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. 2002 Sep;13(9):3203–3217. doi: 10.1091/mbc.02-05-0086

α4β1 Integrin Regulates Lamellipodia Protrusion via a Focal Complex/Focal Adhesion-independent MechanismV⃞

Karen A Pinco 1, Wei He 1, Joy T Yang 1,*
Editor: Mary C Beckerle1
PMCID: PMC124153  PMID: 12221126

Abstract

α4β1 integrin plays an important role in cell migration. We show that when ectopically expressed in Chinese hamster ovary cells, α4β1 is sufficient and required for promoting protrusion of broad lamellipodia in response to scratch-wounding, whereas α5β1 does not have this effect. By time-lapse microscopy of cells expressing an α4/green fluorescent protein fusion protein, we show that α4β1 forms transient puncta at the leading edge of cells that begin to protrude lamellipodia in response to scratch-wounding. The cells expressing a mutant α4/green fluorescent protein that binds paxillin at a reduced level had a faster response to scratch-wounding, forming α4-positive puncta and protruding lamellipodia much earlier. While enhancing lamellipodia protrusion, this mutation reduces random motility of the cells in Transwell assays, indicating that lamellipodia protrusion and random motility are distinct types of motile activities that are differentially regulated by interactions between α4β1 and paxillin. Finally, we show that, at the leading edge, α4-positive puncta and paxillin-positive focal complexes/adhesions do not colocalize, but α4β1 and paxillin colocalize partially in ruffles. These findings provide evidence for a specific role of α4β1 in lamellipodia protrusion that is distinct from the motility-promoting functions of α5β1 and other integrins that mediate cell adhesion and signaling events through focal complexes and focal adhesions.

INTRODUCTION

Cell migration is essential for a variety of biological events, including embryonic development, wound healing, inflammation, and metastasis of malignant cells. Cell migration along a substratum is regulated by extracellular signals transduced into cells partly through adhesive interactions between the cell and its surrounding extracellular matrix (ECM). Integrins, the major receptors that mediate cell–ECM interactions (Hynes, 1992), play important roles in regulating cell motility.

Integrins are a large family of heterodimeric cell adhesion receptors. Many integrins, including α5β1 and αVβ3, mediate cell-ECM adhesion by forming junctional complexes called focal adhesions, which bind extracellularly to specific ECM components and intracellularly to cytoskeletal proteins and signaling molecules. In cultured adherent cells, such as fibroblasts, focal adhesions play key roles in regulating motility (Lauffenburger and Horwitz, 1996; Horwitz and Parsons, 1999). When fibroblasts begin to migrate on an ECM substratum, small nascent focal complexes assemble in plasma membrane protrusions at the leading edge of the cell. These complexes grow larger and subsequently recruit α5β1 and other integrins as they evolve into highly organized focal adhesions (Laukaitis et al., 2001). As a cell moves forward, focal adhesions not only act as anchors but also function as nucleation and activation sites for signaling proteins, which in turn activate an intracellular signaling network, leading to actin cytoskeletal reorganization and generation of cell motility (Lauffenburger and Horwitz, 1996).

α4β1, a member of the integrin family, is not localized in focal adhesions in most cell types, yet this integrin also plays important roles in cell migration. α4β1 binds to an alternatively spliced V25 (also called CS-1) region of fibronectin (FN) (Wayner et al., 1989; Guan and Hynes, 1990) instead of the RGD sequence that is recognized by α5β1 and other integrins localized to focal adhesions (Pytela et al., 1985). α4β1 also binds to vascular cell adhesion molecule-1 (VCAM-1), a member of the immunoglobulin superfamily (Osborn et al., 1989; Elices et al., 1990). α4β1 is expressed in many migratory cell types in vivo, including neural crest cells and their derivatives (Sheppard et al., 1994; Kil et al., 1998; Pinco et al., 2001), smooth muscle cells of newly formed blood vessels (Sheppard et al., 1994), hematopoietic cell lineages (Neuhaus et al., 1991), and epicardial progenitor cells (Pinco et al., 2001). Furthermore, the migration of neural crest cells and hematopoietic cells on FN can be disrupted in culture by antibodies that specifically inhibit binding between α4β1 and FN (Yednock et al., 1992; Kil et al., 1998; Testaz et al., 1999), and progenitor cells fail to migrate on the heart to form the epicardium in mouse embryos deficient in α4β1 (Sengbusch et al., 2002).

Although an important role for α4β1 in cell migration has been well documented, questions remain as to how α4β1 promotes cell migration. Because α4β1 is not localized in focal adhesions in most cell types and has a ligand-binding specificity different from integrins in focal adhesions, this integrin may promote cell migration by a mechanism distinct from that of α5β1 and other integrins in focal adhesions. This idea is also supported by an observation that the cytoplasmic tails of α4 and α5 subunits confer different cellular activities with the α4 tail conferring migratory activities and the α5 tail conferring adhesive activities (Chan et al., 1992; Kassner et al., 1995).

In this article, we examined the migratory behaviors of Chinese hamster ovary (CHO) cells ectopically expressing α4β1, by using a scratch-wound assay. Our data show that α4β1 plays a unique role in promoting lamellipodia protrusion through a focal complex/focal adhesion-independent mechanism.

MATERIALS AND METHODS

Construction of Plasmids

For expressing green fluorescent protein (GFP)-tagged α4 integrin in CHO cells, we constructed a plasmid pQN4G. To construct this plasmid, upstream human α4 cDNA (Takada et al., 1989; obtained from American Type Culture Collection, Rockville, MD) and downstream mouse α4 cDNA (Neuhaus et al., 1991; a generous gift from Dr. Martin Hemler, Dana-Farber Cancer Institute, Boston, MA) were joined at a unique and conserved KpnI site, and the 3′ end of this chimeric α4 cDNA was ligated to the 5′ end of GFP cDNA by insertion into PGBI25-fN1 GFP plasmid vector (Quantum Biotechnologies, Montreal, Quebec, Canada). The fusion protein's expression was driven by a cytomegalovirus promoter. The pQN4Y991AG plasmid was the same as pQN4G except that the tyrosine at position 1093, equivalent to position 991 in human α4 cDNA product, of the α4 tail region was mutated to alanine by polymerase chain reaction.

Purified Ligands

Mouse plasma FN was purchased from Invitrogen (Carlsbad, CA). A recombinant FN fragment containing FN type III repeats 12–15 and the CS-1 region and recombinant soluble VCAM-1 (Lobb et al., 1991) were provided by Richard Hynes (Massachusetts Institute of Technology, Cambridge, MA) and Roy Lobb (Biogene, Cambridge, MA), respectively.

