Characterization of E-cadherin Endocytosis in Isolated MCF-7 and Chinese Hamster Ovary Cells

THE INITIAL FATE OF UNBOUND E-CADHERIN*

Andrew D. Paterson {ddagger} §, Robert G. Parton {ddagger} § ¶, Charles Ferguson § ¶, Jennifer L. Stow § || and Alpha S. Yap {ddagger} § **

From the {ddagger}School for Biomedical Science, §Institute for Molecular Bioscience, Center for Microscopy and Microanalysis, ||School of Molecular and Microbial Science, The University of Queensland, St. Lucia, Brisbane, Australia 4072

Received for publication, January 6, 2003 , and in revised form, March 18, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The endocytosis of E-cadherin has recently emerged as an important determinant of cadherin function with the potential to participate in remodeling adhesive contacts. In this study we focused on the initial fate of E-cadherin when it predominantly exists free on the cell surface prior to adhesive binding or incorporation into junctions. Surface-labeling techniques were used to define the endocytic itinerary of E-cadherin in MCF-7 cells and in Chinese hamster ovary cells stably expressing human E-cadherin. We found that in this experimental system E-cadherin entered a transferrin-negative compartment before transport to the early endosomal compartment, where it merged with classical clathrin-mediated uptake pathways. E-cadherin endocytosis was inhibited by mutant dynamin, but not by an Eps15 mutant that effectively blocked transferrin internalization. Furthermore, sustained signaling by the ARF6 GTPase appeared to trap endocytosed E-cadherin in large peripheral structures. We conclude that in isolated cells unbound E-cadherin on the cell surface is predominantly endocytosed by a clathrin-independent pathway resembling macropinocytotic internalization, which then fuses with the early endosomal system. Taken with earlier reports, this suggests the possibility that multiple pathways exist for E-cadherin entry into cells that are likely to reflect cell context and regulation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cadherin cell adhesion molecules support cell-cell adhesion and recognition in most tissues of the body (1, 2, 3). E-cadherin is the predominant cadherin found in epithelia, where it localizes to specialized adherens junctions between cells, as well as being found more generally in the lateral plasma membrane where cells come into contact with one another. Like other classical cadherins, E-cadherin is a key determinant of tissue organization both during development and in post-embryonic life (2). Of note, E-cadherin contributes to morphogenetic patterning in the early embryo and during organogenesis, where it participates in dynamic adhesive contacts that may be different from those found in classical adherens junctions (4, 5, 6, 7). Conversely, loss of E-cadherin function is an important step in epithelial tumor progression to invasiveness and metastasis (8).

In many instances, the morphogenetic influence of E-cadherin and other classical cadherins is critically influenced by changes in the cell surface expression of these molecules. For example, quantitative as well as qualitative changes in cadherin expression determine cell sorting both in cell culture models (9) as well as in the early embryo (10). Indeed, both quantitative and qualitative changes in cadherin expression are subject to developmental regulation (5, 6). Down-regulation of E-cadherin, by either transcriptional silencing or protein degradation, also occurs during epithelial-mesenchymal transitions during organogenesis and tumorigenesis (11). However, many very dynamic morphogenetic events entail regulated changes in cadherin activity that are not attributable to alterations in either the overall level of protein expression, or to changes in the repertoire of cadherins expressed at the cell surface. This is exemplified by cell-upon-cell locomotion in the Drosophila ovary (7) and in Xenopus embryos (12), where cadherin adhesions provide the traction for cells to move upon one another, but must also be regulated and remodeled to allow translocation to occur.

Post-Golgi trafficking of E-cadherin has recently emerged as an alternative mechanism to support dynamic changes in cadherin activity. We found that polarized Madin-Darby canine kidney epithelial cell monolayers display some internalization of cell surface E-cadherin (13). This entered an early endosomal compartment and was then recycled back to the cell surface relatively quickly (time scale of 15–30 min) without undergoing degradation. Importantly, this post-Golgi trafficking pathway contributed to the remodeling of cell-cell contacts: blocking the recycling of internalized E-cadherin back to the cell surface significantly perturbed the ability of cells to re-establish cadherin-based cell-cell contacts that had been disrupted by manipulation of extracellular calcium. We postulated that recycling of E-cadherin might provide a mechanism for cadherinbased contacts to be rapidly remodeled during morphogenesis and tissue reorganization. Indeed, inhibition of dynamin activity blocked morphogenesis in the early Xenopus embryo, closely correlated with disturbances in C-cadherin trafficking (14).

