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J. Biol. Chem., Vol. 282, Issue 11, 8545-8556, March 16, 2007

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IQ-domain GTPase-activating Protein 1 Regulates -Catenin at Membrane Ruffles and Its Role in Macropinocytosis of N-cadherin and Adenomatous Polyposis Coli^*

From the Westmead Institute for Cancer Research, University of Sydney, Westmead Millennium Institute at Westmead Hospital, Westmead, New South Wales 2145, Australia

Received for publication, November 3, 2006 , and in revised form, January 8, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
-Catenin is an integral component of E-cadherin dependent cell-cell junctions. Here we show that -catenin co-localizes with IQ-domain GTPase-activating protein 1 (IQGAP1), adenomatous polyposis coli (APC), and N-cadherin at actin-positive membrane ruffles in NIH 3T3 fibroblasts. We used deletion mapping to identify the membrane ruffle-targeting region of -catenin, localizing it to amino acids 47-217, which overlap the IQGAP1 binding site. Knockdown by small interference RNA (siRNA) revealed IQGAP1-dependent membrane targeting of -catenin, APC, and N-cadherin. Transient overexpression of IQGAP1 or N-cadherin increased -catenin at membrane ruffles. IQGAP1/APC regulates cell migration, and using a wound healing assay we demonstrate that siRNA-mediated loss of -catenin also caused a modest reduction in the rate of cell migration. More significantly, we discovered that -catenin is internalized by Arf6-dependent macropinocytosis near sites of membrane ruffling. The -catenin macropinosomes co-stained for APC, N-cadherin, and to a lesser extent IQGAP1, and internalization of each binding partner was abrogated by siRNA-dependent knockdown of -catenin. In addition, -catenin macropinosomes co-localized with the lysosomal marker, lysosome associated membrane protein 1, consistent with their recycling by the late endosomal machinery. Our findings expand on current knowledge of -catenin function. We propose that in motile cells -catenin is recruited by IQGAP1 and N-cadherin to active membrane ruffles, wherein -catenin mediates the internalization and possible recycling of the membrane-associated proteins N-cadherin and APC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
-Catenin is a multifunctional protein with roles in cell-cell adhesion and as a mediator of nuclear signaling in the Wnt pathway and in various types of cancer (1). -Catenin is involved in cell adhesion through its interaction with E-cadherin at cell-cell junctions (2), and as a transcriptional activator it binds/activates the lymphoid enhancing factor-1/T-cell factor transcription factors to induce genes involved in cell transformation (3). Cytoplasmic -catenin is normally subject to strict regulation by the APC²/axin/glycogen synthase kinase-3 degradation complex, which promotes -catenin turnover through the proteasome (1). Disruption of -catenin degradation results in increased -catenin protein levels and an altered distribution of -catenin, which shifts from membrane and cytoplasm to the nucleus, culminating in changes typical of the transformed cellular phenotype including enhanced cell migration and increased proliferation. Despite an increase in our knowledge concerning -catenin subcellular distribution, nuclear transport, and turnover (3, 4), our understanding of how membrane-associated -catenin is internalized and recycled is rather poor.

Membrane-localized -catenin is mostly bound to E-cadherin at adherens junctions, structures that mediate cell-cell adhesion and maintain epithelial cell contact, polarity, and communication (5). The adherens junctions are positioned at the basolateral membrane, and their formation is dependent on the homophilic binding of the transmembrane protein E-cadherin. -Catenin together with -catenin tethers adherens junctions to the actin cytoskeleton (2, 6). The formation and disassembly of adherens junctions is a dynamic process linked to tissue remodeling and tumor cell invasion (7). The loss of adherens junctions can result in disruption of apical-basal polarity and loss of the epithelial phenotype and is a contributing factor to the so-called epithelial-to-mesenchymal transition, a shift in cellular phenotype associated with the progression of epithelial-derived cancers (8). The loss of -catenin and E-cadherin from the cell surface is emerging as a key event in this process, especially in breast and colon carcinomas where E-cadherin down-regulation occurs through multiple mechanisms (7-9). In addition to the reduced anchorage of cells caused by loss of E-cadherin, they can also acquire a more migratory phenotype through the release of -catenin from the membrane and its translocation to the nucleus, where it activates genes involved in cell transformation and migration (1, 3).