Cells, Transfections, and Cell Culture

CHO cells were maintained in DMEM containing 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), l-glutamine, and antibiotics. CHO-α4 cells (Kassner et al., 1995), provided by Martin Hemler (Dana-Farber Cancer Institute), were maintained in F-12 (Invitrogen) containing 10% FBS, l-glutamine, antibiotics, and 0.4 mg/ml G418 (Life Invitrogen). CHOB2, CHOB2-α4, and CHOB2-α5 cell lines, provided by Cary Wu (University of Pittsburgh, Pittsburgh, PA) and Michael DiPersio (Albany Medical College, Albany, NY), were maintained in minimal essential medium-α (Invitrogen) containing 10% FBS, l-glutamine, antibiotics, and 0.4 mg/ml G418. CHO cells were transfected with pQN4G and pQN4Y991AG by using Lipofectin/Optimem (Invitrogen) following manufacturer's instructions. CHO-α4/GFP and CHO-α4Y991A/GFP clones were selected in F-12 containing 0.8 mg/ml G418 and screened for α4/GFP and α4Y991A/GFP expression by using fluorescence-activated cell sorting. Stably transfected cell lines were maintained using the initial concentration of G418.

Analysis of Lamellipodia Protrusion and α4-Positive Puncta Formation at Edges of Scratch-Wounds

For the studies using time-lapse microscopy, cells were plated onto glass bottom Microwell dishes (MatTek, Ashland, MA) coated with 10 μg/ml FN or VCAM-1 for 2 h at 37°C. At confluence, the cell monolayer was scraped with a Pipetman tip to generate scratch-wounds. The wounded surface was washed with phosphate-buffered saline (PBS) and then returned to serum-containing medium. After 2-h incubation, media were replaced with Leibovitz's L-15 medium (Invitrogen) containing 10% FBS. Migration at the wound edge was monitored by phase or fluorescence microscopy by using an Axiovert 135 TV microscope (Carl Zeiss, Thornwood, NY) equipped with a temperature controller (Harvard Apparatus, Holliston, MA). Cell movement was recorded with a charge-coupled device camera (Roper Photometrics, Trenton, NJ) by using IPLab-Spectrum software (Scanalytics, Fairfax, VA). The last frame of each time-lapse movie was analyzed for the percentage of cells at wound edges that protruded broad lamellipodia.

For studies using regular microscopy, cells were plated on tissue culture plates or coverslips coated with 10 mg/ml FN and scratch-wounded as described above. Nonoverlapping fields were photographed at designated time points by phase (250×) or fluorescence microscopy (630×). The percentage of cells at wound edges that protruded broad lamellipodia or formed α4-positive puncta was scored using the phase or fluorescence micrographs, respectively. This method was also used in an antibody perturbation experiment on the fanning activity of CHOB2-α4 cells. In this experiment, an anti-α4 antibody P1H4 (Chemicon International, Temecula, CA), which is identical to a functional blocking antibody, P4C2, was added to the cells at 25 mg/ml. The cells were preincubated with the antibody for 2 h at room temperature, plated on FN and cultured in the presence of the antibody before and after scratch-wounding. At the 2-h time point, the cells at wound edges were photographed and scored for the percentage of cells at wound edges that protruded broad lamellipodia.

Flow Cytometry

Flow cytometry analysis was performed as described by Hildreth et al. (1999) with some modifications. Washed cells were resuspended at 2 × 106 cells/ml in PBS, containing 5% normal goat serum (Vector Laboratories, Burlingame, CA) and 1% bovine serum albumin (BSA) (PBS/NGS/BSA), and blocked on ice for 20 min. Cells (100 μl) were mixed with 100 μl of one of the following primary antibodies at 20 μg/ml: mouse anti-α4 (α4−PUJ1; Upstate Biotechnology, Lake Placid, NY), mouse anti-hamster α5, PB1 (Brown and Juliano, 1985), or mouse anti-hamster β1, 7E2 (Brown and Juliano, 1988). PB1 and 7E2 were provided by Rudy Juliano (Department of Pharmacology, University of North Carolina, Chapel Hill, NC). After 45 min on ice, cells were washed with PBS and resuspended in 100 μl of PBS/normal goat serum/BSA containing 20 μg/ml of either fluorescein- or R-phycoerythrin–conjugated secondary antibodies (BioSource International, Camarillo, CA). After 45 min and a final wash with PBS, cells were resuspended in 0.5 ml of 2% paraformaldehyde in PBS and analyzed on a FACStar Plus with an Innova-90 laser (Coherrent, Santa Clara, CA) exciting at 488 nm wavelengths and running at 100 mW.

Adhesion Assay

The adhesion assay was performed as in Yang and Hynes (1996) with the following modifications. Triplicate wells of 96-well plates were coated with 10 μg/ml FN, CS-1, or VCAM-1 at 37°C for 2 h. Then 5 × 104 cells were plated per well and allowed to adhere in a tissue culture incubator. After 15 min, nonadherent cells were removed by submerging the plate in PBS and shaking off the cells. Seven nonoverlapping high-power fields (200×) along the diameter of each well were photographed, and the number of adherent cells per field was counted.

Migration Assays

For the scratch-wound cell migration assay, the cells were plated on wells of 24-well tissue culture plates coated with 10 mg/ml FN and scratch-wounded as described above to generate scratch-wounds 0.28–0.56 mm in width. Scratch-wounds were allowed to heal in medium containing 10% FBS in a tissue culture incubator. Photographs were taken at designated time points with a phase microscope (Nikon, Melville, NY). By using the photographs, the distance cells migrated was calculated as a percentage of wound closure. For each data point, 10–30 nonoverlapping measurements were taken from multiple wells; mean and SDs were calculated from three independent experiments. Rates of cell proliferation were measured by immunohistochemical detection of 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO) incorporation (Morgenbesser et al., 1995). Cells were plated on FN-coated coverslips and wounded as described above. Fifteen minutes before the time point, the culture medium was replaced by medium containing 50 μM BrdU and 10% FBS. After 15-min culturing, cells were washed in PBS, fixed with cold methanol, washed with PBS, and treated with 1.5 M HCl in the tissue culture incubator for 40 min. Cells were then washed with PBS and stained with an anti-BrdU antibody (Sigma-Aldrich). For each cell line, microscopic fields (630×) from three coverslips were photographed. Percentage of cells with BrdU incorporation in each microscopic field was determined and mean and SDs were calculated.

The Transwell cell migration assay was performed as described in Liu et al. (1999) with the following exceptions. Transwell inserts were coated with 10 μg/ml FN in serum-free F-12 for 2 h at 37°C. Media from the top chamber were replaced with 200 μl of cell suspension (1.5 × 105 cells/ml in F-12), and chambers were incubated for 4 h at 37°C.