In addition to being constitutively active in polarized monolayers, E-cadherin internalization and trafficking is also subject to cellular regulation. Growth factors that induce epithelial cell scattering appear to promote E-cadherin internalization (15, 16, 17). A variety of intracellular signaling pathways, which include the Rac (18) and ARF6 GTPases (17) and protein kinase C (19) can also influence cadherin trafficking. In the case of protein kinase C, this affects both internalization of E-cadherin from the membrane as well as its recycling back to the cell surface (19). Finally, E-cadherin recycling may be regulated by the activity state of the cadherin itself: cell-cell contact appeared to withdraw cadherin from the recycling pathway, whereas disruption of cell-cell contacts promoted the internalization of E-cadherin (13).

Taken together, these data indicate that the initial step of cadherin endocytosis is likely to be a key point in cadherin trafficking. This would be consistent with evidence that the endocytosis of a wide range of cell surface molecules is subject to stringent cellular control (20, 21). The precise mechanism for E-cadherin endocytosis is, however, incompletely understood. A wide repertoire of mechanisms exist for cell surface molecules to be internalized, including both classic clathrin-mediated endocytosis (22) and a variety of clathrin-independent pathways (21). Interestingly, recent studies have presented evidence that E-cadherin might be internalized by either clathrin-dependent (13, 23) or clathrin-independent mechanisms (18), suggesting that multiple pathways may also exist for E-cadherin internalization. Many of these studies were conducted in fully confluent cultured cell monolayers, which more closely resemble the situation in mature epithelia with stable adhesive contacts, rather than the dynamic adhesive contacts formed during tissue reorganization. In the latter case, turnover of adhesion is likely to be rapid, requiring mechanisms for unbound cadherin molecules to be cleared away from regions of the cell surface undergoing remodeling (24). In this study we therefore chose to focus on characterizing the initial fate of unbound E-cadherin on the cell surface of isolated MCF-7 and CHO1 cells. Combining surface labeling assays with light and immunoelectron microscopy our experiments identify a clathrin-independent pathway for E-cadherin internalization that is subject to regulation by ARF6.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—MCF-7 cells were grown in Dulbecco's modified Eagle's medium (BioWhittaker), CHO cells in Hams' F-12 (BioWhittaker), and all media were supplemented with 10% (v/v) fetal bovine serum, penicillin, streptomycin, and non-essential amino acids (Invitrogen). CHO cells stably expressing human E-cadherin were used as described previously (25, 26). Cells were incubated at 37 °C, with 5% CO2 in 95% air. Cells used for immunofluorescence were plated on glass coverslips. All transient transfections were carried out with LipofectAMINE Plus (Invitrogen) according to the manufacturer's instructions. Transfected cells were grown for 16–24 h before being used in experiments.

Antibodies—Primary antibodies used were: 1) mouse monoclonal antibody HECD-1 raised against the ectodomain of human E-cadherin (a kind gift from Prof. Masatoshi Takeichi; Kyoto University), 2) human anti-early endosomal antigen-1 (EEA-1; a gift from Dr. Ban Hock Toh, Monash University), 3) rabbit anti-Rab-7 and 4) rabbit anti-HA (both provided by Prof. David James, University of Queensland), 5) rabbit anti-cathepsin D (UBI), 6) rabbit anti-GFP (Molecular Probes), and 7) rabbit anti-{beta}-COP (a kind gift from Dr. Rohan Teasdale, University of Queensland). Secondary antibodies used were: 1) Alexa-488 conjugated goat anti-rabbit IgG (Molecular Probes), 2) Cy3-conjugated donkey antimouse IgG (Jackson Immunoresearch Laboratories), 3) Cy3-conjugated goat anti-mouse IgG F(ab')2 fragment-specific antibodies (Jackson Labs). F(ab') fragments of hECD-1 were generated using the Immunopure IgG1 F(ab') and F(ab')2 preparation kit (Pierce).

Internalization Assay—MCF-7 cultures were passaged 12 h before experiments, and on the day of the assay the cell medium was changed to Hanks' balanced salt solution supplemented with 5 mM Ca2+ and 50 mg/ml bovine serum albumin and cells were allowed to equilibrate for 1 h. Samples were then incubated for 1 h at 4 °C with HECD-1 F(ab') fragments or intact HECD-1 IgG diluted in Hanks' balanced salt solution. Coverslips were washed with ice-cold PBS to remove the unbound antibody and the media was replaced with Hanks' balanced salt solution prewarmed to 37 °C. After incubation at 37 °C for varying periods of time, cells were then washed with PBS and returned to 4 °C. The residual surface-bound antibody was removed by acid washing (0.5 M acetic acid, 0.5 M NaCl; 3x 5 min washes) (27). Cells were washed with PBS before fixation with 4% paraformaldehyde (in 10 mM PIPES, 2 mM MgCl, 2 mM KCl, 300 mM sucrose, and 2 mM EGTA) for 20 min at 4 °C.