More recently, a second membrane localization of cellular -catenin was reported. Etienne-Manneville et al. (10) showed that -catenin was detectable at the leading edge of migrating rat astrocytes; however, this localization pattern has not yet been confirmed or characterized. Although it is possible this atypical membrane staining pattern reflected artifactual antibody staining, it is equally plausible that -catenin does localize to lamellipodia and active ruffling membrane in mesenchymal cells or fibroblasts. The appearance of the -catenin binding partner, APC, was recently reported at such dynamic membrane structures (11), and the membrane association of APC was functionally linked to cell migration. In this study we performed a series of experiments that provide compelling evidence for the localization of -catenin at membrane ruffles. We further show that membrane-associated -catenin correlates with a modest contribution to cell migration and that this unexpected localization pattern is important for the internalization of -catenin and specific binding partners through the process of macropinocytosis. We identified IQGAP1, an important regulator of actin dynamics and cell migration (11, 12), as a key regulator of -catenin at the membrane.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture, Reagents, and Transfection—The following cell lines were used: non-tumor derived epithelial cell lines (canine kidney epithelial cells), HEK-293T (immortalized human embryonic kidney cells), and MCF10A (breast epithelial cell line); breast tumor epithelial cell lines T47D, MCF-7, and MDA.MB.231; mouse fibroblast cell line NIH 3T3. All cell lines were confirmed mycoplasma-free and were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (penicillin and streptomycin) at 37 °C in 5% CO₂ humidified atmosphere. For transfection cells were grown on glass coverslips in 8-well dishes (Nunc) and 12 h post-seeding were transfected with 2 µg of DNA or 3 µg of siRNA per ml of media using Lipofectamine 2000 (Invitrogen); the lipid-DNA mix was left on cells for 6 h before replacing the medium and processing cells 40 h later. Cells were processed for microscopy or wound healing assay. The effect of ARF6 was studied by treating the cells with 30 µM Myr-Arf6 peptide (Calbiochem) for 1 h.

Plasmids—The following plasmids were transfected into NIH 3T3 cells: pEGFP-Rac1 and pEGFP-RhoA constructs were gifts from Dr. Mark Philips (13); human pEGFP-E-cadherin was a gift from Dr. Alpha Yap; murine pEGFP-N-cadherin was a gift from Dr. C. Gauthier-Rouvière; human pASEF-wt and pASEF-ca were gifts from Prof. Tetsu Akiyama (14, 15); human pEGFP-IQGAP-wt and -ct (amino acids 1503-1657) were gifts from Dr. Kozo Kaibuchi (11). Human -catenin-wt-FLAG plasmid and -catenin-218-467-FLAG plasmid were gifts from Dr. Eric Fearon (16) and were used as templates to construct a set of -catenin-GFP vectors in the expression plasmid pEGFP-N1 (Clontech). The -catenin cDNA insert sequences were PCR-amplified using forward and reverse primers (see Table 1) containing KpnI site (in forward primer) and BamH1 (in reverse primer) restriction sites and cloned in-frame into pEGFP-N1. -Catenin-wt-FLAG plasmid was used as template for all constructs except -catenin-218-467-GFP, where -catenin-218-467-FLAG was used as the template. All clones were confirmed by sequencing.

View this table: [in this window] [in a new window]   TABLE 1 Primers used for cloning -catenin-GFP plasmids

Deconvolution Analysis of Microscope Images—For high resolution imaging of cells, an inverted Zeiss Axiovert 200M microscope was used to capture 21 sections of cell samples, each section of 0.35 µm, a z axis range that spanned the apical to basal part of the cell. Images were resolved by iterative deconvolution using the Zeiss software inverse filter algorithm. The deconvolved images selected for presentation in this study correspond to sections 10-15 and represent a mid-plane transsection of the cell.

Dextran Labeling Experiment—For uptake of dextran-FITC (71 kDa; Sigma) or dextran-TRITC (76 kDa; Sigma), cells were transfected with ASEF-ca plasmid. After 30 h cells were incubated with 4 mg/ml dextran for 15 min to 1 h at 37 °C in serum-free media followed by 3 washes with fresh media and PBS (17). Cells were then fixed with 3.7% formalin, stained with -catenin monoclonal antibody or M-APC polyclonal antibody, and analyzed by Zeiss deconvolution microscopy as described above.

