The effect of ARF6–GTP on AJ disassembly is independent of actin cytoskeleton remodeling and phospholipid metabolism

To investigate whether the effect of ARF6 on AJ disassembly could be attributed to its regulatory role in membrane traffic or its effect on actin organization, we utilized an ARF6 triple mutant, ARF6(Q67L:Q37E:S38I), which contained additional mutations in the effector-binding domain of the protein. This effector domain mutant was shown to be incapable of actin remodeling, but did not interfere with ARF6–GTP-mediated membrane cycling (Al-Awar et al., 2000). It is likely that the mutations in the effector loop prevent interaction with key downstream targets required for ARF6-induced peripheral actin remodeling. Similarly to previous observations in HeLa cells, MDCK cells transfected with ARF6(Q67L: Q37E:S38I) did not exhibit prominent actin-rich membrane ruffles at the peripheral edges (data not shown). However, expression of ARF6(Q67L:Q37E:S38I) mimicked the effect of ARF6(Q67L) on AJ disassembly, with the majority of the cadherin label present in perinuclear vesicles in the absence of membrane ruffles (Figure 3). These findings suggest that ARF6-mediated regulation of AJ stability and turnover is independent of its effect on the actin cytoskeleton, and probably occurs by mobilization of AJ components via vesicle traffic.

Open in a separate window

Fig. 3. ARF6(Q67L:Q37E:S38I), an effector domain ARF6 mutant incapable of membrane ruffling, can induce AJ disassembly. (A) Cells transiently expressing HA-tagged ARF6(Q67L:Q37E:S38I) were labeled for E-cadherin (red) and HA (green). Coincident staining appears yellow in the merged image. Transfected cells exhibited a loss of E-cadherin label at the cell junctions with a redistribution of the protein to the perinuclear vesicles in the cytoplasm. (B) Cells were labeled for HA (red) and for transferrin receptor (Tfn-R; red). Coincident staining appears yellow in the merged image. A stacked image of optical sections along the z-axis is shown (left). A magnified image across a single confocal plane at the perinuclear region is shown on the right.

We examined the nature of the perinuclear structures that acquired E-cadherin. Double-labeling experiments revealed substantial co-localization of E-cadherin/ARF6-positive vesicles with endosomal markers, such as the transferrin receptor (Tfn-R), in perinuclear compartments (Figure 3B). No overlapping distribution was observed with antibodies against Golgi, endoplasmic reticulum or lysosomal markers (data not shown). The redistribution of E-cadherin into transferrin receptor-containing endosomes is particularly relevant since cell surface E-cadherin has been shown to be trafficked constitutively along endocytic recycling pathways in MDCK cells (Le et al., 1999).

ARF6(Q67L) stimulates actin remodeling by increasing levels of PIP2 (Honda et al., 1999). Thus, we examined whether the effect of ARF6–GTP on AJs was mediated by its ability to induce locally elevated levels of PIP2. For these studies, we utilized an expression plasmid encoding the pleckstrin homology domain of PLC-δ tagged with GFP (PH–GFP), which has previously been characterized as a marker of PIP2 distribution in cells (Varnai and Balla, 1998). We transfected plasmid encoding PH–GFP into MDCK cells expressing ARF6(Q67L). As shown in the montage in Figure 4A, we found that PH–GFP labeled ARF6–GTP-induced surface ruffles, but did not localize to ARF6–GTP-generated internal endosomal vesicles. Double labeling for E-cadherin and PIP2 also revealed no overlap in E-cadherin and PIP2 distribution in endosomes (Figure 4B). In normal MDCK cells, GFP labeling was diffuse in the cytoplasm with minimal labeling at the cell surface in endogenous protrusions (Figure 4C). These latter findings revealed that the two phenotypes, i.e. the internalization of AJ components and membrane ruffling, can be uncoupled and are probably mediated by distinct downstream effectors and signaling pathways.