Plasmids—The following plasmids were transiently transfected into either MCF-7 or CHO cells: pCB/Dynamin-1 S45N (a kind gift from Dr. Sandy Schmid (28), pEYFP-Cav3 (29), pEGFP-Eps-15/E{Delta}95/295 (kindly provided by Drs. Ben Nichols and Alexandre Benmerah (30, 31)), pXS/Arf-6, pXS/Arf-6 T27N, and pXS/Arf-6 (generous gifts from Dr. Julie Donaldson), pEGFP/Rab5 Q79L (32), and pEGFP/Rab 11 (33).

Immunofluorescence Microscopy—Unless otherwise stated, cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, then permeabilized in 0.25% Triton X-100/PBS. After fixation and permeabilization, samples were washed in PBS, then incubated in blocking buffer (5% (w/v) nonfat dried milk in PBS) for 1 h. Primary and secondary antibodies were diluted in blocking buffer and incubated serially for 60 min at room temperature. After final washing with PBS, samples were mounted in 1% NPG, 50% glycerol/PBS. Images were acquired with an Olympus AX70 microscope and Orca I digital camera (Hamamatsu) using Metamorph Imaging Software (version 4.01, Universal Imaging Corp.). Images were compiled using Adobe Photoshop 6.

Electron Microscopy—To characterize the surface distribution of E-cadherin, isolated MCF-7 cells were fixed with 0.5% glutaraldehyde, then incubated with HECD-1 IgG for 1 h, before sectioning and processing for electron microscopy as previously described (34). Surfacelabeled HECD-1 was detected using 5 nm anti-mouse gold. To quantitate the distribution of surface label, ~400 gold particles were counted from sections of 15 independent cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Itinerary of Surface-labeled E-cadherin Internalization—E-cadherin internalization was assayed with a surface labeling strategy that has been extensively used to characterize endocytosis of the transferrin receptor and many other cell surface proteins (35, 36). For these experiments we used MCF-7 cells, a well differentiated human breast cancer cell line that expresses endogenous E-cadherin. To maximize the rate of internalization, cells were passaged 12 h prior to experiments to obtain sparse, subconfluent cultures. This provided an optically amenable system where cell surface proteins were readily accessible for labeling. To label the surface pool of E-cadherin, cells were incubated at 4 °C with F(ab') fragments or whole IgG of monoclonal antibody HECD-1 directed against the ectodomain of human E-cadherin. After washing to remove unbound antibody, samples were then warmed to 37 °C to allow internalization of surface-labeled E-cadherin to resume. At various times after release of the 4 °C block any residual antibody on the cell surface was stripped by washing with a high-salt/acid buffer (27) and the samples were processed to detect the internalized label by indirect immunofluorescence microscopy.

As shown in Fig. 1A, immediately after labeling at 4 °C, HECD-1 F(ab') staining was found distributed throughout the free surface of cells as well as being concentrated in areas of cell-cell contact. High salt/acid washing immediately after labeling removed almost all staining (Fig. 1B), confirming efficient stripping of the surface-bound label. Within 5 min after warming cells to 37 °C, HECD-1 F(ab') could be detected in peripheral cytoplasmic vesicles that were resistant to surface stripping (Fig. 1C). Vesicular staining of internalized HECD-1 F(ab') increased progressively after release of the 4 °C block, becoming extensively distributed throughout the cytoplasm and in the perinuclear region after 30 min (Fig. 1, DF).



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FIG. 1.
Internalization of surface-labeled E-cadherin. Subconfluent MCF-7 cells were incubated with anti-E-cadherin (HECD-1) F(ab') fragments at 4 °C, then allowed to internalize the surface label at 37 °C for 0–60 min (BF). Samples were processed to identify the label by indirect immunofluorescence microscopy immediately after labeling (A; NW, not washed), or after washing with a high salt/acid solution to strip residual surface F(ab') (BF). Bar is 15 µm.

 

Comparison of E-cadherin Internalization with Clathrin-mediated Endocytosis—To begin to characterize the E-cadherin internalization pathway, we compared the localization of surface-labeled E-cadherin with that of transferrin, a well characterized marker of clathrin-mediated endocytosis (20, 36). Cells were surface-labeled with both HECD-1 and fluorescein-conjugated transferrin and their itineraries of internalization compared at various times after release of the 4 °C block. For these studies we used both whole HECD-1 IgG (Fig. 2, AD) and HECD-1 F(ab') fragments (Fig. 2, E and F) with identical results. After 15–30 min internalization (Fig. 2, BD and F) HECD-1 co-localized extensively in vesicles that were also positive for transferrin, suggesting that at this time internalized E-cadherin and transferrin resided in the same compartment. However, a striking disparity was detected between E-cadherin and transferrin at earlier stages of internalization. After 5 min internalization (Fig. 2, A and E), although E-cadherin and transferrin were readily detected in distinct vesicles, these did not co-localize. This suggested that although the trafficking pathways for these two proteins merged at some stage, their initial entry pathways might be different.



