms of Rap1, Rap2 and R-Ras, which are the substrates of C3G (Figure 3D). Figure 3.Cre-dependent disruption of the C3G gene. (A) MEF-hC3G cells were infected with a recombinant retrovirus, MX-pie (indicated as Cre −) or MX-pie-Cre (Cre +), and selected in DMEM containing 2 μg/ml puromycin for 48 h. A 5 μg aliquot of DNA from the cells and from an irrelevant mouse tail biopsy sample containing the indicated copy numbers of the human C3G genes were digested with XhoI and analyzed by Southern blotting. (B) MEF-hC3G cells were infected with Cre-carrying adenovirus (AxCanCre), Cre-carrying retrovirus (MX-pie-Cre) and control virus (MX-pie) at the multiplicity of infection (MOI) indicated at the top of panels. Forty-eight hours after infection, cells were lysed in lysis buffer and separated by SDS–PAGE, followed by immunoblotting by use of anti-C3G antibody. The filter was reprobed with anti-tubulin monoclonal antibody to confirm that similar amounts of lysates were analyzed (lower panels). (C) Cell lysates of MEF-hC3G were separated by SDS–PAGE, followed by western blotting by use of anti-C3G polyclonal antibody, sc-869, anti-C3G serum 1A or 1B. (D) MEF-hC3G cells were infected with Cre-carrying retrovirus. After 48 h, cells were lysed in lysis buffer and GTP-bound G proteins were collected by use of either GST–Raf-RBD (for Ras and R-Ras) or GST–RalGDS-RBD (for Rap1 and Rap2). The resulting complexes were precipitated by glutathione–Sepharose beads and analyzed by SDS–PAGE and western blotting by use of antibodies. Small aliquots of lysates were analyzed by immunoblotting to confirm a similar level of expression of G proteins. For the detection of GTP-bound R-Ras, MEF-hC3G cells were infected with MSCV-R-Ras retrovirus and maintained in DMEM containing 2 μg/ml puromycin for 48 h. The cells were analyzed as described, except that anti-FLAG antibody was used to detect the expressed R-Ras. Download figure Download PowerPoint Impaired cell attachment and cell adhesion of C3G-deficient cells Although the morphology of Cre-expressing MEF-hC3G cells was indistinguishable from that of the parent MEF- hC3G cells, cell attachment and cell spreading after replating were significantly impaired by C3G deficiency (Figure 4A). The replating efficiency was quantitated by labeling of cells with the fluorescent dye 2′,7′-bis- (2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF, AM). As shown in Figure 4B, C3G enhanced cell attachment to dishes coated with collagen type I or fibronectin (FN), but not those coated with poly-L-lysine. Cell spreading was quantitated by measurement of cell sizes 1 and 3 h after replating. Again, C3G induced cell spreading on the dishes coated with collagen type I, but not with poly-L-lysine (Figure 4C). These results suggested that C3G was required for the cell attachment and cell spreading that were mediated by specific interaction between the extracellular matrices and integrin. Figure 4.Requirement for C3G in cell adhesion and cell spreading. (A) MEF-hC3G cells were infected with MX-pie-Cre or MX-pie. After 48 h, cells were trypsinized, kept in suspension for 1 h and plated on dishes coated with collagen type I. The cells were observed by time-lapse microscopy. We show representative photographs at the indicated time points. (B) MEF-hC3G cells were infected with MX-pie or MX-pie-Cre. After 48 h, cells were trypsinized and labeled with BCECF, AM. The cells were plated on 96-well black-colored plates coated with the reagents indicated at the bottom and incubated for 1 h at 37°C in a CO2 incubator. Cells were washed three times with HBSS, and the fluorescence intensity was measured at excitation and emission wavelengths of 488 and 530 nm, respectively. Plating efficiency is shown as the average for three wells, with the SE. (C) MEF-hC3G cells infected with MX-pie or MX-pie-Cre were plated to dishes coated with collagen (Col-I) or poly-L-lysine (PLL). Twenty EGFP-positive cells were photographed after 1 and 3 h and measured for size. Average and SE are shown. Download figure Download PowerPoint Cell attachment-induced Rap1 activation in C3G-deficient cells To understand the mechanism by which C3G modulated cell attachment and cell spreading, we examined the activation of Rap1 after replating (Figure 5A). Rap1 was activated rapidly upon cell attachment in the untreated MEF-hC3G cells, whereas Rap1 activation was significantly attenuated in the MEF-hC3G cells infected with MX-pie-Cre. C3G activates the Rap and R-Ras subfamilies in vitro and in 293T cells (Ohba et al., 2000a,b). Therefore, we next proceeded to examine the effect of C3G deficiency on Rap2 and R-Ras (Figure 5B). The results are summarized as follows: (i) Rap1, Rap2 and R-Ras were all activated upon attachment of MEF-hC3G cells to the culture dishes; (ii) C3G deficiency inhibited the activation of Rap1 upon cell attachment, but not that of Rap2: neither of these phenomena was affected by the type of extracellular matrix; (iii) C3G deficiency inhibited R-Ras activation partially, suggesting the involvement of other GEF(s) in cell attachment-induced R-Ras activation. These results indicated that lack of Rap1 activation