Western Blot Analysis—Cells were resuspended in protein extraction buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, supplemented with protease inhibitor mix (Roche Diagnostics) and shock-frozen in liquid nitrogen. After a quick thaw at 37 °C, cells were refrozen in liquid nitrogen, thawed on ice for 20 min, and cleared of insoluble components by centrifugation at 13,000 rpm at 4 °C for 15 min. The supernatant containing total protein was quantified using the Bio-Rad protein assay. Cell extracts were then denatured at 95 °C for 5 min in sample buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 0.01% bromphenol blue, 10% -mercaptoethanol, 5% SDS), and 40 µg of proteins were separated on a 7.5% SDS-poly-acrylamide gel and transferred onto nitrocellulose membranes (Millipore). Membranes were treated in blocking solution (5% dry milk in PBS containing 0.2% Tween 20) and incubated with primary antibody at room temperature for 2 h followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:5000; Sigma) for 1 h at room temperature. Proteins were visualized by ECL (Amersham Biosciences). Prestained broad range molecular weight marker (Bio-Rad) was used as the molecular size standard.

RNA Interference—Double-stranded 21-mer RNA oligonucleotides homologous to sequences in mouse IQGAP1 or -catenin (see Table 2) were purchased as purified duplexes (Qiagen-Xeragon Inc). Cells at medium density were transfected with 3 µg of RNA duplexes in 1 ml of Dulbecco's modified Eagle's medium using Lipofectamine for 6 h and either harvested 48 h post-transfection for image analysis or analyzed by time-lapse microscopy.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
-Catenin Is Localized at Membrane Ruffles in Migrating Cells—Our laboratory has an ongoing interest in the subcellular localization and intracellular trafficking of -catenin (18-20). The steady-state localization of -catenin in the nucleus and at cell-cell junctions is well documented (1). -Catenin has been reported at cell membrane lamellipodia (10), and we have detected -catenin at lamellipodia, filopodia, and short membrane ruffles; however, such localization patterns remain poorly characterized. We tested for non-adherens junction membrane staining of -catenin in a panel of cell lines using the wound healing assay, which permits analysis of actively migrating cells. Confluent monolayers of different epithelial cell lines were scratched and allowed to heal for 6 h then immunostained with antibody to -catenin and with fluorescent-tagged phalloidin to detect actin (Fig. 1A and supplemental Fig. S1). In canine kidney epithelial cells, visible -catenin was primarily restricted to cell-cell junctions, consistent with the fact that these cells form extremely tight and stable cell-cell junctions and are not highly motile. In the breast cancer epithelial cell lines T47D and MCF-7, -catenin was detected both at cell-cell junctions and also at membrane ruffles in migrating cells at the wound edge. The staining of -catenin was more biased toward actin-positive membrane ruffles in NIH 3T3 fibroblasts (Fig. 1B). To confirm specificity of the membrane staining of -catenin, we treated NIH 3T3 cells with siRNA to silence -catenin and observed a consequent loss of staining from cell-cell junctions, the nucleus and cytoplasm, and also from the membrane ruffles. In contrast, a control siRNA had no effect on -catenin staining patterns, and the actin staining pattern was unaffected, indicating that membrane ruffles are a genuine site of -catenin accumulation.

To further test the localization of -catenin at membrane ruffles, we transfected NIH 3T3 cells with plasmids encoding different factors known to modulate actin dynamics and induce membrane ruffling; a constitutively active form of Rac1 GTPase (13, 21), the Rac1 GTPase exchange factor, ASEF (14) and a constitutively active form of ASEF (15), and stained cells for actin and -catenin. Both Rac1 and ASEF increased the proportion of transfected cells with actin-positive membrane ruffles by 1.5-2.5-fold, and a corresponding increase in cellular -catenin was detected at these membrane structures (Fig. 1C). The strongest effect was elicited by constitutively active ASEF (ASEF-ca), which induced a 3-fold increase in actin-positive ruffles and a 2.5-fold increase in -catenin-positive ruffles. Thus, the localization of -catenin at membrane ruffles is associated with induction of these migratory structures.

View larger version (59K): [in this window] [in a new window]   FIGURE 1. -Catenin localizes at membrane ruffles. A, different epithelial cell lines were grown to confluence and wounded. Cells were allowed to heal for 6 h then immunostained with-catenin monoclonal antibody and Texas Red secondary conjugate to detect membrane staining by fluorescence microscopy. MDCK, Madin-Darby canine kidney cells. B, NIH 3T3 cells were transfected with control (top panel) and -catenin (bottom panel) siRNA. 48 h later confluent cells were wounded, and the cells were allowed to heal for 6 h before fixation and staining for -catenin (as above) and actin (fluorescein-labeled phalloidin). The % of actin ruffle-positive cells displaying -catenin positive membrane ruffles after each siRNA treatment is shown in the graph. C, -catenin localizes to ASEF-induced membrane ruffles. NIH 3T3 cells were transfected with HA-tagged ASEF (wild-type or constitutively active forms), GFP, or GFP-Rac1L61 expression plasmids. At 30 h post-transfection, cells were immunostained with anti-HA antibody (for ASEF-transfected cells) and Alexa Fluor-488-conjugated secondary antibody, with -catenin and Texas Red-conjugated secondary antibody or TRITC-conjugated phalloidin. Cell images are shown in the left panel, and quantification of ASEF-ca-transfected cells for localization of -catenin or actin at membrane ruffles is shown at the right. The values are the mean ± S.E. from 2-3 experiments, scoring at least 100 cells per slide.

