October 2006
Volume 47, Issue 10
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Cornea  |   October 2006
Role of Rac1 in Fibronectin-Induced Adhesion and Motility of Human Corneal Epithelial Cells
Author Affiliations
  • Kazuhiro Kimura
    From the Departments of Ocular Pathophysiology and
  • Koji Kawamoto
    Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
  • Shinichiro Teranishi
    Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
  • Teruo Nishida
    From the Departments of Ocular Pathophysiology and
    Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan.
Investigative Ophthalmology & Visual Science October 2006, Vol.47, 4323-4329. doi:10.1167/iovs.05-1508
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      Kazuhiro Kimura, Koji Kawamoto, Shinichiro Teranishi, Teruo Nishida; Role of Rac1 in Fibronectin-Induced Adhesion and Motility of Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2006;47(10):4323-4329. doi: 10.1167/iovs.05-1508.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. The fibronectin–integrin system plays an important role in adhesion and migration of corneal epithelial cells and thereby contributes to epithelial wound healing. The role of Rac1, a member of the Rho family of GTPases, in the intracellular signaling responsible for regulation of the adhesion and motility of corneal epithelial cells by fibronectin was examined.

methods. Simian virus 40–transformed human corneal epithelial (HCE) cells were plated on fibronectin or on bovine serum albumin as a control. Cell motility was monitored by time-lapse video microscopy. The actin cytoskeleton and focal adhesions were detected by staining of cells with rhodamine-phalloidin and antibodies to phosphotyrosine, respectively. The activation of Rac1 and phosphorylation of its effector PAK were evaluated with a pull-down assay and immunoblot analysis, respectively. The effects of mutant forms of Rac1 were determined by cell transfection.

results. HCE cells plated on fibronectin manifested greater levels of cell adhesion and motility than did those plated on bovine serum albumin. Fibronectin also induced the accumulation of F-actin and the formation of focal adhesions at the cell periphery as well as the activation of Rac1 and the phosphorylation of PAK. Expression of the dominant negative mutant Asn17Rac1 inhibited the effects of fibronectin on cell adhesion and motility, the actin cytoskeleton, and focal adhesions. Expression of the constitutive active mutant Val12Rac1 mimicked the effects of fibronectin on F-actin and focal adhesions.

conclusions. Rac1 is necessary for the promotion of HCE cell adhesion and motility by fibronectin. It therefore probably plays an important role in corneal wound healing.