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FIG. 2.
Comparison of E-cadherin and clathrin-mediated uptake pathways. AF, comparison of E-cadherin internalization with transferrin endocytosis. Isolated MCF-7 cells were incubated with either HECD-1 IgG (AD; E-Cad IgG, red) or HECD-1 F(ab') (E and F; E-Cad F(ab'), red) and fluorescently labeled transferrin (Tfn, green) at 4 °C, then allowed to internalize for 5–60 min, before residual label was stripped and the samples processed for indirect immunofluorescence microscopy. Bars are 3 (AD) and 5 µm (E and F). G and H, comparison of internalized E-cadherin with endogenous clathrin immunostaining. MCF-7 cells were labeled with HECD-1 F(ab') (red) that was allowed to internalize for 15 min before fixation and staining for endogenous clathrin (green) by dual-label indirect immunofluorescence microscopy. The peripheral region highlighted in G is displayed in H. Bar is 8 µm (G).

 

To pursue this we compared staining for endogenous clathrin with that of surface-labeled E-cadherin after 15 min internalization. Clathrin stained in two major patterns: a perinuclear distribution consistent with Golgi-associated clathrin and more diffuse peripheral puncta that likely represent both clathrincoated pits and coated vesicles (Fig. 2G). Little internalized E-cadherin co-localized with clathrin (Fig. 2G). This was most apparent at the cell periphery where E-cadherin and clathrin were clearly identified in quite separate structures (Fig. 2H).

Internalized E-cadherin Enters the Early Endosomal Compartment—We then sought to characterize the relationship between internalized E-cadherin and the endosomal compartment. After 15–30 min internalization we found that many vesicles containing internalized HECD1-F(ab') also stained for the early endosomal marker, EEA-1 (Fig. 3B). However, at 5 min we could detect no co-localization between EEA-1 and HECD1-F(ab'), which labeled quite distinct vesicles (Fig. 3A). In contrast, as has generally been reported (33, 37), after 5 min many transferrin-positive vesicles were identified that also stained for EEA-1 (Fig. 3, C and D). This suggested that E-cadherin was internalized via an EEA-1-negative intermediary compartment before entering the early endosomal system.



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FIG. 3.
Internalized E-cadherin enters the early endosomal system. Isolated MCF-7 cells were surface-labeled with HECD-1 F(ab') fragments (A, B, E, and F) or with fluorescently labeled transferrin (C and D), then allowed to internalize the label for 5–15 (AD) or 30 min (E and F). After internalization, residual surface label was stripped and the samples processed for fluorescence microscopy. Internalized E-cadherin (A, B, E, and F; red) or transferrin (C and D; red) was compared with endogenous EEA-1 (AD; green), transiently expressed Rab 5 (Q79L) (E; green) or transiently expressed GFP-Rab 11 (F; green). Bars are 4 (AD) and 10 µm (E and F).

 

To confirm that E-cadherin was trafficked into an early endosomal compartment, we examined the pattern of cadherin uptake in cells transiently expressing a Rab5 mutant (Q79L) that causes fusion of early endosomes (33, 37). As shown in Fig. 3E, endocytosed E-cadherin accumulated in enlarged vesicles that coincided precisely with the localization of the Rab5 mutant transgene. This is consistent with previous reports that expression of this mutant causes cargo to accumulate in enlarged early endosomes (37). To assess whether E-cadherin could also enter the recycling endosomal compartment, we compared the localization of internalized E-cadherin with that of Rab11, a key marker of recycling endosomes (36). After 30 min internalization, we found that a proportion of surface-labeled E-cadherin was found in vesicles that also stained for transiently transfected Rab 11-GFP (Fig. 3F).

Taken together, these data indicated that internalized E-cadherin was trafficked to the early endosomal system, where at least a proportion was able to access recycling endosomes. In contrast, internalized E-cadherin was not found in Rab 7-positive vesicles (Fig. 4A), part of the late endosomal system. Nor was any co-localization seen in cells stained for cathepsin D (Fig. 4B), a protease found in lysosomes, or for {beta}-COP, a marker of the Golgi apparatus (Fig. 4C).