Transiently Expressed N-cadherin and IQGAP1 Increase -Catenin at Membrane Ruffles—In contrast to APC, which regulates -catenin turnover (1, 3), N-cadherin and IQGAP1 independently locate at free membrane and are potential regulators of -catenin targeting to membrane ruffles. Indeed, when transiently expressed in cells, GFP-tagged forms of both N-cadherin and IQGAP1 stimulated -catenin staining at ruffles relative to the effect seen with E-cadherin (Fig. 2, B and C) or a GFP control (Fig. 1 and supplemental Fig. S4).

View larger version (35K): [in this window] [in a new window]   FIGURE 2. -Catenin co-localizes with IQGAP1, APC, and N-cadherin at membrane ruffles. A, NIH 3T3 cells were transfected with pASEF-ca (but not stained) and immunostained for endogenous -catenin and its partner proteins IQGAP1, APC, and N-cadherin. Cells were analyzed by fluorescence microscopy as shown. The % cells showing co-localization with partner proteins is shown at right. B, NIH 3T3 cells were transfected with plasmids encoding GFP-tagged E-cadherin, N-cadherin (50), and IQGAP1 (11) and stained for -catenin. C, the % cells with -catenin at ruffles was scored and graphed as the mean ± S.E. Data are from two experiments scoring 200 cells per sample.

View larger version (48K): [in this window] [in a new window]   FIGURE 3. -Catenin localization at membrane ruffles requires amino acids 47-217. A, schematic diagram of -catenin constructs compared for subcellular localization. The IQGAP1 and APC/N-cadherin binding domains of -catenin are indicated. B, NIH 3T3 cells were co-transfected with pASEF-ca-HA and different -catenin-GFP constructs. After 30 h cells were immunostained with anti-HA antibody and Texas Red conjugate secondary antibody. 200 co-transfected cells were scored per slide for the localization of ASEF and -catenin-GFP at membrane ruffles. Values shown are mean ± S.E. from two experiments. C, representative microscopy images of membrane staining for transiently expressed ASEF and -catenin-GFP fusions.

Interestingly, an internal deletion of the arm domain (amino acids 218-467) that prevents binding to APC and N-cadherin did not impair -catenin localization at ruffles. Thus, although N-cadherin may boost -catenin at the free membrane, it is not essential. These results indicate that residues 47-217 are critical for localization of -catenin at membrane ruffles. This sequence contains the binding site for IQGAP1 (amino acids 123-183; Ref. 24), implicating IQGAP1 as a primary anchor or regulator of -catenin at membrane ruffles.

View larger version (36K): [in this window] [in a new window]   FIGURE 4. IQGAP1 contributes to the localization of APC, -catenin, and N-cadherin at membrane ruffles. A, NIH 3T3 cells were transfected with control or IQGAP1 siRNA as detailed under "Materials and Methods." After 48 h total protein was extracted, and 40-µg samples were analyzed by Western blot using anti-IQGAP1 or actin monoclonal antibody. B, NIH 3T3 cells were transfected with control or IQGAP1 siRNA and immunostained with anti-IQGAP1 antibody and Texas Red-conjugated secondary antibody. The partner protein, APC, -catenin, or N-cadherin was immunostained with primary antibody and Alexa Fluor-488-conjugated antibody (images are shown here for -catenin staining). C, cells were scored for the presence of IQGAP1 (control siRNA cells) or absence of IQGAP1 (IQGAP1 siRNA) at membrane ruffles and the proportion of cells in each case that displayed membrane ruffle staining of the three partners. A total of 200-300 cells were scored for each sample, and values shown are the mean ± S.E. from two experiments.