Open in a separate window

Fig. 4. ARF6–GTP-generated endosomal vesicles are devoid of PIP2. (A) HA-tagged ARF6(Q67L)-expressing cells were transfected with plasmid encoding PH–GFP, and 24 h post-transfection cells were fixed and labeled with anti-HA monoclonal antibody to determine ARF6 distribution (red). Images were merged and coincident staining appears yellow. Merged images of horizontal x/y sections along the z-axis are shown [1 (top)–6 (bottom)]. An overlapping distribution of PH–GFP (i.e. PIP2) and ARF6(Q67L) was detected in surface ruffles, but not in ARF6(Q67L)-positive perinuclear vesicles. (B) ARF6(Q67L)-expressing cells were transfected with plasmid encoding PH–GFP and labeled for E-cadherin. Coincident staining for E-cadherin (red) and PH–GFP (green) appears yellow. Merged images of a single optical section at the perinuclear plane are shown. (C) Cells infected with retrovirus alone were transfected with plasmid encoding PH–GFP, and 24 h post-transfection, cells were fixed and viewed under a confocal microscope. PH–GFP label was mainly diffuse in the cytoplasm and some label was seen at the cell surface.

Expression of ARF6–GTP has no effect on cell polarity

We investigated whether ARF6(Q67L)-induced disassembly of AJs was accompanied by a loss of cell polarity. To this end, cells infected with retrovirus encoding HA-tagged ARF6(Q67L) were grown on transwell filter units, and localization of ARF6–GTP was compared with the distribution of other apical and basolateral membrane proteins. Images along the x/z-axis showed that ARF6(Q67L) localized to the basolateral membrane and cytoplasm, since its distribution partially overlapped with transferrin receptors, a basolateral marker (Figure 5A), and not with GP-135, an apical membrane marker (Figure 5B and D), or with ZO-1, a component of the apical tight junctions (Figure 5C). The cells were polarized and exhibited microvilli at the apical end. No labeling for ARF6–GTP was observed in the tips of the microvilli (Figure 5D). The localization of ARF6–GTP to the basolateral membrane and cytoplasm may reflect a specific role for ARF6 in the constitutive recycling of E-cadherin from AJs to the basolateral membrane, as has been described previously (Le et al., 1999). These studies also revealed that despite the loss of AJs, cells expressing ARF6(Q67L) retained their polarized distribution of apical and basolateral membrane proteins.

Open in a separate window

Fig. 5. ARF6 activation does not perturb cell polarity. MDCK cells infected with retrovirus encoding HA-tagged ARF6(Q67L) for transient protein expression were grown on transwell filters, fixed, double labeled as indicated below, and processed by immunofluorescence microscopy. Cells were viewed using a confocal imaging system. Cells were double labeled with anti-HA rabbit polyclonal antibody (green) and with mouse monoclonal antibodies (red) to either Tfn-R (A), gp135 (B and D) or ZO-1 (C). Merged images of vertical (x/z) sections are shown (A–C). A horizontal x/y section at a single confocal plane at the apical microvilli is also shown (D). Coincident red and green staining appears yellow.

While the above data clearly demonstrate the basolateral distribution of ARF6–GTP, an earlier study by Altschuler et al. (1999) reported that ARF6–GTP localized exclusively to the apical plasma membrane of MDCK cells. At this point, the reason for this disparity is still unclear. However, in addition to the MDCK cell line used in this study, we have found that ARF6–GTP localizes to the lateral plasma membrane in other epithelial cell lines including MCF-7 and T-47D (data not shown).

ARF6(T27N), the dominant-negative ARF6 mutant, enhances cell–cell adhesion

To assess further the importance of the ARF6 GTPase cycle in cell–cell adhesion, we analyzed cells expressing HA-tagged ARF6(T27N), the dominant-negative ARF6 mutant defective in GTP binding, and cells expressing HA-tagged wild-type ARF6. Transient and stable expression of ARF6(T27N) and wild-type ARF6 in MDCK cells was achieved using the neomycin-based retroviral system described above. We observed that ARF6(T27N)-expressing cells formed tight colonies that appeared to be even more compact than those formed by cells infected with retrovirus alone. Confocal images of these cells along the x/y-and x/z-axis revealed that the majority of ARF6(T27N) localized predominantly to the AJs (Figure 6A and B). A small fraction of the label was also seen at subapical membrane compartments.