Cell adhesion and migration play important roles in the healing of corneal epithelial wounds. 1 2 In response to epithelial wounding, the remaining corneal epithelial cells attach to and migrate over the fibronectin matrix expressed at the wound site, to cover the defective area. 3 4 These corneal epithelial cells express proteins of the integrin family that bind fibronectin and other components of the extracellular matrix. 5 6 Adhesion of cells to fibronectin induces the clustering of integrins and the assembly of focal adhesion complexes that are linked to the actin cytoskeleton. 7 8 The outside-in signaling triggered by the fibronectin–integrin interaction also elicits changes in the structure of the leading edge of migrating cells. Several groups have performed time-lapse microscopy to analyze cell behavior on fibronectin. 9 10 11 With the use of time-lapse cinematography, we have also shown in an organ culture system that corneal epithelial cells manifest active ruffling at the leading edge during migration. 12  
Members of the Rho family of proteins regulate the adhesion and migration of various cell types. 13 14 This protein family includes Rho, Cdc42, and Rac, each of which cycles between an active, GTP-bound form and an inactive, GDP-bound form. 15 16 These proteins function as switches in intracellular signaling and thereby regulate reorganization of the actin cytoskeleton. 15 17 18 In general, Rho regulates the formation of stress fibers, whereas Cdc42 and Rac1 regulate that of filopodia and lamellipodia, respectively. 15 19 20 The active forms of Rho family GTPases interact with several effector proteins. 14 21 The downstream targets of activated Rac1 include p21-activated kinase (PAK), WASP (or N-WASP), IQGAP, and ACK. 19 21 22 23 The GTP-bound form of Rac1 binds to members of the PAK family and thereby stimulates their kinase activity and induces their autophosphorylation. 24 25 Moreover, activated PAK isoforms or constitutively active mutants induce the formation of lamellipodia and filopodia by triggering rearrangement of the actin cytoskeleton. 21 26  
We have shown that a fibronectin matrix promotes the adhesion and migration of corneal epithelial cells in vitro. 27 Furthermore, we have found that lysophosphatidic acid, an activator of Rho signaling, stimulates corneal epithelial cell migration in a manner sensitive to Y-27632, 28 29 an inhibitor of the Rho effector ROCK. Rho has also been shown to be required for reorganization of the actin cytoskeleton and tyrosine phosphorylation in the avian corneal epithelium. 30 Whereas Rho appears to participate in the regulation of corneal epithelial migration, the possible role of other Rho family proteins in the adhesion or migration of corneal epithelial cells has remained unknown. We have therefore now examined whether Rac1 contributes to the regulation of corneal epithelial cell adhesion or migration by fibronectin. 
Methods
Reagents, Antibodies, and Plasmids
A mixture of Dulbecco’s modified Eagle’s medium and nutrient mixture F-12 (DMEM/F-12) as well as a serum-free, general purpose cell culture medium (Opti-MEM), fetal bovine serum, trypsin-EDTA, gentamicin, and a lipophilic transfection reagent [Lipofectamine 2000] were obtained from Invitrogen-Gibco (Carlsbad, CA); fibronectin from Roche (Basel, Switzerland); cresyl violet from Nacalai Tesque (Kyoto, Japan); bovine serum albumin (BSA), bovine insulin, cholera toxin, human recombinant epidermal growth factor, Nonidet P-40 (NP-40), and a protease inhibitor cocktail from Sigma-Aldrich (St. Louis, MO); plastic culture dishes (35- or 100-mm-diameter) and 96-well plates from Corning (Corning, NY); and 35-mm glass-bottom culture dishes from Iwaki (Tokyo, Japan). 
Mouse monoclonal antibodies to phosphotyrosine and to Rac1 were from UBI (Temecula, CA); rabbit polyclonal antibodies to αPAK or to the phospho-Ser141 forms of PAK1/2/3 from Santa Cruz Biotechnology and Biosource (Camerillo, CA), respectively; mouse monoclonal antibody to Myc or green fluorescent protein (GFP) from Covance (Vienna, CA) and MBL (Nagoya, Japan); Alexa Fluor 488–labeled goat antibodies to mouse immunoglobulin G and rhodamine-phalloidin from Invitrogen; glutathione-conjugated Sepharose 4B beads, horseradish peroxidase–conjugated goat secondary antibodies, enhanced chemiluminescent [ECLPlus] detection reagents from GE Healthcare (Little Chalfont, UK); and the plasmid pEGFP-C1, which encodes enhanced green fluorescent protein (EGFP), from BD-Clontech (Palo Alto, CA). The plasmids pEFBOS-myc (for expression of Myc epitope–tagged proteins), pEFBOS-myc-Val12Rac1, and pEFBOS-myc-Asn17Rac1 were kindly provided by Yoshimi Takai (Osaka University, Osaka, Japan), 31 32 33 and pEGFP-Asn17Rac1 was constructed as described previously. 34 35 36  
Cells and Cell Culture
Simian virus 40-immortalized human corneal epithelial (HCE) cells were provided by the RIKEN Bioresource Center (Tsukuba, Japan). The cells were originally established and characterized by Kaoru Araki-Sasaki. 37 They were passaged in supplemented hormonal epithelial medium (SHEM), which comprises DMEM/F-12 supplemented with 15% heat-inactivated fetal bovine serum, bovine insulin (5 μg/mL), cholera toxin (0.1 μg/mL), human recombinant epidermal growth factor (10 ng/mL), and gentamicin (40 μg/mL). 
For experiments, HCE cells were cultured for 24 hours in unsupplemented DMEM/F-12, isolated by treatment with trypsin-EDTA, suspended in the same medium, and plated at a density of 2 × 104 cells per 35-mm dish, 2 × 106 cells per 100-mm dish, or 2 × 103 cells per well in 96-well plates, all of which had been coated with fibronectin (10 μg/mL) plus 1% BSA or with 1% BSA alone (control). 
Transfection
HCE cells were transfected for 3 hours with 0.5 μg of Rac1 plasmids mixed with 4 μL of transfection reagent (Lipofectamine 2000; ;Invitrogen) in 2 mL of cell culture medium (Opti-MEM; Invitrogen). After the medium was replaced with DMEM/F-12, the cells were incubated for an additional 14 hours before the experiments. Transfection efficiency was examined by counting cells expressing EGFP protein by confocal microscopy. 
Cell Adhesion Assay
HCE cells (transfected or not) were transferred to 96-well plates that had been coated with fibronectin plus BSA or with BSA alone. After incubation for 45 minutes, the cells were washed twice with Ca2+- and Mg2+-free phosphate-buffered saline [PBS(−)], fixed for 15 minutes at 37°C with 3.7% formalin in PBS(−), and stained with 1% cresyl violet The number of attached cells was determined by observation with a phase-contrast microscope (Carl Zeiss Meditec GmbH, Hallbergmoos, Germany). 
Cell Motility Assay
HCE cells (transfected or not) were plated on 35-mm glass-bottomed culture dishes that had been coated with fibronectin plus BSA or with BSA alone. The cells were cultured in unsupplemented DMEM/F-12. After they were plated for 6 hours, the cells were supplemented with 25 mM HEPES in unsupplemented DMEM/F-12 and 10 to 30 cells per field was monitored for 6 hours in a humidified chamber with 5% CO2 at 37°C with the use of a fluorescence inverted microscope (Axioscope; Carl Zeiss Meditec GmbH). Video images were collected with a charge-coupled device camera at 5-minute intervals for 6 hours. The positions of nuclei were tracked to quantify cell motility with the use of commercial software (Move-tr/2D ; Library, Tokyo, Japan). 
Immunofluorescence Microscopy