Silencing of -Catenin Reduces APC and N-cadherin, but Not IQGAP1, at Membrane Ruffles—NIH 3T3 cells were treated with -catenin siRNA, resulting in >90% specific loss of -catenin protein (Fig. 5A). These cells were then stained and assessed for protein localization at membrane ruffles as described above for the IQGAP1 knockdown (Fig. 4). We found that -catenin silencing had no effect on IQGAP1 at membrane ruffles, whereas it abolished staining of APC and N-cadherin in 40-50% of cells (Fig. 5B). These data indicate that IQGAP1 acts upstream of -catenin. The knockdown of either IQGAP1 or -catenin reduced APC and N-cadherin at ruffles, suggesting that IQGAP1 may co-operate with -catenin to enlist membrane recruitment of specific partners.

The Loss of -Catenin Retards Cell Migration in a Wound Healing Assay—We investigated the possibility that -catenin contributes to cell migration in a wound healing assay. NIH 3T3 cells were transfected with control or -catenin siRNA, and 72 h post-transfection cells were wounded, and cells at the wound edge were imaged by time-lapse microscopy. A Zeiss Axiovert 200M live cell imaging system was used to track 8 different wound regions every hour over a 15-h period. For each region three points were selected for post-image analysis, and the distance migrated by cells every hour was measured using Zeiss Axiovision software. After the wound-heal experiment, cells were fixed and stained for -catenin and actin to confirm the siRNA-mediated loss of -catenin. Using this approach we observed a slight retardation of wound healing in -catenin siRNA-transfected cells relative to control siRNA-treated cells (Fig. 6). The most consistent effect was observed within the first 6 h, suggesting that loss of -catenin slows the response to wounding and the initiation of cell movement. It has previously been suggested that -catenin might contribute to cell migration or invasion through its nuclear transcriptional activity; however, a transcription-independent link between -catenin and cell migration has also been suggested (23). We note that membrane ruffle-associated -catenin forms a significant fraction of the -catenin pool in migrating 3T3 cells, leading us to propose that the membrane-associated fraction of -catenin may contribute to cell migration in this assay. However, because the change in cell migration activity was relatively modest, it was evident that -catenin might in fact be recruited to membrane ruffles for another reason.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. siRNA silencing of -catenin reduces APC and N-cadherin at membrane ruffles. A, NIH 3T3 cells were transfected with control or -catenin siRNA, and efficacy of the -catenin knock-down was confirmed by Western blot as shown. B, cells transfected with control or -catenin siRNA were co-stained for IQGAP1, APC, or N-cadherin and scored for membrane ruffle staining (as shown in legend to Fig. 4). A total of 200-300 cells were scored for each sample, and values shown are the mean ± S.E. from two experiments.

View larger version (60K): [in this window] [in a new window]   FIGURE 6. Silencing of -catenin slows cell migration. NIH 3T3 cells were treated with control or -catenin siRNA (as in Fig. 5). A, cells were allowed to reach confluence (72 h), then wounded and analyzed over a 15-h period. Migration of cells at the wound edge was recorded by time-lapse microscopy every hour using a Zeiss Axiovision 200M system (see "Materials and Methods"). The distance migrated by cells was measured at 24 distinct positions along the wound. The average distance migrated was plotted against time, with values shown representing the mean ± S.E. from two different experiments. B, representative phase contrast images of migrating cells at different times post wounding. Cells moved in the direction of arrows.

To determine whether the particles we observed are macropinosomes, we transfected NIH 3T3 cells with pASEF-ca and 48 h later incubated these cells with the fluid phase pinocytic marker, TRITC-conjugated dextran, then fixed the cells after 1 h and immunostained for -catenin. Deconvolution analysis of microscopy cell images was used to show that surface-labeled dextran was internalized from the membrane into the cytoplasm of the cells and that the internalized dextran co-localized with -catenin in the cytoplasmic particles (Fig. 7C). This is strong evidence that the -catenin is indeed being internalized by macropinocytosis at the site of membrane ruffles. We also observed co-localization between dextran and APC in macropinosome particles (Fig. 7C).

We further tested some breast tumor epithelial cell lines for the presence of -catenin macropinosomes. -Catenin was frequently observed at membrane ruffles and in macropinosomes. -Catenin macropinosomes were seen in 11-13% of cells in the cell lines T47D, MCF7, and MDA.MB.2321 (Fig. 7A and supplemental Fig. S5).