Open in a separate window

Fig. 6. Subcellular distribution of ARF6(T27N) and wild-type ARF6. (A) Cells expressing HA-tagged ARF6(T27N) were double labeled with anti-HA rabbit polyclonal antibody (green) and anti-E-cadherin mouse monoclonal antibody (red). An image across a single confocal plane at the cell junctions is shown to emphasize the localization of ARF6(T27N) at the cell junctions. (B) Cells expressing HA-tagged ARF6(T27N) were double labeled for HA (green) and for either E-cadherin (red, shown on the left) or gp135 (red, shown on the right). Coincident red and green staining appears yellow. Merged images taken along the x/z-axis are shown. ARF6(T27N) localizes to the AJs of MDCK cells; a small fraction of label was also seen at the lateral membrane and subapical region. (C) Cells transiently expressing HA-tagged wild-type (wt) ARF6 were labeled for HA (green) and for either E-cadherin (red, left) or gp135 (red, right). Coincident red and green staining appears yellow. Merged images taken along the x/z-axis are shown. Wild-type ARF6 localizes to the AJs, the lateral membrane and subapical membranes.

Expression of wild-type ARF6 had no effect on cell morphology and was identical to normal untransfected MDCK cells in cell culture (not shown). Images of ARF6 distribution along the x/z-axis revealed that the wild-type protein was localized to the AJs and to the subapical region of the cell (Figure 6C). Thus, the distinct and unique localization patterns of ARF6 and its mutants defective in GTP binding and hydrolysis suggest that the ARF6 GTPase shuttles between the AJs and vesicular compartments in the cytoplasm, and probably regulates the traffic of proteins between these compartments.

To substantiate our findings obtained by morphological investigations, we determined whether the detergent-solubility properties of β-catenin (an AJ component) were altered in cell lines expressing wild-type ARF6 or its mutants, ARF6(Q67L) and ARF6(T27N). For these experiments, we used the Triton X-100 solubility assay, in which cells were extracted with ‘CSK’ buffer containing 0.5% Triton X-100, and the fraction of β-catenin that partitioned into the Triton-insoluble (cytoskeleton-associated) fraction was monitored using immunoblot analysis. As seen in Figure 7, in the case of cells expressing wild-type ARF6, approximately half of the cellular β-catenin was present in the insoluble fraction. A similar distribution was observed in untransfected MDCK cells (not shown). However, in cells expressing ARF6(T27N), most of the β-catenin was cytoskeleton associated (resistant to Triton X-100 solubilization), whereas in cells expressing the ARF6–GTP mutant, the majority of β-catenin was detergent soluble. Thus, the differences in subcellular distribution and phenotype of wild-type ARF6 and its mutants defective in GTP hydrolysis and binding were reflected in the detergent-solubility properties of β-catenin in cell lines expressing the various mutant forms of ARF6. Moreover, the increased association of β-catenin with the cytoskeletal fraction in ARF6(T27N)-expressing cell lines indicates that an inhibition of nucleotide exchange on ARF6 prevents the turnover of AJs, and thereby stabilizes the epithelial phenotype.

Open in a separate window

Fig. 7. Effect of the ARF6 GTPase cycle on the detergent-solubility properties of β-catenin. Cells stably expressing either ARF6, ARF6(Q67L) or ARF6(T27N) were lysed in CSK-A buffer containing 0.5% Triton X-100, centrifuged, and Triton-soluble (s) and -insoluble (i) fractions were analyzed for β-catenin distribution using immunoblotting procedures.