HCE cells cultured for 45 minutes on 35-mm glass-bottomed culture dishes coated with fibronectin plus BSA or with BSA alone were fixed for 15 minutes at 37°C with 3.7% formalin, washed with PBS(−), and incubated for 1 hour at room temperature with 1% BSA in PBS(−). The cells were then incubated for 1 hour with antibodies to phosphotyrosine (1:200 dilution in PBS(−) containing 1% BSA), washed with PBS(−), and incubated for 1 hour with Alexa Fluor 488–conjugated goat secondary antibodies (1:1000 dilution) and rhodamine-phalloidin (1:200 dilution) in PBS(−) containing 1% BSA. They were then examined with a laser confocal microscope (LSM5; Carl Zeiss Meditec GmbH). For analysis of transfected cells, the cells were stained with rhodamine-phalloidin or with antibodies to phosphotyrosine and Alexa Fluor 594–conjugated goat anti-mouse secondary antibodies. 
Pull-Down Assay for GTP-Rac1
HCE cells cultured for 45 minutes in 100-mm dishes coated with fibronectin plus BSA or with BSA alone were lysed in 0.5 mL of a solution containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 10% glycerol, 1% NP-40, 1 mM NaF, 1 mM EGTA, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000g for 15 minutes, and the resultant supernatants (200 μg of protein) were incubated for 2 hours at 4°C with 20 μg of glutathione S-transferase (GST) or a GST fusion protein containing the Cdc42/Rac binding domain (CRIB) of PAK1 immobilized on glutathione–Sepharose 4B beads. 36 The beads were washed three times with lysis buffer, and bound GTP-Rac1 was then detected by immunoblot analysis with antibodies to Rac1. 
Immunoblot Analysis
HCE cells plated on 100-mm dishes coated with fibronectin plus BSA or with BSA alone were incubated for 45 minutes or HCE cells transfected with Rac1 plasmids and then lysed in 0.5 mL of a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, and 1% protease inhibitor cocktail. The lysates were centrifuged at 15,000g for 15 minutes, and the resultant supernatants (20 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel. The separated proteins were transferred to a nitrocellulose membrane, which was then exposed to 5% skim milk for 1 hour at room temperature before incubation for 1 hour with antibodies to αPAK, the phospho-Ser141 of PAK1/2/3, Rac1, myc, and GFP at a 1:500 dilution in washing buffer (20 mM Tris-HCl [pH 7.4], 5% skim milk, 0.1% Tween 20). The membrane was washed in washing buffer, incubated for 1 hour at room temperature with horseradish peroxidase–conjugated goat secondary antibodies (1:1000 dilution in washing buffer), washed again, incubated with chemiluminescent detection reagents for 5 minutes, and then exposed to film. 
Statistical Analysis
Quantitative data are presented as the mean ± SD. Differences were analyzed with the Dunnett test. P <0.05 was considered statistically significant. 
Results
We first examined the effects of fibronectin on the adhesion and motility of HCE cells. The number of cells that adhered to plates coated with fibronectin plus BSA was approximately 10 times that apparent with plates coated with BSA alone (Fig. 1A) . Time-lapse video microscopy also revealed that fibronectin markedly increased the motility of HCE cells (Fig. 1B)
We next investigated the effects of fibronectin on the actin cytoskeleton and focal adhesions by immunofluorescence microscopy (Fig. 2) . HCE cells plated on fibronectin plus BSA appeared more flattened and spread out than did those plated on BSA. Cells cultured on fibronectin also manifested a thick rim of F-actin staining in a dense meshwork pattern, especially at what appeared to be the leading edge. Costaining with antibodies to phosphotyrosine revealed numerous small dotlike structures, presumably corresponding to focal adhesions, associated with the bundles of F-actin at the cell periphery (Fig. 2) . In contrast, cells plated on BSA exhibited only a thin rim of F-actin staining and only a few small dotlike structures reactive with the antibodies to phosphotyrosine at the cell periphery. These results thus suggest that fibronectin induces the formation of membrane ruffles and focal adhesions through reorganization of the actin cytoskeleton. 
To examine the expression of endogenous Rac1 protein in HCE cells, we performed immunoblot analysis with anti-Rac1 antibody. As shown in Figure 3A , we detected the expression of endogenous Rac1 protein in HCE cells. Moreover, we confirmed the expression of Asn17Rac1, a dominant negative mutant of Rac1, and Val12Rac1, a dominant active mutant of Rac1 after transfection by immunoblot analysis with anti-myc or anti-GFP antibody (Fig. 3B) . To investigate transfection efficiency, we transfected cultured corneal epithelial cells with control plasmid-carrying GFP and observed them by confocal microscopy. As shown in Figure 3C , transfection efficiencies were ∼60%. Then, to examine the activation status of Rac1 in HCE cells, we performed a pull-down assay for GTP-Rac1 with a GST fusion protein containing the CRIB domain of PAK1. The amount of GTP-Rac1 in cells cultured on fibronectin plus BSA was markedly increased compared with that in cells maintained on BSA (Fig. 4A) . The total amount of Rac1 in HCE cells did not differ between the two culture conditions. The quantitative analysis revealed that the amount of GTP-Rac1 in cells cultured on fibronectin plus BSA increased significantly (Fig. 4B) . These results indicated that fibronectin induces the activation of Rac1 in these cells. 
We also investigated the effect of fibronectin on the phosphorylation of PAK in HCE cells. Compared with cells plated on BSA, those plated on fibronectin plus BSA exhibited an increased level of PAK phosphorylation (Fig. 5A) . The quantitative analysis revealed that the level of PAK phosphorylation in cells plated on fibronectin increased 1.5-fold, a significant increase (Fig. 5B) . The abundance of PAK in the cells did not differ between the two culture conditions. These data thus demonstrate that the interaction of HCE cells with fibronectin results in activation of PAK, the Rac1 effector. 
To examine directly the role of Rac1 in the effects of fibronectin on HCE cells, we transfected the cells with an expression vector for Asn17Rac1, a dominant negative mutant of Rac1. Immunofluorescence microscopy revealed that expression of this mutant protein markedly inhibited the effects of fibronectin on the accumulation of F-actin and the formation of focal adhesions at the cell periphery (Fig. 6) . These results suggest that Rac1 mediates the induction by fibronectin of the formation of membrane ruffles and focal adhesions in HCE cells. 
We next examined the role of Rac1 in the effects of fibronectin on HCE cell adhesion and motility. The extents of both cell adhesion to and cell motility on fibronectin were greatly reduced in cells transfected with the Asn17Rac1 vector compared with those in cells transfected with a control vector (Fig. 7) . These results thus indicate that Rac1 is necessary for the promotion of HCE cell adhesion and motility by fibronectin. 
Finally, we investigated the effects of expression of Val12Rac1, a constitutively active form of Rac1, in HCE cells plated on BSA. Expression of Val12Rac1 induced the accumulation of F-actin and increased the number of dotlike structures reactive with antibodies to phosphotyrosine at the periphery of HCE cells (Fig. 8) . The phenotype induced by expression of this active form of Rac1 in cells plated on BSA was thus similar to that of nontransfected cells plated on fibronectin plus BSA, consistent with the notion that Rac1 is sufficient for the induction of this phenotype by fibronectin. 