-Catenin Macropinocytosis Is Regulated by Arf6 GTPase—The Arf6 GTPase has been implicated in endocytosis and actin remodeling (27), E-cadherin internalization (28, 29), and macropinocytosis (17). Arf6 must cycle between GTP and GDP-bound forms to function properly, and the overexpression of either constitutively active or dominant negative mutants can inhibit its activity by preventing its GTP/GDP binding cycle (27). Palacois et al. (28) reported that GTP-bound Arf6 can induce disassembly of adherens junctions and the internalization/endocytosis of E-cadherin. They also observed Arf6-mediated ruffling of the lateral membrane, which is implicated in its role in facilitating macropinocytosis (30). We hypothesized that a block in the Arf6 GTPase cycle might affect macropinocytosis of -catenin in NIH 3T3 cells. To address this, we tested a myristoylated synthetic peptide corresponding to the N-terminal region of Arf6, which was previously reported to block Arf6 activities in various cellular processes including the aluminum fluoride induced activation of phospholipase D and release of -arrestin protein (31, 32). Cells were transfected with the ASEF-ca plasmid to induce ruffling, and 30 h later cells were treated with the myrArf6 (2-13) peptide and stained with antibodies against Arf6 and -catenin (Fig. 7D). Microscopic analysis revealed that Arf6 peptide treatment caused a 67% reduction in -catenin staining at membrane ruffles and in -catenin positive macropinosomes relative to vehicle-treated cells. A similar result was observed for human T47D breast cancer cells (see supplemental Fig. S6). We conclude that macropinocytosis of -catenin from membrane ruffles is regulated by Arf6 GTPase.

View larger version (59K): [in this window] [in a new window]   FIGURE 7. -Catenin undergoes macropinocytosis at membrane ruffles. A, immunostaining of NIH 3T3 cells and T47D breast cancer cells revealed the frequent detection of -catenin-positive particles in the cytoplasm, often near the site of membrane ruffles. B, to determine whether the macropinosome-like particles correlated with membrane ruffling, NIH 3T3 cells were transfected with pEGFP or pASEF-ca, and the transfected cells were analyzed for both -catenin particles and membrane ruffle formation. Data shown are the mean ± S.E. from three experiments. Number cells scored shown in parentheses. C, to test if the -catenin particles co-stain with the uptake of fluid-phase fluorescent dextran, ASEF-transfected NIH 3T3 cells were incubated with dextran conjugated TRITC for 1 h and then stained with -catenin or M-APC antibodies and Alexa Fluor-488-conjugated secondary antibody. In each case 30 different z-stack images were captured at 0.3-µm step size collected on a Zeiss Axiovert 200M microscope and deconvolved using Zeiss software. This analysis confirmed co-localization of -catenin and APC with dextran in cytoplasmic macropinosomes. D, to test for regulation by Arf-6 GTPase, ASEF-transfected NIH 3T3 cells were treated with 33 µM myr-Arf6-peptide (2-13) or vehicle alone (Me2SO (DMSO)) for 1 h, stained with -catenin antibody, and then scored by microscopy for -catenin at membrane ruffles or in macropinosomes. Data shown are the mean ± S.E. from three experiments.

IQGAP1 Regulates -Catenin Macropinocytosis—We scored for the presence of -catenin macropinosomes after knockdown of IQGAP1. An 70% reduction in cells with -catenin-positive macropinosomes was observed after IQGAP1 siRNA treatment compared with cells treated with control siRNA (Fig. 9A). This is consistent with the idea that loss of IQGAP1 reduces targeting of -catenin to membrane ruffles, indirectly affecting its ability to be internalized through macropinocytosis. To further show that IQGAP1 regulates -catenin internalization, we transiently expressed different GFP-fusion proteins in NIH 3T3 cells. Overexpressed GFP-IQGAP1 increased the formation of -catenin-positive macropinosomes, whereas expression of the C-terminal dominant negative sequence of IQGAP1 (IQGAP1-ct) reduced -catenin macropinocytosis (Fig. 9B). The overexpression of GFP-N-cadherin had little effect on -catenin macropinosomes.

-Catenin Regulates Macropinocytosis of APC, IQGAP1, and N-cadherin—Does -catenin itself contribute to macropinocytosis? To address this question, NIH 3T3 cells were treated with control siRNA or -catenin siRNA to assess the impact of -catenin silencing on macropinosome formation. The -catenin siRNA treatment reduced visible -catenin-positive macropinosomes in cells by 84% compared with cells treated with control siRNA (Fig. 9C). It is interesting to note that this knockdown impacted modestly on the overall macropinocytosis process, as staining for actin-positive macropinosome particles was reduced by 28% compared with control. More striking was the specific effect on the -catenin binding partners APC, N-cadherin, and IQGAP1; cells with visible staining of these partners at macropinosomes were reduced by 80-90% after -catenin siRNA treatment (Fig. 9C). In contrast, knockdown of -catenin had little effect on IQGAP1 staining at membrane ruffles (Fig. 5). These data imply a possible chaperone/scaffolding role for -catenin in the IQGAP1-dependent internalization of N-cadherin and APC.