ARF6 regulates hepatocyte growth factor/scatter factor-induced AJ disassembly

Since the studies described above suggested that the ARF6 GTPase cycle regulates AJ turnover, we investigated the importance of ARF6 activation in a physiologically relevant disassembly of AJs. To this end, we examined the effect of dominant-negative ARF6 expression in cells treated with hepatocyte growth factor (HGF)/scatter factor. HGF is involved in the invasive behavior of several tumor cell types, and treatment of epithelial cells with HGF leads to the activation of the receptor tyrosine kinase c-met. HGF-induced signaling induces the disruption of cell–cell adhesion by promoting the internalization of E-cadherin (Stella and Comoglio, 1999). As stated above, E-cadherin is constitutively internalized and then recycled back to the basolateral surface (Le et al., 1999). For our investigations, untransfected MDCK cells and those stably transfected with ARF6(T27N) were incubated with HGF for varying time intervals, and E-cadherin distribution was monitored. In untransfected cells, consistent with previously reported findings, E-cadherin was internalized upon HGF treatment; there was a dose-dependent increase in internalization with increased exposure to HGF (not shown). Figure 8A shows the distribution of E-cadherin in normal MDCK cells and in cells expressing ARF6(T27N), respectively, after 4 h of HGF treatment. As shown, expression of the dominant-negative ARF6 mutant ARF6(T27N) completely blocked HGF-induced cadherin internalization and breakdown of AJs. Furthermore, by phase-contrast microscopy we found that cells stably expressing ARF6(T27N) abolished HGF-mediated cell scatter (data not shown). These findings suggest that ARF6 plays a critical role in the internalization of E-cadherin and mediates HGF-induced AJ disassembly.

Open in a separate window

Open in a separate window

Open in a separate window

Fig. 8. Effect of dominant-negative ARF6 on HGF- and Ca²⁺ depletion-mediated AJ disassembly. (A) Untransfected MDCK cells or cells stably expressing ARF6(T27N) were grown on coverslips and incubated with 5 ng/ml HGF for 4 h. Cells were fixed and labeled for E-cadherin (green), and stained for actin (red). As shown, ARF6(T27N) expression blocks HGF-induced internalization of E-cadherin. (B) HGF induces the subcellular redistribution of endogenous ARF6. Normal MDCK cells or cells stably expressing either ARF6(Q67L) or ARF6(T27N) were lysed in CSK-A buffer, resolved on 14% SDS gels and the Triton-soluble fraction was analyzed for endogenous ARF6 distribution with anti-ARF6 monoclonal antibody using immunoblotting procedures. Band density was measured using the enhanced UltroScan XL Laser Densitometer (Pharmacia). As a loading control, the membrane was also probed for γ-tubulin expression. HGF treatment led to a marked increase in soluble ARF6. In cells expressing ARF6(Q67L), similar amounts of soluble ARF6 were detected in the presence and absence of HGF, while expression of ARF6(T27N) blocked the redistribution of ARF6 into the soluble pool. Exogenous ARF6(Q67L) runs at a higher molecular weight than the endogenous protein (arrow). Exogenous ARF6(T27N) was not detected on the blot since most of the ARF6(T27N) was present in the insoluble fraction (not shown). The data shown are representative of four independent experiments. (C) Normal MDCK cells (left) and those expressing wild-type ARF6 (center) or ARF67(T27N) (right) were incubated with EDTA (2.5 mM) at 37°C for 15 min. Cells were fixed and labeled for E-cadherin (green) and stained for actin (red). Merged images are shown; coincident staining appears yellow. ARF6(T27N) expression blocks the Ca²⁺ depletion-induced collapse of E-cadherin into the cytoplasm.

Next we asked whether we could assess the activation profile of endogenous ARF6 in response to HGF treatment using the detergent-solubility assay described above. We asked whether HGF treatment would increase the soluble pool of endogenous ARF6. To this end, untransfected MDCK cells were exposed to HGF for 1 h, after which cells were subjected to detergent-based fractionation as described above and the endogenous ARF6 protein associated with the detergent-soluble fraction was determined. As shown in Figure 8B, we observed a marked increase in soluble ARF6 after HGF treatment. When the assay was performed in cell lines stably expressing the dominant-negative ARF6 mutant ARF6(T27N), HGF-induced redistribution of endogenous ARF6 into the soluble pool was inhibited. In contrast, a majority of the endogenous ARF6 pool was soluble in cells expressing ARF6(Q67L); the latter is likely to be a reflection of the ‘activated ARF6’ phenotype. These results suggest that HGF induced the translocation of endogenous ARF6 protein to the detergent-soluble fraction, a process that was blocked by dominant-negative ARF6. Thus, ARF6 activation lies downstream of c-met receptor activation.