Discussion
Fibronectin induced the accumulation of F-actin and the formation of focal adhesions at the periphery of HCE cells and promoted both the adhesion and motility of these cells. It also increased the amount of the active, GTP-bound form of Rac1 and induced the phosphorylation of PAK, an Rac1 effector, in HCE cells. The effects of fibronectin on reorganization of the actin cytoskeleton, the formation of focal adhesions, and cell adhesion and motility in HCE cells was inhibited by the expression of a dominant negative mutant of Rac1, whereas those on the actin cytoskeleton and focal adhesions were mimicked by the expression of a constitutively active form of Rac1. These observations thus indicate that signaling by Rac1 mediates the effects of fibronectin on cell adhesion and motility in HCE cells. 
Fibronectin induces the assembly of integrins and elicits intracellular signaling that leads to the accumulation of F-actin and the formation of focal adhesions linked to the actin cytoskeleton in various cell types, effects that promote both cell adhesion and migration. 38 39 40 41 Fibronectin is expressed at the site of corneal stroma at wounds and promotes both the adhesion and migration of corneal epithelial cells in vivo and in vitro. 6 42 We have now shown that Rac1 is activated by fibronectin and mediates the effects of this extracellular matrix protein on the adhesion and motility of HCE cells. A role for Rac1 in the effects of fibronectin on cell adhesion and motility has been demonstrated in other epithelial cells, including those of breast, 43 keratinocytes, 44 and A549 human lung adenocarcinoma cells. 45  
A report of a recent study showed that, with the use of the knockdown method with small interfering (siRNA) and stable cell mutants, Rac1 activity has an effect on cell migration in fibroblast and mammary epithelial cells. 43 Rac activity promoted the formation of peripheral lamellae and increased random migration and then decreasing Rac1 activity suppressed peripheral lamellae and changed the cell migration pattern from random to directionally persistent. Conversely, several groups have reported that the decrease in Rac1 by gene ablation and RNA interference causes defective cell migration in macrophages and neutrophils. 46 47 Other studies have shown that strong overexpression of the dominant active form of Rac1 protein disrupts cell migration. 48 49 In our experiment, we showed that Rac1 activity was necessary for cell motility and the formation of peripheral lamellae induced by fibronectin in corneal epithelial cells with the dominant negative form. The Rac1 activity was very important for cell migration; however, a very high or low level of Rac1 activity induced cell immobility and proper Rac1 activity in respective cell type was necessary for cell migration. 
Most cells migrate randomly to explore their local environments with the peripheral lamellae. 50 51 The cell migration speed is high, even though the direction is not decided. However, directional cell migration is necessary in special situations, such as development, tissue repair, and inflammation. In other words, cells migrate directionally to reach the biological objective region as smoothly as possible in these situations. Directional migration may be involved in multiple steps, including the change of cytoskeleton, cell adhesion, and signal transduction mediated by many cytokines and growth factors. 52 53 54 Thus, the mechanism regulating whether cells migrate either randomly or directionally is important for understanding the cell migratory process. 
Rac GTPases include three isoforms: Rac1, Rac2, and Rac3. 55 56 The three distinct Rac isoforms share a very high sequence identity (up to 90%). Rac1 is ubiquitously expressed, and Rac2 was expressed in the myeloid-lineage. Rac2 is different from Rac1 and Rac3 in tissue distribution and biochemical properties. 57 In this study, we focused on Rac1 to clarify the molecular mechanism of increase in cell migration and adhesion by fibronectin in human corneal epithelial cells, because Rac1 was ubiquitously expressed and much evidence of involvement in cell adhesion and migration has been reported. 55 56 57 58 Recently, it was reported that Rac3 establishes its 48 ability to promote membrane ruffling, transformation, and activation of c-Jun transcriptional activity in the same way as Rac1. 59 60 61 However, Rac3 differs from other Rac proteins only in the carboxyl terminus 55 and downstream effector pathways of Rac3 overlap partially but not completely with those used by Rac1. 62 These results suggest that the Rac3 function is even more essential for molecular characterization; however, Rac3 as well as Rac1 may also be involved in cell migration and adhesion in human corneal epithelial cells on fibronectin. 
Rho family GTPases regulate cell adhesion and migration mediated by reorganization of the actin cytoskeleton in several epithelial cell types. 63 64 We previously showed that lysophosphatidic acid induces the migration of corneal epithelial cells in a manner dependent on activation of Rho. 28 In the current study, Rac1 was activated by fibronectin in HCE cells and mediated the effects of fibronectin on the adhesion and motility of these cells. These findings demonstrate that not only Rho but also Rac1 participates in the regulation of the adhesion and migration of corneal epithelial cells by fibronectin. Both Rho and Rac1 were found to contribute to migration of and wound closure by lung epithelial cells, 65 and both Cdc42 and Rac1 (but not Rho) are necessary for the rapid spreading of mammary epithelial cells on fibronectin. 66 These observations suggest that different Rho family GTPases mediate regulation of cell adhesion and migration in a cell type– or stimulus-specific manner. 
PAK is activated by direct interaction with the GTP-bound forms of Rac1 or Cdc42. 67 Expression of dominant active forms of Rac1 and Cdc42 have been shown to induce the phosphorylation and activation of PAK in epithelial cells. 68 69 The plating of NIH 3T3 cells on dishes coated with fibronectin or with antibodies to β1 integrin has also been shown to induce cell spreading mediated by the activation of Rac and Cdc42, increasing the kinase activity and autophosphorylation of PAKs. 25 We have now shown that fibronectin induces phosphorylation of PAK in HCE cells. Rac mediates changes in epithelial cell shape during dorsal closure in Drosophila, 70 and a Drosophila homologue of PAK was found to localize to focal adhesions and to be enriched at the leading edge of embryonic epithelial cells undergoing dorsal closure. 71 Together, these observations suggest that Rac1-PAK signaling may be activated at focal adhesions and at the edge of membrane ruffles during the migration of corneal epithelial cells on fibronectin. 
Our present results thus suggest that Rac1-PAK signaling mediates the regulation of cell adhesion and motility by fibronectin in HCE cells. Further characterization of the mechanism of this regulation may provide a better understanding of corneal epithelial wound healing as well as a basis for the development of new treatments for corneal epithelial wounds. 
 