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FIG. 4.
Comparison of E-cadherin internalization with alternate trafficking pathways. MCF-7 cells (AC) or CHO cells stably expressing E-cadherin (D) were allowed to internalize surface-bound HECD-1 F(ab') for 30 min before processing for immunofluoresence microscopy. The localization of internalized E-cadherin (red) was compared with that of endogenous Rab 7 (A, green), endogenous cathepsin D (B, green), endogenous {beta}-COP (C, green), or transiently expressed Cav3-YFP (D, green). Bars are 12 (AC) and 4 µm (D).

 

E-Cadherin Is Not Internalized by Caveolae in Isolated CHO Cells—It was recently suggested that E-cadherin may be internalized through a caveolar trafficking pathway, based on the striking observation that sustained Rac signaling in keratinocytes caused E-cadherin to accumulate in large caveolin-positive cytoplasmic vacuoles (18). To investigate this possibility in our system, we compared the localization of internalized E-cadherin and caveolin, an essential component of caveolae. Because MCF-7 cells have low levels of endogenous caveolin we used a CHO cell line that stably expresses human E-cadherin (hE-CHO cells) and which forms caveolae. hECHO cells were transiently transfected with caveolin-3 YFP (Cav3-YFP), a construct that is a faithful marker of caveolae at the light microscopic level (29). hE-CHO cells effectively endocytosed surface-bound HECD-1 (both whole IgG and F(ab') fragments), displaying a pattern of vesicular staining qualitatively similar to that seen in MCF-7 cells (Fig. 4D). Although Cav3-YFP was clearly detected in punctate structures consistent with caveolae, no co-localization with E-cadherin was seen at any stage during our endocytosis assays (Fig. 4D). It thus seemed unlikely that caveolae were principally responsible for E-cadherin internalization in our model system.

Characterization of E-cadherin Internalization by Immunoelectron Microscopy—To further examine the initial step by which E-cadherin is internalized we employed electron microscopy in an attempt to identify distinct surface structures associated with E-cadherin (Fig. 5). Cells were fixed with glutaraldehyde, and the cell surface was then labeled with monoclonal antibody HECD-1 followed by 5 nm anti-mouse gold: under these conditions, only surface E-cadherin is accessible for immunolabeling. Labeling was found throughout the entire cell surface. Although most labeling was found on the undefined plasma membrane (83.8 + 5.3%, mean ± S.E., n = 406 gold particles counted), a significant proportion of gold particles were found in prominent invaginations of the plasma membrane (15.3 ± 5.1%), possibly indicative of ruffling activity or macropinocytosis (Fig. 5, A and B). Labeling for E-cadherin in clathrin-coated pits, in contrast, was extremely rare (1.0 ± 0.4% of surface gold; Fig. 5, C and D). Similar results were obtained after surface labeling cells for E-cadherin with both anti-cadherin and anti-mouse gold prior to allowing internalization of the E-cadherin complex for various times at 37 °C (results not shown). These observations indicated that E-cadherin did not show detectable concentrations in clathrin-coated pits relative to the general plasma membrane.



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FIG. 5.
Immunoelectron microscopic visualization of surface E-cadherin. Cells were fixed with 0.5% glutaraldehyde, immunolabeled for surface E-cadherin, and then processed for electron microscopy. Note that under these labeling conditions only surface E-cadherin is accessible for immunolabeling. Gold particles are circled in panels AC. Labeling is evident within large invaginations of the cell surface (panel A and higher magnification of boxed area in panel D) and is particularly high in areas rich in surface projections (low magnification overview in B). Low labeling is evident in clathrin-coated pits (arrow-heads in C and D). Bars, 200 nm.

 

Molecular Determinants of E-cadherin Endocytosis—To characterize the molecular regulation of E-cadherin internalization in isolated cells we first examined the potential role of dynamin, a GTPase implicated in various forms of endocytosis (22, 28). MCF-7 cells were transiently transfected with a well characterized dominantly interfering mutant (S45N) of dynamin. Consistent with its established role in clathrin-mediated endocytosis (22, 28), expression of dynaminS45N effectively inhibited transferrin uptake in MCF-7 cells (Fig. 6, C and D). In these studies we quantitated uptake of surface label by scoring the number of cells that displayed a characteristic pattern of widely distributed, vesicular uptake throughout the cytoplasm. The vast majority of cells expressing dynaminS45N showed no vesicular staining of internalized transferrin whatsoever (Fig. 6E). Expression of dynaminS45N also reduced the proportion of cells displaying the characteristic diffuse vesicular pattern of E-cadherin uptake (from 80.9 ± 3.4%, mean ± S.E., in controls to 35.0 ± 7.4% in transfected cells; Fig. 6, A, B, and E), although to a lesser extent than transferrin (from 90.7 ± 1.7% in controls to 4.7 ± 0.8% in transfected cells). This indicated that dynamin activity contributed to E-cadherin internalization, although less critically than was associated with transferrin uptake.