Further support for the role of ARF6 activation in E-cadherin internalization came from the demonstra tion that expression of dominant-negative ARF6, ARF6(T27N), blocked the redistribution of E-cadherin into the cytoplasm upon disassembly of the AJs induced by Ca²⁺ depletion (Figure 8C). In untransfected cells and in cells expressing wild-type ARF6, E-cadherin was redistributed to the perinuclear cytoplasm upon depletion of extracellular Ca²⁺. In contrast, in cells expressing ARF6(T27N), a majority of the E-cadherin remained at the cell surface despite the loss of Ca²⁺-dependent, E-cadherin-based cell–cell adhesion.

Effect of the ARF6 GTPase cycle on epithelial cell migration

Taken together, the studies described above suggest that ARF6 activation might serve to promote the migratory potential of epithelial cells. Thus, we examined the migratory capacity of cells stably expressing either wild-type ARF6, ARF6(Q67L), ARF6(Q67L:Q37E:S38I) or ARF6(T27N), compared with untransfected MDCK cells, using a transwell filter migration assay. For these experiments, cells were seeded on top of transwell filters and migration of cells toward chemoattractant in the lower chamber was monitored. Assessment of the number of cells that migrated revealed that ARF6(Q67L)-expressing cells exhibited a dramatic increase in cell migration compared with control untransfected cells or cells expressing wild-type ARF6. The effect of the ARF6–GTP mutant on cell migration was observed only 12–16 h after cells were seeded on filters. No change in migratory capacity was observed at earlier time points (within 6–8 h). This observation is significant because it implies that the effect of ARF6–GTP on epithelial cell migration is probably not due to a direct effect on the ‘migration machinery’ per se (such as integrin receptors or other focal adhesion components), but rather due to its effect on AJs. In contrast to the effect of ARF6–GTP, expression of dominant-negative ARF6(T27N) blocked the basal migratory capacity of MDCK cells (Figure 9), suggesting a requirement for ARF6 activation in MDCK cell migration. Finally, cells expressing the activated mutant of ARF6, incapable of membrane ruffling, did not lead to increased migratory capacity of MDCK cells (Figure 9). This result implies that AJ disassembly is not sufficient for increased cell migration, and that other factors, such as cytoskeletal remodeling, are also required.

Open in a separate window

Fig. 9. The ARF6 GTPase cycle modulates epithelial cell migration. Cells stably expressing either wild-type ARF6, ARF6(Q67L), ARF6 triple mutant [ARF6(TM)] or ARF6(T27N) were grown on transwell filters and their migratory potential was assessed in the presence or absence of HGF as indicated. The number of cells/field that migrated through the filter after 16 h was counted. The mean of six separate fields is shown. The data are representative of four independent experiments.

We also examined the effect of the ARF6 mutants described above on HGF-induced increase in migratory potential. As shown in Figure 9, HGF enhanced the migratory potential of MDCK cells to a similar extent to ARF6(Q67L). In contrast to the lack of enhanced migratory capacity of cells expressing the ARF6 effector domain mutant, HGF treatment of these cells promoted cell migration to the same extent as ARF6(Q67L), suggesting that HGF may stimulate other signaling events that function in concert with ARF6–GTP to promote cell migration. However, expression of ARF6(T27N) abrogated HGF-induced cell migration. Taken together, the above studies indicate a requirement for ARF6 activation during ‘basal and stimulated’ epithelial cell migration.

ARF6–GTP-induced AJ disassembly and actin remodeling: effects uncoupled

In addition to its well characterized role in actin remodeling, PIP2 has also been shown to have a role in various membrane traffic events (Martin, 1998). For instance, endosomal vesicles accumulate in nerve terminals lacking synaptojanin, an inositol-5-phosphatase that releases phosphate and metabolizes PIP2 (Cremona and De Camilli, 2001). Consistent with previously reported findings in other cell types (Honda et al., 1999), we observed elevated levels of PIP2 in regions of membrane ruffles in MDCK cells expressing activated ARF6. However, there was no accumulation of PIP2 on ARF6–GTP vesicles that contained internalized AJ components. Thus, the accumulation of endocytic vesicles probably occurs independently of the effects of ARF6 on phospholipid metabolism. Further support for this contention is our finding that cadherin internalization and AJ disassembly can occur by expression of an activated ARF6 mutant incapable of membrane ruffling. Interestingly, labeling for PIP2 in these transfected cells was similar to the staining observed in untransfected cells (F.Palacios and C.D’Souza-Schorey, unpublished observations). The above findings demonstrate that the ARF6–GTP effector pathways that mediate AJ disassembly and ruffling of the lateral membrane are distinct in epithelial cells.