Figure 1.
 
Effects of fibronectin on the adhesion and motility of HCE cells. (A) Cell adhesion. Cells were transferred in 96-well culture plates that had been coated with either fibronectin (10 μg/mL) plus 1% BSA or 1% BSA alone and were incubated for 45 minutes. Adherent cells were then counted. (B) Cell motility. The motility of cells cultured in 35-mm glass-bottomed dishes that had been coated with fibronectin plus BSA or BSA as in (A) was monitored by time-lapse video microscopy 6 hours after plating. The motility rate was expressed as a percentage of that for cells plated on BSA alone (control). Data are presented as the mean ± SD of results of three independent experiments. *P < 0.05 versus the corresponding result for cells plated on BSA alone (Dunnett test).
Figure 1.
 
Effects of fibronectin on the adhesion and motility of HCE cells. (A) Cell adhesion. Cells were transferred in 96-well culture plates that had been coated with either fibronectin (10 μg/mL) plus 1% BSA or 1% BSA alone and were incubated for 45 minutes. Adherent cells were then counted. (B) Cell motility. The motility of cells cultured in 35-mm glass-bottomed dishes that had been coated with fibronectin plus BSA or BSA as in (A) was monitored by time-lapse video microscopy 6 hours after plating. The motility rate was expressed as a percentage of that for cells plated on BSA alone (control). Data are presented as the mean ± SD of results of three independent experiments. *P < 0.05 versus the corresponding result for cells plated on BSA alone (Dunnett test).
Figure 2.
 
Effects of fibronectin on the actin cytoskeleton and focal adhesions in HCE cells. Cells were plated on 35-mm glass-bottomed culture dishes that had been coated with fibronectin plus BSA (bottom) or with BSA alone (top). They were then incubated for 45 minutes, fixed, and stained both with antibodies to phosphotyrosine (pTyr, green) to detect focal adhesions and with rhodamine-phalloidin (red), to detect actin filaments. Data are representative of results of three independent experiments. Scale bar, 10 μm.
Figure 2.
 
Effects of fibronectin on the actin cytoskeleton and focal adhesions in HCE cells. Cells were plated on 35-mm glass-bottomed culture dishes that had been coated with fibronectin plus BSA (bottom) or with BSA alone (top). They were then incubated for 45 minutes, fixed, and stained both with antibodies to phosphotyrosine (pTyr, green) to detect focal adhesions and with rhodamine-phalloidin (red), to detect actin filaments. Data are representative of results of three independent experiments. Scale bar, 10 μm.
Figure 3.
 
The expression of endogenous Rac1 and the EGFP-tagged dominant negative and myc-tagged dominant active form of Rac1 in human corneal epithelial cells. (A) HCE cell cultures were prepared and the cell lysate subjected to immunoblot analysis with antibody to Rac1. Lysis buffer without cell lysate was used as a negative control. (B) HCE cells were cultured for 14 hours after transfection of pEGFP-Asn17Rac1 and pEFBOS-myc-Val12Rac1, and cell lysate was prepared and subjected to immunoblot analysis with antibody to myc and GFP. Cell lysate from cells without transfection was used as the negative control. HCE cells were transfected with pEGFP-C1 and cultured for 24 hours, and expression of EGFP was checked by fluorescence microscopy. Quantification of transfection efficiency was performed. Data are representative of results of three independent experiments.
Figure 3.
 
The expression of endogenous Rac1 and the EGFP-tagged dominant negative and myc-tagged dominant active form of Rac1 in human corneal epithelial cells. (A) HCE cell cultures were prepared and the cell lysate subjected to immunoblot analysis with antibody to Rac1. Lysis buffer without cell lysate was used as a negative control. (B) HCE cells were cultured for 14 hours after transfection of pEGFP-Asn17Rac1 and pEFBOS-myc-Val12Rac1, and cell lysate was prepared and subjected to immunoblot analysis with antibody to myc and GFP. Cell lysate from cells without transfection was used as the negative control. HCE cells were transfected with pEGFP-C1 and cultured for 24 hours, and expression of EGFP was checked by fluorescence microscopy. Quantification of transfection efficiency was performed. Data are representative of results of three independent experiments.
Figure 4.
 
Effect of fibronectin on the activation status of Rac1 in HCE cells. (A) Cells were plated on 100-mm culture dishes that had been coated with fibronectin plus BSA or with BSA alone. They were then incubated for 45 minutes, lysed, and subjected to a pull-down assay for GTP-Rac1. Precipitated GTP-Rac1 (lanes 3, 4, 7, and 8) as well as cell lysates (lanes 1, 2, 5, and 6) derived from two separate culture dishes were examined by immunoblot analysis with antibodies to Rac1. Odd-numbered lanes contain GST fusion protein and even-numbered lanes contain GST fusion protein with the CRIB domain of PAK1. (B) The ratios of GTP-loaded Rac1 to total Rac1 were determined by densitometry of the bands on immunoblot analysis. The amount of GTP-Rac1 estimated by densitometry was normalized to the total amount of corresponding protein in cell lysate. Results are presented as the ratio of GTP-Rac1 precipitated with GST in the cells cultured on BSA. The data are expressed as relative values after normalization against the ratio in the cells in suspension and are representative of results of three independent experiments. *P < 0.05 versus precipitated Rac1 with GST-incubated cells lysate from cell on BSA (Dunnett test).
Figure 4.
 