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FIG. 6.
E-cadherin internalization requires dynamin activity. MCF-7 cells transiently expressing a dominant-negative dynamin 1 mutant (K45N) were allowed to internalize HECD-1 IgG (E-Cad; A and B) or fluorescently labeled transferrin (Tfn; C and D) for 30 min before processing for fluoresence microscopy. Transfected cells were identified by co-staining for the HA-epitope tag (A and C; arrows). Expression of the mutant transgene abolished the diffuse vesicular staining characteristic of both internalized E-cadherin (B) and internalized transferrin (D). E, to quantitate changes in internalization we counted the proportion of cells with diffuse vesicular staining in untransfected cells and in cells expressing the dynamin mutant (DynDN).

 

To specifically test whether E-cadherin might be internalized by a clathrin-dependent pathway, we then perturbed the function of EPS-15, a crucial component of clathrin-mediated endocytosis (22). In agreement with previous studies (30, 31), transient expression of the EPS-15E{Delta}95/295 mutant reduced the uptake of transferrin by ~70% compared with control cells (Fig. 7, A, B, and E). In contrast, EPS-15E{Delta}95/295 had no detectable effect on E-cadherin endocytosis (Fig. 7, CE). After 30 min internalization, cells expressing EPS-15E{Delta}95/295 displayed a distinct vesicular pattern of endocytosed E-cadherin (Fig. 7D), identical to that seen in control cells (not shown). This strongly suggested that E-cadherin might be internalized by a clathrin-independent pathway in isolated MCF-7 cells.



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FIG. 7.
E-cadherin internalization in isolated MCF-7 cells is not affected by inhibition of EPS-15 activity. MCF-7 cells transiently expressing a dominant-negative EPS-15E{Delta}95/295 mutant (EPS-15 DN; arrows) were allowed to internalize fluorescently labeled transferrin (A and B) or HECD-1 F(ab') (C and D) for 30 min before being processed for fluorescence microscopy. Expression of EPS-15E{Delta}95/295 inhibited the uptake of transferrin (A and B) but not uptake of E-cadherin (C and D). Endocytosis was quantitated (E) by counting the proportion of cells displaying a diffuse vesicular pattern of label in untransfected cells and in cells expressing EPS-15E{Delta}95/295.

 

E-Cadherin Endocytosis Is Sensitive to the ARF6 GTPase— The ARF6 GTPase defines a clathrin-independent pathway for endocytosis and trafficking of a variety of membrane proteins (38, 39). Recently, ARF6 signaling was reported to influence the surface localization of E-cadherin in Madin-Darby canine kidney cell monolayers (17, 23). Accordingly, we tested the effect of manipulating ARF6 signaling on E-cadherin endocytosis in our assay system. MCF-7 cells were transiently transfected with ARF6Q67L, a mutant that is defective in GTPase activity and predicted to be locked in the active GTP-bound state (40). Consistent with previous reports (40), we found that expression of ARF6Q67L did not affect transferrin uptake in isolated MCF-7 cells (Fig. 8F). However, it potently perturbed the pattern of E-cadherin internalization. Cells expressing wild-type ARF6 (Fig. 8A) showed a characteristic pattern of diffusely distributed E-cadherin vesicles after 30 min internalization that was identical to that seen in untransfected cells (not shown). In contrast to these controls, little vesicular staining of E-cadherin was detected in cells expressing ARF6Q67L (Fig. 8, C and D). Internalization was not totally abrogated, however, although the vast majority of cells failed to show diffuse vesicular internalization, E-cadherin labeling was commonly detected in large peripheral structures that were resistant to acid/salt stripping (Fig. 8D). In contrast, expression of an ARF6T27N mutant had no apparent effect on E-cadherin internalization (Fig. 8B).



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FIG. 8.
ARF6 GTPase activity is necessary for traffic of E-cadherin after initial endocytosis. MCF-7 cells transiently expressing wild-type ARF6 (A, Wt), dominant-negative ARF6T27N (B), or the ARF6Q67L mutant lacking GTPase activity (CF) were allowed to internalize HECD-1 F(ab') for 30 min before processing for fluorescence microscopy. All cells shown expressed the appropriate transgenes as identified by specific immunolabeling for the epitope tag. Wild-type ARF6 (A) and ARF6T27N (B) did not affect E-cadherin internalization. Expression of ARF6Q67L prevented internalization of E-cadherin into diffuse small vesicles (C), which instead accumulated in large peripheral structures (D, detail of marked region in C). The large peripheral structures (arrows) did not label for endogenous EEA-1 (green, E) or internalized fluorescently labeled transferrin (green, F). Bar is 12 µm.