Cell Surface Biotinylation and Immunoprecipitation

CHO-α4 cells were surface biotinylated by resuspending at 5 × 106 cells/ml in cell wash buffer (50 mM Tris pH 7.5, 0.15 M NaCl, 1 mM CaCl2, and 5 mM MgCl2) and incubating with EZ-Link NHS-LC-biotin (Pierce Chemical, Rockford, IL) for 60 min at room temperature. Cells were lysed for 15 min at 4°C in ice-cold extraction buffer (0.5% NP-40, 2 mM phenylmethylsulfonyl fluoride, and 0.02 mg/ml aprotonin in cell wash buffer). For coimmunoprecipitation studies, CHO-α4/GFP and CHO-α4Y991A/GFP cells were washed three times with PBS and lysed in ice-cold lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100, 0.05% Tween 20, 2 μg/ml aprotinin, and 0.5 μg/ml leupeptin) for 30 min at 4°C. The cell lysates were cleared with protein-G agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) and subjected to immunoprecipitation by using an anti-α4 antibody (5B10G; Upstate Biotechnology) and protein-G agarose beads. Immunoprecipetates were analyzed on Western blots for the presence of α4/GFP, α4Y991A/GFP, and paxillin, by using an anti-GFP antibody (Molecular Probes, Eugene, OR) and an anti-paxillin antibody (Transduction Laboratories, Lexington, KY), respectively.

Fluorescence and Confocal Microscopy

Cells were plated on glass coverslips coated with 10 mg/ml FN and scratch-wounded. At 3 h or otherwise designated time points after scratch-wounding, the coverslips were collected. For GFP fluorescence, cells were washed three times in PBS, fixed for 15 min in 4% paraformaldehyde (Fluka Chemical, Ronkonkoma, NY) in PBS, and mounted. For immunofluorescence staining, cells were washed three times in PBS, fixed for 15 min in 4% paraformaldehyde in PBS, permeabilized for 15 min in 0.5% NP-40 (Sigma-Aldrich) in PBS, and incubated with antibodies against paxillin (349 from Transduction Laboratories; and 165, a gift from Christopher Turner, SUNY Upstate Medical University, Syracuse, NY) at 37°C. After 30 min, coverslips were washed three times in PBS, incubated with a secondary antibody (BioSource International) at 37°C for 30 min, and washed three times in PBS. Both primary and secondary antibodies were diluted in 10% normal goat serum in PBS. Fluorescent images were obtained using an Axioskop 2 microscope (Carl Zeiss) in conjuction with a Coolsnap fx charge-coupled device camera (Photometrics, Tuscson, AZ) controlled by IPLab-Spectrum software. Confocal images were obtained using the Oz confocal laser scanning microscope system (Noran, Middleton, WI), with Intervision software, version 6.5, on a Silicon Graphics O2 platform.

Online Supplemental Material

The online version of this article contains QuickTime movies that accompany Figures 1, 2, 5, and 7. The speed of the movies is 60× faster than real time. Videos 1–5 accompany Figure 1, videos 6–8 accompany Figure 2, video 9 accompanies Figure 5, and video 10 accompanies Figure 7. Online supplemental material is available at www.molbiolcell.org

Figure 1.

Figure 1

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Time-lapse microscopy of CHO, CHO-α4, and CHO-α4/GFP cells at wound edges. CHO cells on FN (row A), CHO-α4 cells on FN (row B), CHO-α4/GFP cells on FN (row C), CHO-α4 cells on VCAM-1 (row D), and CHO−α4 cells on RGD (row E) were plated and wounded. Two hours after wounding, the cells were photographed every 2 min for 2 h. Four frames of each cell type at 0-, 40-, 80-, and 120-min time points are shown. Bar, 50 μm.

Figure 2.

Figure 2

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Time-lapse microscopy of CHOB2, CHOB2-α5 and CHOB2-α4 cells at wound edges. CHOB2 cells (row A), CHOB2-α5 cells (row B), and CHOB2-α4 cells (row C) were plated and wounded. Two hours after wounding, the cells were photographed every 2 min for 2 h. Three frames of each cell type at 0-, 40-, 80-, and 120-min time points are shown. Bar, 50 μm.

Figure 5.

Figure 5

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Time-lapse microscopy of α4/GFP on the surface of CHO-α4/GFP cells migrating into a scratch-wound. CHO-α4/GFP cells were plated and wounded. After 3 h, the cells with fanning activity were photographed under a fluorescence microscope at 2-min intervals for 26 min. Three frames at low magnification (a–c) and nine frames at high magnification (d–l) are shown representing this time period. Asterisks are fixed reference marks to highlight the movement of the cell. Note that α4/GFP fluorescence was very strong in ruffles (arrowheads). As the ruffles flattened out, α4/GFP fluorescence was seen as puncta along the leading edge of small lamellipodia protrusions (e and k, arrows). As the leading edge continued to extend forward, the puncta of fluorescence stayed at a fixed position behind of the new cell front (f, arrow), and they disappeared when the cell front formed new ruffles (j and l, arrowheads). Bars, 10 μm.

Figure 7.

Figure 7

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Y991A mutation enhances cell fanning and migration into the scratch-wound. (A) CHO-α4/GFP and CHO-α4Y991A/GFP cells were plated on FN and wounded. Two hours after wounding, the cells were photographed every 2 min for 2 h. Four frames of both cell types at 0-, 40-, 80-, and 120-min time points are shown. Bar, 50 μm. (B) CHO-α4/GFP and CHO-α4Y991A/GFP cells were plated on FN-coated coverslips and wounded. Percentages of cells at the wound edge (n = ∼300) with fanning activity were scored at 0.5-, 1-, 2-, and 3-h time points.

RESULTS

α4β1 Integrin Promotes Lamellipodia Protrusion When Ectopically Expressed in CHO Cells

It was previously shown that CHO cells express α5β1 but not α4β1 (Schreiner et al., 1989; Kassner and Hemler, 1993). Using a Transwell assay, Martin Hemler and colleagues have shown that when α4β1 is ectopically expressed in CHO cells, this integrin enhances cell motility (Kassner et al., 1995). To determine how α4β1 promotes cell motility, we used a scratch-wound assay and time-lapse microscopy to examine the migratory behaviors of a CHO cell line that stably expresses α4 (CHO-α4 cells) (Kassner et al., 1995). In the scratch-wound assay, the CHO-α4 cells and the parental CHO cells were plated on FN-coated dishes; as the cells formed a confluent monolayer, a scratch-wound was made in the monolayer to induce cell migration into the wound. Two hours later (2-h time point), the cells at wound edges were imaged by time-lapse microscopy. As shown in Figure 1, A and B (Videos 1 and 2), CHO-α4 cells and the parental CHO cells displayed very different migratory behaviors. CHO cells at wound edges migrated as a mass. While the monolayer of CHO cells pushed toward the scratch-wound, individual cells protruded short-lived membrane extensions in random directions with little persistent polarity toward the wound. No prominent lamellipodia were observed (Figure 1A). In contrast, some CHO-α4 cells at the wound edge migrated into the wound as individual cells by forming fan-shaped broad lamellipodia with a persistent polarity toward the wound (Figure 1B). At the 4-h time point (2 h after starting to take the movies) 21% of all the cells at the wound edges exhibited this “fanning” behavior (Figure 3). We also performed the scratch-wound assay and photographed the cells at the 12- and 18-h time points. At these later time points, the majority of the CHO-α4 cells at wound edges had the fan shape, whereas none of the CHO cells at wound edges did (our unpublished data). We generated several stable cell lines expressing α4, which was tagged with GFP (CHO-α4/GFP, see below for characterization of these cell lines), and found that all of the α4/GFP-expressing cell lines also had the fanning phenotype (Figure 1C and Video 3) to a similar degree (Figure 3). Therefore, the fanning phenotype was not due to a cloning artifact.