Effect of fibronectin on the activation status of Rac1 in HCE cells. (A) Cells were plated on 100-mm culture dishes that had been coated with fibronectin plus BSA or with BSA alone. They were then incubated for 45 minutes, lysed, and subjected to a pull-down assay for GTP-Rac1. Precipitated GTP-Rac1 (lanes 3, 4, 7, and 8) as well as cell lysates (lanes 1, 2, 5, and 6) derived from two separate culture dishes were examined by immunoblot analysis with antibodies to Rac1. Odd-numbered lanes contain GST fusion protein and even-numbered lanes contain GST fusion protein with the CRIB domain of PAK1. (B) The ratios of GTP-loaded Rac1 to total Rac1 were determined by densitometry of the bands on immunoblot analysis. The amount of GTP-Rac1 estimated by densitometry was normalized to the total amount of corresponding protein in cell lysate. Results are presented as the ratio of GTP-Rac1 precipitated with GST in the cells cultured on BSA. The data are expressed as relative values after normalization against the ratio in the cells in suspension and are representative of results of three independent experiments. *P < 0.05 versus precipitated Rac1 with GST-incubated cells lysate from cell on BSA (Dunnett test).
Figure 5.
 
Effect of fibronectin on PAK phosphorylation in HCE cells. (A) Cells were plated on 100-mm culture dishes that had been coated with fibronectin plus BSA (lanes 2 and 4) or with BSA alone (lanes 1 and 3). They were then incubated for 45 minutes, lysed, and subjected to immunoblot analysis with antibodies to αPAK or to the phospho-Ser141 forms of PAK1/2/3. (B) The ratios of phosphorylated PAK to total PAK were determined by densitometry of the bands on immunoblot analysis. Data are representative of results of three independent experiments. *P < 0.05 versus cells on BSA (Dunnett test).
Figure 5.
 
Effect of fibronectin on PAK phosphorylation in HCE cells. (A) Cells were plated on 100-mm culture dishes that had been coated with fibronectin plus BSA (lanes 2 and 4) or with BSA alone (lanes 1 and 3). They were then incubated for 45 minutes, lysed, and subjected to immunoblot analysis with antibodies to αPAK or to the phospho-Ser141 forms of PAK1/2/3. (B) The ratios of phosphorylated PAK to total PAK were determined by densitometry of the bands on immunoblot analysis. Data are representative of results of three independent experiments. *P < 0.05 versus cells on BSA (Dunnett test).
Figure 6.
 
Effects of a dominant negative form of Rac1 (Asn17Rac1) on the actin cytoskeleton and focal adhesions in HCE cells plated on fibronectin. Cells transfected with pEGFP-Asn17Rac1 or pEGFP-C1 (control vector) were plated on dishes coated with fibronectin plus BSA and analyzed by immunofluorescence microscopy as in Figure 2 . Red fluorescence: staining with rhodamine-phalloidin or with antibodies to phosphotyrosine; green fluorescence: transfected cells expressing EGFP. Data are representative of results in three independent experiments. Scale bar, 10 μm.
Figure 6.
 
Effects of a dominant negative form of Rac1 (Asn17Rac1) on the actin cytoskeleton and focal adhesions in HCE cells plated on fibronectin. Cells transfected with pEGFP-Asn17Rac1 or pEGFP-C1 (control vector) were plated on dishes coated with fibronectin plus BSA and analyzed by immunofluorescence microscopy as in Figure 2 . Red fluorescence: staining with rhodamine-phalloidin or with antibodies to phosphotyrosine; green fluorescence: transfected cells expressing EGFP. Data are representative of results in three independent experiments. Scale bar, 10 μm.
Figure 7.
 
Effects of a dominant negative mutant of Rac1 (Asn17Rac1) on the adhesion and motility of HCE cells plated on fibronectin. Cells transfected with pEGFP-Asn17Rac1 or pEGFP-C1 (control vector) were plated on fibronectin plus BSA and analyzed for adhesion (A) or motility (B) as in Figure 1 . Data are the mean ± SD of results of three independent experiments. *P < 0.05 versus cells transfected with the control vector (Dunnett test).
Figure 7.
 
Effects of a dominant negative mutant of Rac1 (Asn17Rac1) on the adhesion and motility of HCE cells plated on fibronectin. Cells transfected with pEGFP-Asn17Rac1 or pEGFP-C1 (control vector) were plated on fibronectin plus BSA and analyzed for adhesion (A) or motility (B) as in Figure 1 . Data are the mean ± SD of results of three independent experiments. *P < 0.05 versus cells transfected with the control vector (Dunnett test).
Figure 8.
 
Effects of a constitutively active form of Rac1 (Val12Rac1) on the actin cytoskeleton and focal adhesions in HCE cells plated on BSA. Cells transfected with pEGFP-C1 (0.1 μg) and 0.5 μg of either pEFBOS-myc-Val12Rac1 or pEFBOS-myc (control vector) were plated on BSA and analyzed by immunofluorescence microscopy as in Figure 2 . Red fluorescence: staining with rhodamine-phalloidin or with antibodies to phosphotyrosine; green fluorescence: transfected cells expressing EGFP. Data are representative of three independent experiments. Scale bar, 10 μm.
Figure 8.
 