View larger version (40K): [in this window] [in a new window]   FIGURE 8. -Catenin co-localizes with actin, APC, IQGAP1, and N-cadherin at macropinosomes. NIH 3T3 cells were transfected with ASEF-ca and stained with antibodies to -catenin and the different partner proteins. Co-staining at macropinosomes was scored (see arrows in the cell images) and quantified. Values shown in graph are mean ± S.E. from two experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
-Catenin is a mediator of the wnt signaling pathway and is generally regarded to have two major sites of action in the cell; that is, at membrane adherens junctions and in the nucleus (1, 38). In this study we show that -catenin also accumulates at membrane ruffles in actively migrating cells and identified the -catenin binding partner, IQGAP1, as a key regulator of this process. IQGAP1 is known to modulate cell migration (11, 12), and the specific silencing of -catenin likewise displayed a reproducible, albeit modest, influence on the rate of cell migration. The main consequence of -catenin recruitment to membrane ruffles was its rapid internalization through ruffle-induced macropinocytosis. This macropinocytic pathway was regulated by Arf6 and IQGAP1. We propose, based on data reported in this study, that -catenin may act as a linker or scaffold for the IQGAP1-dependent internalization and recycling of specific binding partners including APC and N-cadherin (our findings are summarized in Fig. 10B). The concept of -catenin acting as a chaperone or scaffold for the internalization of membrane-associated proteins provides a new context for its targeting to these dynamic membrane regions.

-Catenin at Membrane Ruffles—-Catenin is usually described as an integral component of cell adherens junctions and plays a key role in the nucleus as a transcriptional regulator and activator of cell transformation in several cancers (3, 38). In addition to cell-cell membrane junctions, -catenin has been detected at other membrane locations including microtubule-dependent membrane protrusions (39) and actin-dependent membrane ruffles (10). We recently demonstrated a role for -catenin in regulating APC localization at membrane protrusions (20). Here we addressed the question of why -catenin localized to membrane ruffles. First, we tested its involvement in cell migration by imaging live cells after wounding and tracking the rate of wound closure in NIH 3T3 fibroblasts transfected with control siRNA or -catenin siRNA. The silencing of -catenin delayed the initiation process of wound closure for up to 4-6 h but thereafter did not affect the rate of cell migration significantly. To our knowledge this is the first study to test the impact of -catenin knock-down on fibroblast cell migration. Wong and Gumbiner (23) silenced -catenin in breast tumor epithelial cells and observed a reduction in in vitro invasive ability, but the impact on cell migration was not appraised directly. The knockdown of -catenin in endothelial cells had the opposite effect and increased entry of cells into the wound (40). Previous work with fibroblasts has shown, however, that the overexpression of -catenin in fibroblasts can directly stimulate both in vitro cell migration and invasiveness (41). Because the overexpression of -catenin also correlates with increased transcriptional activity, it remains difficult to conclude whether -catenin influences cell movement directly by its membrane ruffle localization and/or indirectly through its nuclear transcription function.

View larger version (47K): [in this window] [in a new window]   FIGURE 9. IQGAP1 and -catenin regulate macropinocytosis of partner proteins. A, NIH 3T3 cells were transfected with control or IQGAP1 siRNA, and >200 cells were scored for the presence of -catenin or IQGAP1 at macropinosomes. B, cells were transfected with plasmids encoding GFP-fusions of N-cadherin or IQGAP1 (wild-type or ct mutant) and co-stained for -catenin. The proportion of transfected cells with -catenin-positive macropinosomes was scored and graphed. Data are the mean ± S.E. from two experiments. C, cells were transfected with control or -catenin siRNA, then stained for -catenin and partner proteins and processed for microscopy. Cells were scored randomly for the presence of -catenin or partner at macropinosomes. The data shown are the mean ± S.E. from three experiments. The loss of -catenin reduced staining of partners at macropinosomes.

IQGAP1-dependent Macropinocytosis of -Catenin—Membrane ruffling is also important for solute uptake by macropinocytosis. A key finding of this study is that IQGAP1-dependent recruitment of -catenin to membrane ruffles facilitates the macropinocytosis of -catenin and specific binding partners. Ruffle-mediated macropinocytosis provides a mechanism for internalizing large sections of lateral plasma membrane in an actin-dependent but clathrin-independent manner (25, 26, 43). We stimulated membrane ruffle formation by overexpressing the ASEF GTPase exchange factor and observed an increase in -catenin macropinosome formation. Consistent with other studies (28, 29), this process was regulated by Arf6 GTPase. Moreover, the knockdown or competitive inhibition of IQGAP1 diminished -catenin macropinocytosis. It is not yet known whether IQGAP1 regulates -catenin endocytosis in fibroblast cells.