Figure 3.

Figure 3

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Percentage of fanning cells at wound edges. The last frame of each movie in Figures 1 and 2 were analyzed. Cells at the edge of the scratch-wound (n = 30–50) were scored for fanning, and the percentage of these cells was calculated. This was done for each of the following cell lines: CHO, CHO-α4, CHO-α4/GFP, CHO-α4Y991A/GFP, CHOB2, CHOB2-α5, and CHOB2-α4 on a FN substrate as well as for CHO-α4 on both VCAM-1 and RGD.

α4β1 Promotes Lamellipodia Protrusion Independent of α5β1

The CHO-α4 and CHO-α4/GFP cells also express α5β1. Our flow cytometry analyses showed that the expression level of α5 at the cell surface was slightly reduced in these cells (Figure 4A). Thus, the fanning behavior may be promoted directly by α4β1, or indirectly due to decreased expression of α5β1 at the cell surface. To distinguish between these possibilities and determine whether the fanning phenotype depends on α4β1, we tested the CHO-α4 cells in the scratch-wound assay by using an α4β1-specific ligand, VCAM-1, or an α5β1-specific ligand, the RGD peptide, as the substrate. The cells fanned on VCAM-1 (Figure 1D and Video 4) but not on the RGD peptide (Figure 1E and Video 5). At the 4-h time point, CHO-α4 cells plated on RGD alone showed no evidence of fanning (Figure 3). However, 27% of the CHO-α4 cells on VCAM-1 at wound edges exhibited the fanning behavior. Because α4β1 is the only receptor for VCAM-1 in CHO-α4 cells, this result indicates that the binding between α4β1 and VCAM-1 is sufficient for the fanning phenotype, and that the RGD-recognizing integrins, including α5β1, are not sufficient.

Figure 4.

Figure 4

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GFP-tagged α4 integrin is functionally normal. (A) Flow cytometry analysis. Unlabeled CHO cells served as negative controls (column 1). Cells were labeled with either anti-human α4 (column 2), anti-hamster β1 (column 3), or anti-hamster α5 (column 4). Fluorescence intensity is shown in logarithmic scale. (B) α4/GFP, α4, or GFP alone was each transfected into CHO cells. The CHO-α4 cells (a) and the parental CHO cells (c) were stained with an anti-α4 antibody by immunofluorescence. α4/GFP (b) localized at the surface of transfected cell in the same pattern as α4 in the CHO-α4 cells (a). The parental CHO cells had only background staining (c). GFP alone localized diffusely in the nucleus and cytoplasm and did not reach the cell surface (d). Bar, 10 μm. (C) Number of adherent cells per high-power field (n = 7) was determined for each of the triplicate wells for CHO, CHO-α4, and CHO-α4/GFP cells adhering to FN, CS-1, or VCAM-1. The mean values and standard derivations for the triplicates were graphed. Note that the CHO-α4 and CHO-α4/GFP cell lines but not the CHO cells adhered to CS-1 and VCAM-1. (D) Cells were photographed at 0-, 12-, and 18-h time points. The percentage of wound closure was determined (n = 20–30) from at least two (3 for all but α4/GFP) independent experiments. The mean values and SDs were graphed. Note that CHO-α4 and CHO-α4/GFP cells both had a much faster wound closure rate than that of CHO cells, with no significant difference between the wound closure rates of CHO-α4 and CHO-α4/GFP.

To test directly the roles of α4β1 and α5β1 in the fanning phenotype, we took advantage of a CHO-derived cell line, named CHOB2, which expressed a negligible level of α5β1 (Schreiner et al., 1989). It has previously been shown that α5β1, when stably transfected into CHOB2 cells, can rescue the ability of CHOB2 cells to adhere to and migrate randomly on FN (Bauer et al., 1992). When α4β1 is stably expressed in CHOB2 cells, it can also rescue the cells for adhesion and migration (Wu et al., 1995). We compared the α5- and α4-expressing CHOB2 cell lines for their migratory behaviors at edges of scratch-wounds, by using FN as the substrate. The results are shown in Figure 2. CHOB2 cells adhere poorly to FN. To examine their migratory behaviors at wound edges, the cells were plated and scratch-wounded, but the washes were omitted to allow the cells to remain on the dish. These cells (Figure 2A and Video 6) at wound edges did not show any polarization, although the cells were able to protrude membrane extensions in a random manner. CHOB2-α5 cells (Figure 2B and Video 7) adhered and migrated much in the same manner as the CHO cells. At the 4-h time point, CHOB2 and CHOB2-α5 both showed no evidence of fanning at wound edges (Figure 3). In contrast, some CHOB2-α4 cells at wound edges (Figure 2C and Video 8) migrated by forming lamellipodia with persistent polarity toward the wound, although to a lesser extent compared with CHO-α4 cells. At the 4-h time point, 11% of CHOB2-α4 cells at wound edges exhibited the fanning behavior (Figure 3). These results show that 1) α5β1 alone does not promote fanning on FN and α4β1 is required; and 2) α4β1 is sufficient to promote fanning on FN, but optimal fanning on FN also requires α5β1.

To test further whether α4β1 is required for the fanning phenotype, we performed an antibody perturbation experiment, by using an anti-α4 antibody that specifically disrupted binding between α4β1 and FN (Sechler et al., 2000). When this antibody was added to scratch-wounded CHOB2-α4 cells, considerably fewer cells exhibited the fanning activity than did the CHOB2-α4 cells in the absence of the antibody (Table 1). This result indicates that the ligand-binding activity of α4β1 is required for promoting the fanning behavior.

Table 1.

Percentage of cells fanning at the wound edge at the 2-h time point  Total number of cells was 340 and 654 for no antibody and with antibody, respectively.

No antibodyb 7.65%
With antibodyc 1.83%
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a

In the absence of anti-α4 antibody. 

b

In the presence of anti-α4 antibody. 

We conclude that α4β1 and α5β1 play different roles in the migration of CHO cells at wound edges, and α4β1 plays a unique role in promoting lamellipodia protrusion.