Effects of a constitutively active form of Rac1 (Val12Rac1) on the actin cytoskeleton and focal adhesions in HCE cells plated on BSA. Cells transfected with pEGFP-C1 (0.1 μg) and 0.5 μg of either pEFBOS-myc-Val12Rac1 or pEFBOS-myc (control vector) were plated on BSA and analyzed by immunofluorescence microscopy as in Figure 2 . Red fluorescence: staining with rhodamine-phalloidin or with antibodies to phosphotyrosine; green fluorescence: transfected cells expressing EGFP. Data are representative of three independent experiments. Scale bar, 10 μm.
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Figure 1.
 
Effects of fibronectin on the adhesion and motility of HCE cells. (A) Cell adhesion. Cells were transferred in 96-well culture plates that had been coated with either fibronectin (10 μg/mL) plus 1% BSA or 1% BSA alone and were incubated for 45 minutes. Adherent cells were then counted. (B) Cell motility. The motility of cells cultured in 35-mm glass-bottomed dishes that had been coated with fibronectin plus BSA or BSA as in (A) was monitored by time-lapse video microscopy 6 hours after plating. The motility rate was expressed as a percentage of that for cells plated on BSA alone (control). Data are presented as the mean ± SD of results of three independent experiments. *P < 0.05 versus the corresponding result for cells plated on BSA alone (Dunnett test).
Figure 1.
 
Effects of fibronectin on the adhesion and motility of HCE cells. (A) Cell adhesion. Cells were transferred in 96-well culture plates that had been coated with either fibronectin (10 μg/mL) plus 1% BSA or 1% BSA alone and were incubated for 45 minutes. Adherent cells were then counted. (B) Cell motility. The motility of cells cultured in 35-mm glass-bottomed dishes that had been coated with fibronectin plus BSA or BSA as in (A) was monitored by time-lapse video microscopy 6 hours after plating. The motility rate was expressed as a percentage of that for cells plated on BSA alone (control). Data are presented as the mean ± SD of results of three independent experiments. *P < 0.05 versus the corresponding result for cells plated on BSA alone (Dunnett test).
Figure 2.
 
Effects of fibronectin on the actin cytoskeleton and focal adhesions in HCE cells. Cells were plated on 35-mm glass-bottomed culture dishes that had been coated with fibronectin plus BSA (bottom) or with BSA alone (top). They were then incubated for 45 minutes, fixed, and stained both with antibodies to phosphotyrosine (pTyr, green) to detect focal adhesions and with rhodamine-phalloidin (red), to detect actin filaments. Data are representative of results of three independent experiments. Scale bar, 10 μm.
Figure 2.
 
Effects of fibronectin on the actin cytoskeleton and focal adhesions in HCE cells. Cells were plated on 35-mm glass-bottomed culture dishes that had been coated with fibronectin plus BSA (bottom) or with BSA alone (top). They were then incubated for 45 minutes, fixed, and stained both with antibodies to phosphotyrosine (pTyr, green) to detect focal adhesions and with rhodamine-phalloidin (red), to detect actin filaments. Data are representative of results of three independent experiments. Scale bar, 10 μm.
Figure 3.
 
The expression of endogenous Rac1 and the EGFP-tagged dominant negative and myc-tagged dominant active form of Rac1 in human corneal epithelial cells. (A) HCE cell cultures were prepared and the cell lysate subjected to immunoblot analysis with antibody to Rac1. Lysis buffer without cell lysate was used as a negative control. (B) HCE cells were cultured for 14 hours after transfection of pEGFP-Asn17Rac1 and pEFBOS-myc-Val12Rac1, and cell lysate was prepared and subjected to immunoblot analysis with antibody to myc and GFP. Cell lysate from cells without transfection was used as the negative control. HCE cells were transfected with pEGFP-C1 and cultured for 24 hours, and expression of EGFP was checked by fluorescence microscopy. Quantification of transfection efficiency was performed. Data are representative of results of three independent experiments.
Figure 3.
 
The expression of endogenous Rac1 and the EGFP-tagged dominant negative and myc-tagged dominant active form of Rac1 in human corneal epithelial cells. (A) HCE cell cultures were prepared and the cell lysate subjected to immunoblot analysis with antibody to Rac1. Lysis buffer without cell lysate was used as a negative control. (B) HCE cells were cultured for 14 hours after transfection of pEGFP-Asn17Rac1 and pEFBOS-myc-Val12Rac1, and cell lysate was prepared and subjected to immunoblot analysis with antibody to myc and GFP. Cell lysate from cells without transfection was used as the negative control. HCE cells were transfected with pEGFP-C1 and cultured for 24 hours, and expression of EGFP was checked by fluorescence microscopy. Quantification of transfection efficiency was performed. Data are representative of results of three independent experiments.
Figure 4.
 
Effect of fibronectin on the activation status of Rac1 in HCE cells. (A) Cells were plated on 100-mm culture dishes that had been coated with fibronectin plus BSA or with BSA alone. They were then incubated for 45 minutes, lysed, and subjected to a pull-down assay for GTP-Rac1. Precipitated GTP-Rac1 (lanes 3, 4, 7, and 8) as well as cell lysates (lanes 1, 2, 5, and 6) derived from two separate culture dishes were examined by immunoblot analysis with antibodies to Rac1. Odd-numbered lanes contain GST fusion protein and even-numbered lanes contain GST fusion protein with the CRIB domain of PAK1. (B) The ratios of GTP-loaded Rac1 to total Rac1 were determined by densitometry of the bands on immunoblot analysis. The amount of GTP-Rac1 estimated by densitometry was normalized to the total amount of corresponding protein in cell lysate. Results are presented as the ratio of GTP-Rac1 precipitated with GST in the cells cultured on BSA. The data are expressed as relative values after normalization against the ratio in the cells in suspension and are representative of results of three independent experiments. *P < 0.05 versus precipitated Rac1 with GST-incubated cells lysate from cell on BSA (Dunnett test).
Figure 4.
 