 

The accumulation of internalized E-cadherin in large peripheral structures, but not in diffuse vesicles, suggested that ARF6Q67L might act on an early step during E-cadherin endocytosis. To further characterize these peripheral structures we co-labeled cells for E-cadherin and either transferrin or EEA1. We found that in cells expressing ARF6Q67L the cadherincontaining peripheral structures were largely devoid of either endogenous EEA-1 (Fig. 8E) or internalized fluorescently labeled transferrin (Fig. 8F), a pattern analogous to that seen in the early phases of E-cadherin internalization in untransfected cells (Figs. 2 and 3), suggesting that ARF6Q67L might be trapping this early compartment.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study we sought to characterize the process by which unbound surface E-cadherin is internalized in isolated MCF-7 and CHO cells, an assay system chosen to allow us to focus on the fate of E-cadherin that is not incorporated into stable adhesive contacts (13). Several lines of evidence suggest that in our experimental system the initial internalization of E-cadherin during endocytosis principally occurred via a clathrin-independent mechanism. 1) At the earliest phase of internalization, surface-labeled E-cadherin did not co-localize with endocytosed transferrin, a classic marker of clathrin-dependent endocytosis (32, 35). 2) Expression of a dominant-inhibitory EPS 15 mutant had no effect on E-cadherin internalization, despite effective inhibition of transferrin uptake to a degree comparable with that previously reported (30, 31). 3) At the light microscopic level internalized E-cadherin did not co-localize significantly with endogenous clathrin; nor did E-cadherin concentrate in clathrin-coated pits by immuno-EM. Taken together, these features of cadherin internalization resemble the patterns seen with a range of other membrane proteins believed to be internalized by clathrin-independent endocytosis pathways. Notably, endocytosis of the interleukin-2 receptor (a transmembrane protein (41, 42) and glycosylphosphatidyl-inositol-anchored proteins (30) also occurs independently of transferrin and was not inhibited by EPS15 mutants, features identical to those that we observed for E-cadherin.

Recent evidence that C-cadherin (14) and E-cadherin (23) trafficking was perturbed by inhibition of dynamin suggested the possibility that classical cadherins might be internalized via a clathrin-dependent pathway. Indeed, we found that in the current assay E-cadherin endocytosis was significantly inhibited by dynaminS45N, although to a lesser extent than transferrin uptake. However, while dynamin was originally postulated to specifically inhibit clathrin-mediated uptake, it is now clear that this molecule can influence a range of other cellular processes, including movement of the cell surface (43), the clathrin-independent endocytosis of interleukin-2 receptors (41), and internalization via caveolae (44). Taken together, our data suggest strongly that in isolated MCF-7 cells E-cadherin was endocytosed predominantly by a clathrin-independent pathway.

Interestingly, our findings also indicate that after entry into cells E-cadherin quite rapidly entered the early endosomal system, being found in compartments that contained transferrin and EEA-1 and that were sensitive to Rab5. This, too, is consistent with recent evidence that, in addition to classic clathrin-mediated uptake (20, 32), some clathrin-independent endocytosis pathways can also enter the early endosomal system. For example, the folate receptor, a glycosylphosphatidyl-inositol-anchored protein, is internalized by a clathrin-independent mechanism, but is subsequently trafficked into a transferrin-containing compartment before entering recycling endosomes (45). Recently internalized E-cadherin did not, however, appear to enter the late endosomal/lysosomal system. Instead, our observation that internalized E-cadherin could localize with Rab11 in a perinuclear site suggested that at least a proportion of internalized E-cadherin could enter recycling endosomes, a pathway that provides an attractive mechanism for subsequently directing E-cadherin back to the cell surface.

What then might be the nature of this clathrin-independent E-cadherin internalization pathway? An important clue comes from the observation that E-cadherin internalization was strikingly affected by constitutively active ARF6. Although ARF6Q67L blocked the formation of cadherin-containing vesicles, some degree of internalization appeared to transpire, because surface-labeled E-cadherin was commonly detected in large peripheral compartments that resemble those previously described to result from sustained ARF6 signaling (40). Similarly, ARF6Q67L causes other proteins subject to clathrin-independent endocytosis (MHC1, Tac) to accumulate in large peripheral structures, and consequently fail to fuse with early endosomes (42). Furthermore, we found that the large cadherin-containing peripheral structures were devoid of either internalized transferrin or EEA-1, suggesting that they might correspond to the early vesicular compartment we identified. This suggests that ARF6Q67L may affect a very early step after cadherin entry, causing nascent vesicles to fuse and/or prevent their progression to enter the early endosomal system. It was also notable that in our cells E-cadherin labeled prominently in surface invaginations and folds that resembled the ruffles and macropinosomes that ARF6 is reported to regulate (40, 46). Taken together, these data suggest that in isolated MCF-7 cells E-cadherin was being internalized by ARF6-dependent macropinocytosis. Interestingly, dynamin also associates with macropinosomes (47), suggesting a possible mechanism for the role of dynamin in E-cadherin internalization that we observed.