α4 Integrin Is Functional When Tagged with GFP

To study the mechanisms by which α4β1 promotes lamellipodia protrusion, we attached GFP to the cytoplasmic tail of the α4 subunit, producing the α4/GFP fusion protein. Two independent cell lines stably expressing α4/GFP were generated and analyzed. Cells from both cell lines migrated in the same manner as the CHO-α4 cells (Figure 1C and Figure 3). To determine more closely whether the GFP tag interfered with the function of α4β1, we used the CHO-α4 cell line as a control to characterize the GFP-tagged α4 for its surface expression, adhesive activity, and migration-promoting activity. Flow cytometry analysis showed that the surface expression levels of α4/GFP in the transfected cell lines were similar to that of α4 in the control CHO-α4 line (Figure 4A). Both the CHO-α4 and CHO-α4/GFP cell lines also had surface expression levels of β1 similar to CHO cells, yet they had slightly decreased levels of α5 compared with the parental CHO cells. We also compared the surface distribution of α4 with and without the GFP tag (Figure 4B). α4/GFP and α4 were both expressed over the entire cell surface when the cells were plated sparsely, as assayed by GFP fluorescence and anti-α4 immunofluorescence, respectively. α4 and α4/GFP were also both detected within the cells, possibly in the ER and Golgi complexes. In control cells transfected with GFP cDNA alone, GFP was expressed in the cytoplasm and nucleus but not on the cell surface. These data showed that α4/GFP was distributed normally at the cell surface.

Because α5β1 binds to the RGD region of FN (Pierschbacher and Ruoslahti, 1984), and α4β1 binds to the CS-1 region of FN, we predicted that the parental CHO cells, which express α5β1 but not α4β1, should adhere to full-length FN (containing both regions) but not to a fragment of FN that contains only the CS-1 region. In contrast, CHO-α4 and CHO-α4/GFP should adhere to both. Because α4β1 also binds to VCAM-1, we predicted that, if the (α4/GFP)β1 protein was functional, the transfected cells would adhere to VCAM-1, whereas the parental CHO cells would not. Cell adhesion assays on these cells fulfilled our predictions (Figure 4C), demonstrating that (α4/GFP)β1 has normal adhesive activities.

As discussed above, we have shown that CHO-α4 and CHO-α4/GFP cells both display a fanning behavior at edges of scratch-wounds. We also compared their rates of wound closure and found that both CHO-α4 and CHO-α4/GFP cells closed the scratch-wounds much faster than the CHO cells, with the migration rates of CHO-α4 and CHO-α4/GFP cells not significantly different from each other (Figure 4D). In parallel with the wound assay, cell proliferation rates were measured for these cell lines under the same plating and wounding conditions as the cells in the wound assays. Our data showed that CHO, CHO-α4,and CHO-α4/GFP cells proliferated at the same rate (Table 2). Thus, the faster wound-closure rates of CHO-α4 and CHO-α4/GFP cells were not due to a faster proliferation rate but due to a faster migration rate of these cells.

Table 2.

Proliferation rates of CHO, CHO-α4 and CHO-α4/GFP cells in wound assays

% Cells with BrdU*
CHO 58 ± 10
CHO-α4 54 ± 8
CHO-α4/GFP 55 ± 10
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*

Numbers represent the mean and standard deviation of the percentages of cells with BrdU incorporation (n = 523, n = 394, n = 288 cells for CHO, CHO-α4, and CHO-α4/GFP, respectively). There are no statistically significant differences among the three cell lines (p > 0.1). 

In summary, we show that (α4/GFP)β1 has the same localization, adhesive activities, and migration-promoting activities as untagged α4β1. We conclude that (α4/GFP)β1 is functionally normal.

α4β1 Forms Transient Puncta at Leading Edge of Cells That Begin to Protrude Lamellipodia in Response to Scratch-Wounding

Using a fluorescence microscope attached to a time-lapse imaging system, we examined the surface dynamics of (α4/GFP)β1 on the CHO-α4/GFP cells that displayed fanning activity (Figure 5). At low magnification, (α4GFP)β1 was seen over the entire surface of the cells. The α4/GFP fluorescence was particularly strong in membrane ruffles at the leading edge (arrowheads in Figure 5, a–c). At higher magnification, transient α4/GFP-positive puncta were found in cells that had just begun to fan into the scratch-wounds. The α4/GFP-positive puncta were seen in some areas at the leading edge where the ruffles flattened out and the membrane extended into smooth edged but small lamellipodia (arrows in Figure 5, e and k). The puncta were located right along the leading edge of these small lamellipodia. As the lamellipodia continued to extend and a new cell front formed, the puncta stayed at their original positions (arrow in Figure 5f). The extended membrane then began to ruffle again, whereas the α4/GFP puncta gradually disappeared (Figure 5 g, h, and l). As the new cell front was ruffling, α4/GFP fluorescence was again seen in the ruffles (arrowheads in Figure 5, j and l). This sequence of events was repeated continuously as the cell formed a broad lamellipodia and moved forward toward the wound (Video 9). This pattern of surface dynamics may be related to the unique function of α4β1 in promoting broad lamellipodia protrusion.

Disrupting α4/Paxillin Binding Allows a Faster Response of Cells to Scratch-Wounding That Correlates with Formation of α4-Positive Puncta at the Leading Edge

Paxillin is a signaling adaptor protein (Turner, 2000), which binds to the cytoplasmic tail of α4 (Liu et al., 1999). A point mutation at the α4 tail, Y991A, disrupts this binding (Liu et al., 1999). It has been shown that this mutation reduces random cell motility (Liu et al., 1999). We reasoned that, if this mutation affects the α4β1-dependent fanning phenotype as well as the formation of the α4-positive puncta at the leading edge, we would be able to establish a mechanistic relationship between fanning and puncta formation. Therefore, we generated and analyzed two independent CHO cell lines that stably express α4 cDNA carrying this mutation; the mutant α4 was tagged with GFP (the cell lines are referred to as CHO-α4Y991A/GFP). To confirm that the Y991A mutation disrupted the paxillin-binding in the CHO-α4Y991A/GFP cells, we performed a coimmunoprecipition experiment. We showed that the amount of paxillin that coimmunoprecipitated with the mutant α4 was much less than that coimmunoprecipitated with the wild-type α4 (Figure 6B). We also showed that both α4/GFP and α4Y991A/GFP remained intact when expressed in CHO cells (Figure 6A). To confirm that the Y991A mutation reduces random motility as reported by Ginsberg and colleagues (Liu et al., 1999), we compared the abilities of the CHO-α4/GFP and CHO-α4Y991A/GFP cells to migrate in the Transwell assay. We found that the motility of CHO-α4Y991A/GFP cells on FN was reduced by ∼55% (Figure 6C).

Figure 6.