Effect of fibronectin on the activation status of Rac1 in HCE cells. (A) Cells were plated on 100-mm culture dishes that had been coated with fibronectin plus BSA or with BSA alone. They were then incubated for 45 minutes, lysed, and subjected to a pull-down assay for GTP-Rac1. Precipitated GTP-Rac1 (lanes 3, 4, 7, and 8) as well as cell lysates (lanes 1, 2, 5, and 6) derived from two separate culture dishes were examined by immunoblot analysis with antibodies to Rac1. Odd-numbered lanes contain GST fusion protein and even-numbered lanes contain GST fusion protein with the CRIB domain of PAK1. (B) The ratios of GTP-loaded Rac1 to total Rac1 were determined by densitometry of the bands on immunoblot analysis. The amount of GTP-Rac1 estimated by densitometry was normalized to the total amount of corresponding protein in cell lysate. Results are presented as the ratio of GTP-Rac1 precipitated with GST in the cells cultured on BSA. The data are expressed as relative values after normalization against the ratio in the cells in suspension and are representative of results of three independent experiments. *P < 0.05 versus precipitated Rac1 with GST-incubated cells lysate from cell on BSA (Dunnett test).
Figure 5.
 
Effect of fibronectin on PAK phosphorylation in HCE cells. (A) Cells were plated on 100-mm culture dishes that had been coated with fibronectin plus BSA (lanes 2 and 4) or with BSA alone (lanes 1 and 3). They were then incubated for 45 minutes, lysed, and subjected to immunoblot analysis with antibodies to αPAK or to the phospho-Ser141 forms of PAK1/2/3. (B) The ratios of phosphorylated PAK to total PAK were determined by densitometry of the bands on immunoblot analysis. Data are representative of results of three independent experiments. *P < 0.05 versus cells on BSA (Dunnett test).
Figure 5.
 
Effect of fibronectin on PAK phosphorylation in HCE cells. (A) Cells were plated on 100-mm culture dishes that had been coated with fibronectin plus BSA (lanes 2 and 4) or with BSA alone (lanes 1 and 3). They were then incubated for 45 minutes, lysed, and subjected to immunoblot analysis with antibodies to αPAK or to the phospho-Ser141 forms of PAK1/2/3. (B) The ratios of phosphorylated PAK to total PAK were determined by densitometry of the bands on immunoblot analysis. Data are representative of results of three independent experiments. *P < 0.05 versus cells on BSA (Dunnett test).
Figure 6.
 
Effects of a dominant negative form of Rac1 (Asn17Rac1) on the actin cytoskeleton and focal adhesions in HCE cells plated on fibronectin. Cells transfected with pEGFP-Asn17Rac1 or pEGFP-C1 (control vector) were plated on dishes coated with fibronectin plus BSA and analyzed by immunofluorescence microscopy as in Figure 2 . Red fluorescence: staining with rhodamine-phalloidin or with antibodies to phosphotyrosine; green fluorescence: transfected cells expressing EGFP. Data are representative of results in three independent experiments. Scale bar, 10 μm.
Figure 6.
 
Effects of a dominant negative form of Rac1 (Asn17Rac1) on the actin cytoskeleton and focal adhesions in HCE cells plated on fibronectin. Cells transfected with pEGFP-Asn17Rac1 or pEGFP-C1 (control vector) were plated on dishes coated with fibronectin plus BSA and analyzed by immunofluorescence microscopy as in Figure 2 . Red fluorescence: staining with rhodamine-phalloidin or with antibodies to phosphotyrosine; green fluorescence: transfected cells expressing EGFP. Data are representative of results in three independent experiments. Scale bar, 10 μm.
Figure 7.
 
Effects of a dominant negative mutant of Rac1 (Asn17Rac1) on the adhesion and motility of HCE cells plated on fibronectin. Cells transfected with pEGFP-Asn17Rac1 or pEGFP-C1 (control vector) were plated on fibronectin plus BSA and analyzed for adhesion (A) or motility (B) as in Figure 1 . Data are the mean ± SD of results of three independent experiments. *P < 0.05 versus cells transfected with the control vector (Dunnett test).
Figure 7.
 
Effects of a dominant negative mutant of Rac1 (Asn17Rac1) on the adhesion and motility of HCE cells plated on fibronectin. Cells transfected with pEGFP-Asn17Rac1 or pEGFP-C1 (control vector) were plated on fibronectin plus BSA and analyzed for adhesion (A) or motility (B) as in Figure 1 . Data are the mean ± SD of results of three independent experiments. *P < 0.05 versus cells transfected with the control vector (Dunnett test).
Figure 8.
 
Effects of a constitutively active form of Rac1 (Val12Rac1) on the actin cytoskeleton and focal adhesions in HCE cells plated on BSA. Cells transfected with pEGFP-C1 (0.1 μg) and 0.5 μg of either pEFBOS-myc-Val12Rac1 or pEFBOS-myc (control vector) were plated on BSA and analyzed by immunofluorescence microscopy as in Figure 2 . Red fluorescence: staining with rhodamine-phalloidin or with antibodies to phosphotyrosine; green fluorescence: transfected cells expressing EGFP. Data are representative of three independent experiments. Scale bar, 10 μm.
Figure 8.
 
Effects of a constitutively active form of Rac1 (Val12Rac1) on the actin cytoskeleton and focal adhesions in HCE cells plated on BSA. Cells transfected with pEGFP-C1 (0.1 μg) and 0.5 μg of either pEFBOS-myc-Val12Rac1 or pEFBOS-myc (control vector) were plated on BSA and analyzed by immunofluorescence microscopy as in Figure 2 . Red fluorescence: staining with rhodamine-phalloidin or with antibodies to phosphotyrosine; green fluorescence: transfected cells expressing EGFP. Data are representative of three independent experiments. Scale bar, 10 μm.
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