An obligate role for ARF6–GTP-induced membrane internalization during AJ disassembly

A depletion of extracellular Ca²⁺, phosphorylation of the c-met receptor, v-Src-mediated phosphorylation of various junctional components, and the depolymerization of the actin cytoskeleton have all been implicated in AJ disassembly (Gumbiner, 2000). ARF6–GTP-induced disassembly of AJs appears to be independent of v-Src-mediated phosphorylation events. Moreover, the demonstration that expression of the actin remodeling-defective ARF6–GTP mutant was able to induce AJ disassembly rules out actin remodeling as a potential mechanism for the effect of ARF6 on AJs. However, the inhibition of E-cadherin internalization upon HGF treatment or Ca²⁺ depletion, by expression of dominant-negative ARF6, indicates that ARF6 regulates AJ assembly via its effect on membrane transport from the AJs to the perinuclear cytoplasm.

AJ assembly and turnover is a highly dynamic process

E-cadherin molecules at the cell surface are constitutively endocytosed and then recycled back to the lateral membrane (Le et al., 1999). This pool of recycling E-cadherin allows the cell to undergo rapid changes in morphology in response to several stimuli, such as HGF and EGF. The inhibition of HGF-induced internalization of E-cadherin from the cell surface, by dominant-negative ARF6, indicates that ARF6 activation is required for the cytoplasmic redistribution of E-cadherin during AJ disassembly. Furthermore, the block in HGF- and v-Src-induced cell scattering, by dominant-negative ARF6, points toward an obligatory role of ARF6-regulated membrane traffic in AJ disassembly and turnover. Taken together, our findings indicate that the ARF6 GTPase cycle modulates the trafficking of E-cadherin between the lateral membrane and intracellular compartments, and hence can dictate the availability of cycling E-cadherin molecules for assembly into AJs.

The ARF6 GTPase cycle has an essential role in epithelial cell migration

The ability of ARF6–GTP to induce AJ disassembly and membrane ruffling points toward ARF6 as a potential regulator of epithelial cell migration. Consistent with this contention, we have demonstrated that sustained activation of ARF6, by expression of a GTP-bound ARF6 mutant, leads to a dramatic stimulation in the migratory potential of epithelia using transwell filter assays. Expression of dominant-negative ARF6 abolished HGF-induced cell migration. From these studies, we conclude that there is an obligatory requirement for ARF6 activation during HGF migration. As indicated earlier (see Results), the effect of the ARF6–GTP mutant on cell migration was not acute and was observed no earlier than 12 h after cells were seeded on filters. A likely explanation for these observations is that the ARF6–GTP-induced increase in migration is due to its effect on AJ disassembly. The disassembly of AJs may cause a breakdown of other cell–cell junctions, since the assembly of other junctional complexes, such as desmosomes and tight junctions, depends on activation of the cadherin-based system (Gumbiner, 1988; Wheelock and Jensen, 1992; Marrs et al., 1995). This would lead to a marked increase in migratory capacity over a more prolonged time period. The observation that the activated ARF6 mutant incapable of membrane ruffling had no significant effect on migratory capacity indicates that AJ disassembly alone is not sufficient for ARF6-induced migration. Peripheral membrane and cytoskeletal rearrangements may also be required for an overall increase in migratory capacity. However, we cannot rule out the possibility that this ARF6-effector domain mutant is incapable of cellular functions other than surface ruffling which may also be required for cell migration. Interestingly, cells expressing the ARF6-effector domain mutant showed enhanced migratory potential in response to HGF treatment. Thus, HGF probably activates ARF6-independent pathways that act synergistically with the ARF6-effector domain mutant to promote cell migration.