caused a decrease in cell attachment and cell spreading of C3G-deficient cells. Figure 5.Rap1 activation induced by cell adhesion. (A) MEF-hC3G cells were infected with MX-pie-Cre or MX-pie. After 48 h, cells were trypsinized, kept in suspension for 1 h and plated on dishes coated with collagen type I. Cells were lysed at the indicated times. After normalizing on the protein quantity, the level of GTP-Rap1 was analyzed by Bos's method. (B) MEF-hC3G cells were infected with MX-pie or MX-pie-Cre, maintained for 48 h, trypsinized, kept in suspension for 1 h and plated on dishes pre-coated as noted at the top. After 1 h, we harvested cells and examined the levels of GTP-bound Rap1, Rap2 and R-Ras. For the detection of GTP-R-Ras, we used MEF-hC3G cells infected with MSCV-R-Ras retrovirus as described in Figure 3C. Download figure Download PowerPoint Increase in cell motility by C3G deficiency Another phenotype of the C3G-deficient cells that we noticed was an increase in cell motility. To quantitate this observation, we recorded phase-contrast and fluorescence images of MEF-hC3G cells infected with MX-pie or MX-pie-Cre for 8 h with a time-lapse fluorescence microscope and obtained the cell paths with a cell-tracking program (Figure 6A). The velocity of C3G-deficient MEF-hC3G cells was higher than that of the control MEF-hC3G cells on collagen-coated dishes (Figure 6B). The difference was statistically significant by t-test and Welch's test (P <0.01). When we used poly-L-lysine-coated dishes, the velocity of MEF-hC3G cells was not affected by the expression of Cre (data not shown). Similar results were obtained by using different isolates of MEF-hC3G cells. In another cell line derived from C3G+/−/− embryos, we did not find any effect of Cre on the cell motility (data not shown). Figure 6.Increased cell motility of C3G-deficient cells. (A) MEF-hC3G cells infected with MX-pie or MX-pie-Cre were trypsinized, kept in suspension for 1 h and plated on collagen-coated dishes. Starting after 1 h, cell images were collected every 3 min under time-lapse fluorescence microscopy equipped with a cooled CCD camera. Paths of the center of EGFP-positive cells during 8 h recording time were traced with MetaMorph2 software. (B) MEF-hC3G cells or MEF-hC3G cells expressing the proteins listed at the bottom were infected with MX-pie-Cre as indicated. We analyzed the cells as in (A) and obtained the mean velocities of 20 cells for each sample. Mid-line, top and bottom of each box indicate median, 75th quartile and 25th quartile, respectively. Cells that show a significant difference from the control, MX-pie infected cells, by t-test and Welch test (P <0.01) are marked with an asterisk at the top of the box. Note that scales shown on the right are for the cells expressing H-RasV12 used. (C) MEF-hC3G cells prepared as described in (A) were lysed and immunoprecipitated with anti-Crk monoclonal antibody and a mixture of protein G– and protein A–Sepharose. Total cell lysates and immunoprecipitated proteins were separated by SDS–PAGE, followed by immunoblotting with anti-C3G anbibody, anti-phosphotyrosine antibody (PY), anti-p130Cas antibody (Cas) or anti-Crk antibody. Download figure Download PowerPoint We next examined whether the expression of other GEFs for Ras-family G proteins could reduce the cell motility of C3G-deficient MEF-hC3G cells (Figure 6B). As expected, re-introduction of C3G into C3G-deficient MEF-hC3G cells reduced the cell motility to the level of the parent MEF-hC3G cells. Expression of Epac, a cAMP-responsive GEF for Rap1 and Rap2, reduced the cell motility to the level of the parent MEF-hC3G cells only in the presence of a cAMP analog, Sp-cAMPS. CalDAG-GEFI, which is another GEF for Rap1, Rap2 and R-Ras and is constitutively active in many cell types (Yamashita et al., 2000), also reduced the cell motility. In contrast, CalDAG-GEFII, a GEF for the Ras and R-Ras subfamilies, and CalDAG-GEFIII, a pan-Ras GEF, did not reduce the cell motility of C3G-deficient MEF-hC3G cells. We further tested the effect of GTPase-deficient mutants of Ras-family G proteins. Rap1 and Rap2 reduced the cell motility most strongly, and R-Ras did so moderately. GTPase-deficient H-Ras, in contrast, remarkably increased the cell motility. Thus, the effect of C3G deficiency in cell migration was antagonized by the activation of its substrates, Rap1, Rap2 and R-Ras. Finally, because Crk and p130Cas are postulated to function upstream to C3G and to increase cell motility (Ohashi et al., 1999; Uemura and Griffin, 1999; Cho and Klemke, 2000; Yano et al., 2000), we confirmed that the C3G deficiency did not affect cell adhesion-induced phosphorylation of p130Cas or Crk binding to p130Cas (Figure 6C). Discussion Requirement for C3G in early embryogenesis At least eight GEFs have been reported to activate Rap1 in mammalian cells (reviewed by Zwartkruis and Bos, 1999). The mortality of C3G-deficient embryos showed that none of these GEFs could complement the loss of C3G during development. This is not surprising because, except for C3G, Rap1 GEFs are expressed in a more or less tissue-specific manner. Moreover, it should be noted that