Figure 6

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Characterization of CHO-α4Y991A/GFP cells. (A) Tagged GFP was not cleaved from the α4/GFP fusion protein when the protein is expressed in CHO cells. CHO-α4 cells were surface biotinylated, lysed, and immunoprecipitated with an anti-α4 antibody, by which α4 integrin was detected as cleavage fragments of 70 and 80 kDa. CHO-α4/GFP and CHO-α4Y991A/GFP cells were lysed and subjected to immunoblot analysis by using an anti-GFP antibody, which revealed a 100-kDa cleavage fragment of α4/GFP fusion protein (the 70-kDa cleavage fragment of α4 plus 30 kDa of GFP), showing that the tagged GFP was not cleaved from the fusion protein. (B) Y991A mutation reduced binding between α4 and paxillin. CHO-α4/GFP and CHO-α4Y991A/GFP cells were lysed, immunoprecipitated with an anti-α4 antibody, and analyzed by immunoblotting by using anti-paxillin or anti-GFP antibody. Total cell lysate for each cell type was analyzed alongside. Note that the amount of paxillin that coimmunoprecipitated with α4Y991A/GFP was significantly less than that with the wild-type α4/GFP. (C) Transwell cell migration assay with the membrane coated on both sides with FN. The number of cells migrated per field (n = 3) was determined for each of the triplicate wells for CHO-α4/GFP and CHO-α4Y991A/GFP cells. Data were means and standard derivations of triplicate experiments (p < 0.001). Note that the Y991A mutation reduced random cell motility. (D) Cells were photographed at 0-, 6-, and 12-h time points. The percentage of wound closure was determined (n = 10), and the mean values and SDs were graphed (p < 0.01). Note that the Y991A mutation enhanced the wound closure rate.

We then compared the CHO-α4Y991A/GFP and CHO-α4/GFP cells by using the scratch-wound assay. To our surprise, all CHO-α4Y991A/GFP cell lines, when plated on FN and tested in the scratch-wound assay, had a faster wound closure rate than CHO-α4/GFP cells (Figure 6D). The faster wound closure rate was not due to faster cell proliferation, because CHO-α4Y991A/GFP and CHO-α4/GFP cells had the same proliferation rates as assayed under the same plating and wounding conditions as in the wound assays (Table 3). The same results were obtained from two independent cell lines, indicating that the faster wound closure rate was not due to a cloning artifact. This result suggests that the scratch-wound assay and the Transwell assay measure different types of motile activities.

Table 3.

Proliferation rates of CHO-α4/GFP and CHO-α4Y991A/GFP cells in wound assay  The numbers represent the mean and standard deviation of the percentages of cells with BrdU incorporation (n = ≈500). There are no statistically significant differences between the two cell lines (p > 0.1).

% Cells with BrdU
6 h 12 h
CHO-α4/GFP 39 ± 7 52 ± 7
CHO-α4 Y991A/GFP 38 ± 6 43 ± 10
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We suspected that the faster wound closure rate of the mutant cells was likely due to enhanced fanning activity of the CHO-α4Y991A/GFP cells at wound edges. To test this idea, we compared the migratory behaviors of CHO-α4/GFP and CHO-α4Y991A/GFP cells at wound edges by time-lapse microscopy and found that the CHO-α4Y991A/GFP cells indeed displayed a much higher degree of fanning at wound edges (Figure 7A and Video 10). At the 4-h time point (2 h after starting the time-lapse movie), the percentage of the CHO-α4Y991A/GFP cells that exhibited fanning activity exceeded that of CHO-α4 and CHO-α4/GFP cells by at least 45% (Figure 3). To evaluate the fanning activities more closely, the CHO-α4Y991A/GFP and CHO-α4/GFP cells at wound edges (n = ∼300) were photographed at 0.5-, 1-, 2-, and 3-h time points and scored for the percentage of cells that displayed the fanning behavior (Figure 7B). We found that at the 0.5-h time point, the CHO-α4/GFP cells at wound edges had little fanning activity (<5% fanning cells), whereas at this time point, ∼30% fanning CHO-α4Y991A/GFP cells was seen at wound edges (Figure 7B). There was a steady increase of the percentage of fanning cells for both cell types over time. At the 3-h time point, while the percentage of fanning CHO-α4/GFP cells remained low (18.6%), that of fanning CHO-α4 Y991A/GFP cells reached 53.8%. This result showed that the CHO-α4Y991A/GFP cells responded to scratch-wounding faster and fanned earlier than the CHO-α4/GFP cells.

To relate the formation of α4-positive puncta at the leading edge to the fanning activity, we examined a large number of CHO-α4/GFP and CHO-α4Y991A/GFP cells at wound edges and scored the percentage of cells with the α4-positive puncta (n = ∼110) (Figure 8K). The α4-positive puncta were found at the leading edge of both cell types (Figure 8, E and F), and the cells that displayed the α4-positive puncta were largely those that had just begun to fan and migrate into the scratch-wounds, which were frequently found among CHO-a4/GFP and CHO-a4Y991A/GFP cells at the 3-h (Figure 8C) and 0.5-h (Figure 8B) time points, respectively. In these cells, the puncta were again located along the leading edge of small, newly formed lamellipodia protrusions (arrows in Figure 8, E and F). However, the cells that had already formed broad lamellipodia and migrated into the wounds did not display the α4-postive puncta at the leading edge (Figure 8D). Therefore, while the CHO-α4Y991A/GFP cells had fanning activity at earlier time points after scratch-wounding than the CHO-α4/GFP cells, the onset of puncta formation was also earlier in these cells. To compare the fanning activity and puncta formation more closely, we scored CHO-α4/GFP and CHO-α4Y991A/GFP cells at wound edges for the percentage of cells with fanning activity (n = ∼300) at the 0.5- and 3-h time points after scratch-wounding, by using phase micrographs at a lower magnification, which provided a better view of cell morphology at wound edges (Figure 8, G–J). The cells with fanning activity were placed in one of two categories: 1) initiating fanning or 2) having formed broad lamellipodia and migrated into the scratch-wounds. We found that the degree to which cells had initiated fanning at the wound edge correlated with the percentage of cells exhibiting α4-positive puncta. CHO-α4/GFP cells at the 0.5-h time point (Figure 8G) showed little fanning activity at the wound edges and little evidence of puncta formation (Figure 8K). However, by the 3-h time point, when some cells began to fan and move into the wound (Figure 8I), the appearance of puncta increased correspondingly (Figure 8K). On the other hand, CHO-α4Y991A/GFP cells had considerable number of cells initiating fanning at the 0.5-h time point (Figure 8H), and at this time point a corresponding percentage of the cells had the α4-positive puncta (Figure 8K). By the 3-h time point (Figure 8J) when the majority of the CHO-α4Y991A/GFP cells have formed broad lamellipodia and migrated into the scratch-wounds, the percentage of the cells with α4-positive puncta had drastically decreased (Figure 8K). These results show that there is a close correlation between the formation of α4-positive puncta and the initial protrusion of broad lamellipodia into the scratch-wound.

Figure 8.