View larger version (47K): [in this window] [in a new window]   FIGURE 10. -Catenin macropinosomes co-localize with the lysosomal marker, LAMP1. A, NIH 3T3 cells were transfected with ASEF-ca plasmid and stained with antibodies against -catenin and various endocytic markers. Co-localization was first assessed by standard fluorescence microscopy, and then a minimum of 11 cell images for each sample were captured as z-stacks and analyzed by deconvolution software to determine co-localization within the cell. The only antibody to co-stain -catenin-positive macropinosomes was LAMP1, and 38% of cells analyzed showed co-staining within the cytoplasm. This indicates that the macropinosomes are targeted to the lysosome. B, diagram summarizing the data from siRNA and transient expression assays in this study. Proteins (effectors) that stimulated the localization of specific proteins to membrane ruffles or to macropinosomes are indicated.

The cadherins are a family of transmembrane proteins that display variable cellular distribution and function in different cell types. E-cadherin is highly expressed in epithelial cells and promotes cell-cell adhesion (45, 46), whereas N-cadherin is implicated in promoting cell motility and invasion (23, 47, 48). Paterson et al. (29) previously speculated that E-cadherin might be internalized by active macropinocytosis in MCF-7 breast cancer cells, but this was not experimentally verified. In this study N-cadherin co-localized strongly with -catenin in NIH 3T3 cells at both adhering and ruffling membrane, in the cytoplasm in macropinosomes, and to a lesser degree in the nucleus (data not shown). It is possible that the two form an intracellular complex important for regulating their cellular targeting, at least for their co-location at membrane ruffles and their internalization by macropinocytosis as demonstrated here. Indeed, the overexpression of N-cadherin contributed to -catenin recruitment at membrane ruffles, and knockdown of -catenin reduced N-cadherin at ruffles, predicting that the two proteins may form a mutual complex with IQGAP1 at actin filaments just below the membrane surface. Our data implicate -catenin as an intermediate in the IQGAP1-regulated internalization of APC and N-cadherin by macropinocytosis.

Although Rac1 is essential for membrane ruffle formation (49), other small GTPases such as Rah/Rab34 have been implicated in the generation of macropinosomes from ruffled membrane (33). We detected actin in most, but not all -catenin-positive macropinosomes, and this is consistent with the notion that actin coats the macropinosome but comes off as the vesicles move toward the center of the cell (33). There is very little known in regard to internalization and recycling of IQGAP1, N-cadherin, or APC, and it is tempting to speculate that one reason -catenin moves to this dynamic membrane region is to help facilitate the internalization of these factors. It is yet to be determined if -catenin moves to ruffles from a defined pool, although its targeting does not seem to be regulated by phosphorylation (Fig. 3 and data not shown). Further study of these questions should help elucidate new roles for membrane associated -catenin.

	FOOTNOTES

 
^* This work was generously supported by grants from the New South Wales Cancer Council, the Australian Research Council, and the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1—S6.

¹ A Senior Research Fellow of the National Health and Medical Research Council of Australia. To whom correspondence should be addressed: Westmead Millennium Institute, Darcy Rd., P. O. Box 412, Westmead, NSW 2145, Australia. Tel.: 61-2-9845-9057; Fax: 61-2-9845-9102; E-mail: beric_henderson{at}wmi.usyd.edu.au.

² The abbreviations used are: APC, adenomatous polyposis coli; ASEF, APC-stimulated exchange factor; GFP, green fluorescent protein; IQGAP1, IQ-domain GTPase-activating protein 1; LAMP1, lysosome-associated membrane protein 1; PBS, phosphate-buffered saline; siRNA, small interference RNA; ca, constitutively active; wt, wild type; ct, C terminus; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate.

	ACKNOWLEDGMENTS

 
We are extremely grateful for the generosity of Drs. Julie Donaldson, Jenny Stow, Phil Robinson, Rob Parton, Mark Philips, Cécile Gauthier-Rouvière, and Alpha Yap in supplying plasmids and antibody reagents. We thank Dr. Alpha Yap for early discussions about the macropinocytosis pathway and Michael Johnson for technical assistance.

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