A role for ARF6 in cadherin trafficking was also recently identified in Madin-Darby canine kidney cell monolayers, where it was reported that ARF6Q67L potentiated E-cadherin internalization while ARF6T27N inhibited internalization (23). In our assays, however, ARF6 activity did not appear to impinge on the actual step of internalization because the T27N mutant had no apparent effect on endocytosis. It is possible that this disparity in findings reflects a difference in the action of ARF6 in isolated cells, as used in our studies, compared with mature polarized monolayers. In our assay system surface E-cadherin is predicted to be free and unoccupied by ligand, whereas in mature epithelial monolayers the majority of cadherin would be expected to be engaged in homophilic binding. Importantly, not only does contact appear to influence cadherin internalization (13), but homophilic ligation also activates cadherin signaling (26, 48). Notably, ARF6 can affect both phosphoinositide signaling and the activity of the Rac GTPase (39, 49, 50), signals that E-cadherin activates to modulate adhesion (26, 51). It is therefore possible that ARF6 influences not only the process of cadherin endocytosis itself, but also the mechanisms of cadherin engagement and/or signaling that support surface adhesion. This would have implications for the adhesiveness, and hence availability for endocytosis, of E-cadherin in monolayers but not in isolated cells. We are currently investigating the possibility that ARF6 may affect E-cadherin signaling and adhesive recognition independently of its effects on the cadherin internalization process.

In summary, our findings identify a novel clathrin-independent uptake pathway for E-cadherin in MCF-7 cells and suggest that this may be the dominant endocytosis mechanism when E-cadherin is free on the cell surface in isolated cells. Our current findings do not exclude the possibility that clathrinmediated cadherin endocytosis also occurs in these cells. A small proportion of E-cadherin was identified in clathrincoated pits and, moreover, if internalization through clathrincoated pits was very rapid, it might not have been detectable with our techniques. Nor can we exclude the possibility that differences in cell types studied influence the pathways of endocytosis. The MCF-7 cell line used in our experiments, although relatively well differentiated, derives from a human tumor, in contrast to the Madin-Darby canine kidney cells used in other studies (13, 17). Our current results, taken together with our previous findings (13, 19) and those of other investigators (15, 16, 17, 18), thus suggest that there may be multiple pathways for cadherin internalization in different cellular contexts. For example, fully confluent epithelial monolayers, where the majority of E-cadherin is incorporated into adhesive contacts, might be characterized by low levels of clathrin-mediated cadherin endocytosis. This would contrast with the more abundant uptake of unligated cadherins seen in isolated cells that occurs via the clathrin-independent, ARF-6-regulated pathway we have now identified. Indeed, the endocytosis pathway identified in our experiments may be most directly relevant to biological contexts where a significant proportion of cellular E-cadherin is free, particularly dynamic morphogenetic processes, including cell migration and wound healing (7, 12, 51). These are situations where rapid remodeling of cadherin adhesive contacts is likely to be physiologically active. More detailed understanding of the mechanism(s) of E-cadherin endocytosis will thus contribute to understanding how tissue context influences cadherin activity.


    FOOTNOTES
 
* This work was supported by the National Health and Medical Research Council (Australia) and the Wellcome Trust (United Kingdom). 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. Back

** Senior International Research Fellow of the Wellcome Trust. To whom correspondence should be addressed: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia 4072. Tel.: 61-7-33462013; Fax: 61-7-33462101; E-mail: a.yap{at}mailbox.uq.edu.au.

1 The abbreviations used are: CHO, Chinese hamster ovary; EEA-1, early endosomal antigen-1; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid. Back


    ACKNOWLEDGMENTS
 
We thank our colleagues for the kind gifts of reagents; Marita Goodwin with assistance in compiling some of the statistics; Jitu Mayor, Rohan Teasdale, Tam Luan Le, and Kevin Miranda for many helpful discussions;, and all our colleagues in the laboratory for their advice, encouragement, and support.



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 MATERIALS AND METHODS
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 DISCUSSION
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