Figure 8

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α4-Positive puncta are present in cells that begin to protrude broad lamellipodia. CHO-α4/GFP (A, C, E, G, and I) and CHO-α4Y991A/GFP (B, D, F, H, and J) cells were plated on FN-coated coverslips and scratch-wounded. At 0.5- (A, B, F, G, and H) and 3-h (C, D, E, I, and J) time points, cells were fixed and analyzed by fluorescence (A–F) and phase microscopy (G–J). E and F were higher magnification of C and B, respectively. The histogram (K) shows the percentage of cells (n = ∼110) with the α4-positive puncta, and the percentages of cells (n = ∼300) that begin to fan and of those that have migrated into the scratch-wounds. Note that at the 0.5-h time point, CHO-α4/GFP cells (A and G) showed little fanning activity, whereas CHO-α4Y991A/GFP cells (B and H) had already initiated fanning. By the 3-h time point, although CHO-α4/GFP cells (C and I) had just begun to fan, many CHO-α4Y991A/GFP cells (D and J) had already formed broad lamellipodia and migrated into the scratch-wounds. The percentage of cells with the presence of α4-positive puncta closely correlated with the percentage of cells initiating fanning (K), in which the puncta were located along the leading edge of small lamellipodia protrusions (E and F, arrows). Asterisks (*) in C and B and arrowheads in E and F indicate locations of α4-positive puncta. Bar, 10 μm.

α4β1 Colocalizes with Paxillin Partially in Leading Edge Ruffles But Is Not Localized in Focal Adhesions and Focal Complexes

To understand how the paxillin-binding regulates the lamellipodia-promoting activity of α4β1, we performed immunofluorescence studies to determine whether α4β1 and paxillin colocalize at the leading edge. It has been shown by others that in the CHO cells that do not express α4β1, paxillin is localized in focal adhesions, focal complexes, and ruffles (Nakamura et al., 2000). In migrating CHO cells, paxillin is recruited into newly formed focal complexes near the leading edge (Laukaitis et al., 2001). We found that paxillin was also localized in these areas in CHO-α4/GFP cells. (α4/GFP)β1 partially colocalized with paxillin in ruffles (Figure 9J, arrow). At the leading edge of cells migrating into scratch-wounds, (α4/GFP)β1 was localized in puncta as seen in the time-lapse studies (Figure 9F, arrows), but these α4-positive puncta clearly did not colocalize with the paxillin-positive focal complexes, which are also seen at the leading edge. (α4/GFP)β1 was also absent from most of the paxillin-positive focal adhesions (Figure 9, C and F). A very small number of paxillin-positive focal adhesions overlapped with some α4-positive spots (Figure 9F, arrowhead), but given that the majority of focal adhesions had no α4 staining, we conclude that α4β1 is not localized in focal adhesions. We also examined the CHO-α4Y991A/GFP cells by paxillin staining and GFP fluorescence and found that the localization patterns of α4 and paxillin were not altered by the Y991A mutation (our unpublished data). Paxillin and the mutant α4β1 were both found in ruffles, although our biochemical data clearly showed that the binding between α4β1 and paxillin was drastically reduced by the Y991A mutation (Figure 6B).

Figure 9.

Figure 9

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α4β1 is localized in ruffles but not in paxillin-positive focal complexes/focal adhesions. CHO-α4/GFP cells were wounded and allowed to heal for 3 h, after which the cells were fixed, stained with an anti-paxillin antibody, and analyzed by fluorescence (A–F) or confocal (G–J) microscopy. Paxillin (A, D, and G, single exposures) was localized in focal adhesions and ruffles. α4/GFP was localized in ruffles and puncta at the leading edge (B, E, and H, single exposures). Paxillin and α4/GFP were colocalized in ruffles, giving yellow color in double exposures (pointed by arrowheads in C, I, and J). An edge view of ruffles show that paxillin and α4/GFP colocalize partially in the ruffles (J, oblique edge view of I). However, α4/GFP did not localize with paxillin-positive focal complexes/focal adhesions (C and F, double exposures), and paxillin did not localize in α4-positive puncta (pointed by arrows in F). The area marked between two asterisks (*) in J shows artifact colors of reflection from glass coverslips beneath the cell. Bar, 5 μm.

DISCUSSION

Cell migration is an integrated process involving multiple steps, including membrane protrusion, formation of stable attachments near the leading edge of the protrusion, forward locomotion of the cell body, release of adhesions, and cell rear retraction (Lauffenburger and Horwitz, 1996). The assembly and disassembly of focal adhesions play critical roles in the formation and release of stable attachments of the cell to its substratum (Webb et al., 2002). The assembly of focal adhesions involves sequential recruitment of adhesion components, including integrins such as α5β1, into nascent focal complexes at the leading edge of membrane protrusions (Laukaitis et al., 2001). The integrins in focal complexes and focal adhesions not only provide anchors for the cells to generate motile force (Smilenov et al., 1999) but also their adhesive activities, when modulated, can regulate migration speed (DiMilla et al., 1993; Cox et al., 2001). Furthermore, when the cell forms membrane protrusions, these protrusions are stabilized by focal complexes that mediate stable cell-substratum adhesion. This relatively stable adhesion allows persistent membrane protrusions but is not required for the initial formation of the protrusions (Bailly et al., 1998).

In this article, we provided evidence for a role of α4β1 integrin in the formation of membrane protrusions that is independent of focal complexes and focal adhesions. We show that α4β1 promotes broad lamellipodia protrusion when ectopically expressed in CHO cells that do not express this integrin endogenously, whereas α5β1 does not have this effect. This protrusion-promoting activity of α4β1 is consistent with an observation that the protrusive activity of T lymphocytes on FN can be inhibited by an anti-α4 antibody but not an anti-α5 antibody (Szabo et al., 1995). In migrating cells, while α5β1 is recruited into focal complexes (Laukaitis et al., 2001), we show that α4β1 forms transient puncta at the leading edge, which do not colocalize with focal complexes and focal adhesions. It is likely that the α4-positive puncta contribute to the lamellipodia protrusion activity of the cells, because the Y991A mutation in the cytoplasmic tail of α4 results in an earlier onset of α4-positive puncta formation as well as earlier initiation of lamellipodia protrusion in response to scratch-wounding. We found that the α4-positive puncta formed along the leading edge of small protrusions before they developed into broad lamellipodia, supporting a role of these puncta in the initiation stage of lamellipodia formation. The dynamic nature of the α4-positive puncta strongly suggests that the puncta result from transient clustering of the α4β1 molecules.