Mutagenesis

ARF6(Q67L:Q37E:S38I) was generated using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions. HA-tagged ARF6(Q67L) cDNA was used as template and the following oligonucleotides (5′-GTTGAAGCTGGGCGAGAT CGTGACCACCATTC-3′ and 5′-GAATGGTGGTCACGATCTCGCCCAGCTTCAAC-3′) encoding mutations (underlined) were used as primers. The sequence was verified, and ARF6(Q67L:Q37E:S38I) cDNA was subcloned into pLZRS-IRES-neo for the generation of retrovirus, as described above.

Cell culture, transfection and generation of stable cell lines

MDCK II cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin and streptomycin. For cell transfections, 1 ml of freshly thawed retrovirus-containing supernatant mixed with 1% DOTAP liposomal transfection reagent (Boehringer Mannheim) was layered onto the cells in 35 mm dishes, and infection was allowed to proceed for 4 h at 37°C. Cells were gently washed in phosphate-buffered saline and then incubated in normal growth media. Twenty-four hours post-incubation, cells were trypsinized and seeded onto glass coverslips or on 0.4 µm pore size clear polyester transwell filters (Corning-Costar), followed by incubation for another 12–24 h. Stable cell lines expressing ARF6 wild-type and mutant cDNAs were generated by retroviral transduction, followed by selection of transfected cells using G418. Expression of the ARF6 proteins was less than a fold above endogenous protein expression (Figure 12).

Open in a separate window

Fig. 12. Expression of wild-type ARF6, ARF6(Q67L) and ARF6(T27N) in MDCK cells using retroviral expression. Lysates of MDCK cells stably expressing either HA-tagged wild-type ARF6, ARF6(Q67L) or ARF6(T27N) were resolved on SDS gels, transferred to nitrocellulose membrane and probed using immunoblotting procedures for either total ARF6 expression or for exogenous ARF6 using anti-HA monoclonal antibody. Expression of ARF6 in transfected cells was barely a fold above endogenous protein expression. Note that unlike the data in Figure 8B, exogenous protein did not migrate at a higher molecular weight compared with endogenous ARF6 since a lower percentage SDS gel was used.

MDCK cell lines stably transfected with a temperature-sensitive mutant of v-src (Birchmeier and Birchmeier, 1993) were grown at 41°C (non-permissive temperature for pp60^v-src activity) in DMEM supplemented with 10% fetal bovine serum. Cells were then switched to 35°C for 4 h for experimentation at permissive temperatures. For the generation of ARF6(T27N)-expressing stable cell lines, cells were then transfected using the retroviral transfection system and further selected using G418 as described earlier.

Immunofluorescent labeling

Procedures used for immunofluorescent staining and microscopy were conducted as described previously (Boshans et al., 2000). In the case of filter-grown cells, immunolabeling was conducted in a transwell chamber and both sides of the filter were labeled as indicated. Anti-E-cadherin mouse monoclonal antibody (3B8) was a kind gift from Dr Warren Gallin (University of Alberta). The anti-mouse monoclonal antibody against ZO-1 (R26.4C) was obtained from the hybridoma bank, University of Iowa. Anti-β-catenin mouse monoclonal antibody was obtained from Transduction Laboratories. Anti-mouse monoclonal and anti-rabbit polyclonal antibodies against HA were obtained from BabCo. Anti-transferrin receptor mouse monoclonal was from Zymed. Anti-gp135 (3F2) was a kind gift of Dr George Ojakian. Rhodamine–phalloidin was obtained from Molecular Probes.

Cell migration assays

MDCK cells stably expressing either ARF6, ARF6(Q67L), ARF6(T27N) or ARF6(Q67L:Q37E:S38I) were grown in normal growth medium on 8 µm pore size polycarbonate transwell filters (Costar). Migration of cells into the lower chamber containing conditioned medium (Kleinman and Jacob, 1998) was followed every 4 h. After incubation overnight, the cells in the lower chamber were fixed and counted by phase-contrast microscopy. In a separate set of experiments, migration assays were carried out as described, except that 20 ng/ml HGF (Calbiochem) was added to medium in the upper chamber, 2 h after seeding cells on filters.