While α4β1 is sufficient and required for CHO cells to protrude broad lamellipodia in response to scratch-wounding, under the same conditions CHO cells expressing endogenous or exogenous α5β1 only randomly protrude short-lived membrane extensions. This result indicates that α5β1 is not sufficient to promote broad lamellipodia protrusion, but optimal lamellipodia protrusion of CHO-α4 cells requires α5β1. This result is consistent with α5β1 playing a role in stabilizing broad lamellipodia after they are formed. We propose that transient clustering of α4β1 molecules at the leading edge mediates strong but transient adhesion of the leading edge membrane to the ECM substrate, whereas the focal complexes evolve into focal adhesions that mediate stable adhesion. Both transient and stable adhesions are required for optimal protrusive activity, but the transient clustering of α4β1 may be the primary adhesive event that initiates broad lamellipodia protrusion in response to scratch-wounding.

Lamellipodia are broad membrane extensions of cells comprised of a planar meshwork of actin filaments (Small et al., 1999). The formation of lamellipodia involves proteins that regulate actin dynamics, such as vasodilator-stimulated phosphoprotein (Reinhard et al., 1992), Wiskott-Aldrich syndrome protein (Rohatgi et al., 1999; Winter et al., 1999), and the Arp2/3 complex (Machesky and Gould, 1999), and their upstream regulators, including Rac (Hall, 1998) and its effector p21-activated kinase (Bagrodia and Cerione, 1999). Broad lamellipodia protrusion does not necessarily require cell-substratum contacts. When stimulated by soluble chemoattractants, some cell types can protrude broad lamellipodia rapidly in the absence of any contact with the substratum (Bailly et al., 1998). But lamellipodia protrusion stimulated by mechanical cues does require cell-substratum contact (Pelham and Wang, 1997). We speculate that scratch-wounding somehow generated a mechanical cue at the wound edge, which induced clustering of α4β1 molecules at the leading edge of the cells. The clustering event not only provides transient contact of the leading edge to the substratum but also may activate a signaling cascade leading to actin cytoskeletal reorganization and the formation of broad lamellipodia. The cytoplasmic domain of α4 can be phosphorylated (Han et al., 2001) and may play an active role in these signaling events. Therefore, we propose that while the integrins in focal complexes/focal adhesions mediate cell-substratum adhesion to stabilize protrusions, α4β1 may act at the leading edge as a mediator for sensing migratory cues to initiate polarized protrusions. Besides α4β1, other integrins may play a similar role at the leading edge. For example, activated αVβ3 can be recruited to the leading edge of cells when stimulated with fibrinogen or basic fibroblast growth factor, and this recruitment is required for directional motility (Kiosses et al., 2001). It is conceivable that integrin clustering and activation may play a similar role at the leading edge in regulating polarized membrane protrusions.

We show that the lamellipodia-promoting activity of α4β1 is negatively regulated by paxillin. This negative regulation is likely to occur in the ruffles at the leading edge where paxillin and α4β1 colocalize. We propose that the binding between paxillin and the α4 tail prevents α4β1 in the ruffles from clustering at the leading edge. This negative regulation may be relieved by an inside-out signaling pathway, which is activated in response to scratch-wounding. We found that, after scratch-wounding, the cells expressing α4Y991A began to fan sooner than the cells expressing wild-type α4, suggesting that (α4Y991A)β1 may be constitutively active in its lamellipodia-promoting activity due to reduced paxillin binding.

Interestingly, the Y991A mutation reduces random motility of the cells in Transwell assays while enhancing lamellipodia protrusion in response to scratch-wounding. Therefore, lamellipodia protrusion and random motility are distinct types of motile activities, which are differentially regulated by interactions between α4β1 and paxillin. Paxillin is a multidomain adaptor protein that is recruited into focal complexes and focal adhesions, where it provides docking sites for cytoskeletal and signaling proteins (Turner, 2000). Furthermore, paxillin has been shown to play essential roles in cell motility, possibly by recruiting signaling components into focal complexes via the PKL-PIX-PAK complex (Turner et al., 1999) and by regulating the turnover of focal adhesions via a FAK-mediated pathway when paxillin and FAK interact in focal adhesions (Hagel et al., 2002). Ginsberg and colleagues show that disrupting the binding between paxillin and α4β1 affects the kinetics of FAK phosphorylation. They propose that the α4 tail changes the kinetics of FAK phosphorylation through α4/paxillin interactions and promotes random cell motility by allowing a more efficient activation of FAK signaling that is primarily mediated by the integrins in focal adhesions, including α5β1 (Liu et al., 1999). Thus, in addition to the cooperative role of α4β1 and α5β1 in promoting lamellipodia protrusion as discussed above, these two integrins may also play a synergistic role in regulating random cell motility, where α4β1 somehow amplifies the α5β1-mediated signaling events although not localized in focal adhesions. In fact, α4β1 can rescue the ability of CHOB2 cells, which lack α5β1, to adhere and migrate on FN (Wu et al., 1995).

Alternatively, paxillin may play a more active role. It has been reported that overexpression of a paxillin LD4 domain deletion mutant, which disrupted binding of paxillin to PKL and localization of PKL to focal adhesions, caused an increase in cell protrusiveness and random motility but an inhibition of the cells to migrate into scratch-wounds (West et al., 2001). Therefore, paxillin/PKL binding and paxillin/α4β1 binding seem to have opposite effects on cell motility, suggesting that paxillin may play an active role in achieving a balance between random and polarized lamellipodia protrusive activities.

In summary, we show that α4β1 can mediate specific events at the leading edge of migrating cells to initiate the formation of broad lamellipodia and that this lamellipodia-promoting activity of α4β1 is independent of focal complexes/focal adhesions. In addition, we demonstrated that lamellipodia protrusion and random motility can be differentially regulated by interactions between α4β1 and paxillin. These findings provide new insight into how cell migration can be regulated by integrin-mediated cell–ECM interactions.

Supplementary Material

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ACKNOWLEDGMENTS

We thank Martin Hemler, Michael Dipersio, and Cary Wu for the CHO-α4, CHOB2-α5, and CHOB2-α4 cell lines; Rudy Juliano for the PB1 and 7E2 antibodies; Christopher Turner for the anti-paxillin antibody; and Richard Hynes and Roy Lobb for the CS-1 and VCAM-1 peptides. We also thank Gina Hamlin (Johns Hopkins School of Medicine Center for Analytical Cytology), Doug Murphy and Michael Delannoy (Johns Hopkins School of Medicine Microscope Facility) Jennifer Sengbusch, Stephen Liu, and James Hildreth for technical assistance. We are grateful to Susan Craig and Richard Hynes for helpful discussions regarding this project, and to Susan Craig, Peter Devreotes, Katherine Wilson, Monn Monn Myat, and Michael Dipersio for helpful suggestions and critical comments on this article. This work was supported by a grant from the American Cancer Society (RPG-98-229-01-DDC).

Abbreviations used:

CHO

Chinese hamster ovary

ECM

extracellular matrix

FN

fibronectin

GFP

green fluorescent protein

VCAM-1

vascular cell adhesion molecule-1

Footnotes

V⃞

Online version of this article contains video material for some figures. Online version available at www.molbiolcell.org.

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.02–05–0086. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.02–05–0086.

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