Index Introduction Materials and Methods Cell culture and antibodies Targeting vectors, gene targeting and Southern blot analysis Expression vectors and transfection Western blot analysis Immunofluorescent staining E-cadherin internalization assay Results Isolation of F9 cells which express only a trace amount of HA-tagged p120 The expression of E-cadherin, β-catenin, plakoglobin and α-catenin was also reduced in p120KI cells Expression of the Arm-repeat domain of p120 is sufficient for normal expression of E-cadherin Large cytoplasmic E-cadherin complex aggregates were detected in p120KI cells Internalization of cell surface E-cadherin in p120KI cells The potential regulatory role of p120 on cytoplasmic E-cadherin trafficking Discussion Isolation of p120KI cells expressing trace levels of p120 molecules Cytoplasmic E-cadherin aggregates Possible involvement of the C-terminus of p120 in trafficking of E-cadherin from the cytoplasm to the plasma membrane or lysosomes Acknowledgements References

Introduction

Cadherins (classical cadherins) are transmembrane cell surface molecules that play a key role in multicellular organization through the establishment of calcium-dependent homophilic interactions at cell-cell contact sites (Takeichi, 1991; Takeichi, 1995). The intracellular domains of cadherins associate with several cytoplasmic proteins including β-catenin (or its homologue, plakoglobin), α-catenin and p120 (also known as p120 catenin). β-catenin (or plakoglobin) interacts directly with both the cytoplasmic tail of cadherin and α-catenin (Barth et al., 1997). α-catenin links the cadherin-catenin complex to the actin cytoskeleton (Knudsen et al., 1995; Nagafuchi et al., 1991; Rimm et al., 1995; Watabe-Uchida et al., 1998). p120 binds directly to the juxtamembrane domain (JMD) of the cadherin tail, but not to α-catenin (Daniel and Reynolds, 1995; Yap et al., 1998). Cadherins are not always located at adhesion junctions. These molecules also spend variable amounts of time in vesicles trafficking to and from the cell surface (Bryant and Stow, 2004). For example, it was reported that E-cadherins are rapidly removed from the plasma membrane and subsequently recycled to sites of new cell-cell contact, when epithelial cells need to become motile such as during gastrulation, developmental morphogenesis or wound healing (Gumbiner, 2000). Therefore, cadherin-based adhesion does not represent a static state, but a dynamic equilibrium between cadherin complexes at the adhesive junctions and those in intracellular vesicles and compartments.

Recently, several reports revealed that p120 plays a set point function for the turnover of cadherins. p120 represents a prototypical member of a subfamily of armadillo (Arm)-domain proteins involved in intercellular adhesion and in nuclei (Anastasiadis and Reynolds, 2000). It was initially discovered as a substrate for Src (Reynolds et al., 1992; Reynolds et al., 1989) and various other tyrosine kinases (Downing and Reynolds, 1991; Kanner et al., 1991), and subsequently shown to interact with the JMD of classical cadherins (Daniel and Reynolds, 1995; Yap et al., 1998). An investigation of p120-deficient SW48 carcinoma cells showed that restoring p120 expression efficiently rescued proper epithelial morphology by stabilizing E-cadherin and increasing its abundance (Ireton et al., 2002). Previous reports showed that the expression of extracellular domain deleted dominant-negative (DN) cadherins typically downregulated endogenous cadherins (Fujimori and Takeichi, 1993; Kintner, 1992; Nieman et al., 1999; Troxell et al., 1999; Zhu and Watt, 1996). Overexpression of mutated DN-VE-cadherin constructs lacking either the p120 or β-catenin binding sites showed that downregulation of endogenous VE-cadherin was caused by sequestration of p120, but not β-catenin, indicating that p120 plays an essential role in the regulation of cadherin stability (Xiao et al., 2003). Recent studies investigating the downregulation of p120 by application of the siRNA technique further indicated that p120 could regulate cadherin expression levels (Davis et al., 2003; Xiao et al., 2003). The rheostatic effect of p120 is exerted partly by the regulation of E-cadherin endocytosis, presumably for the purposes of degradation. However, the mechanism related to the retention of E-cadherin in the endosomal compartment, targeting it for degradation in the lysosomes, or recycling back to the plasma membrane, remains unknown.

Here, by application of a gene trap targeting technique to mouse teratocarcinoma F9 cell line, we obtained a p120 knock-in (p120KI) cell line stably expressing trace levels of exogenous HA-tagged p120, but not endogenous p120. We demonstrated that the expression of E-cadherin, β-catenin, plakoglobin and α-catenin decreased. Internalized E-cadherin was detected as large aggregates in the cytoplasm of p120KI cells. Expression of wild type and various mutant p120 molecules in p120KI cells revealed that the Arm-repeat domain of p120 alone was sufficient in restoring the expression of E-cadherin. It also suggested that the C-terminus of p120 was involved in regulation of the trafficking of internalized E-cadherin.

Materials and Methods

Cell culture and antibodies

Mouse F9 teratocarcinoma cells and their derivatives were cultured in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal calf serum. Culture dishes and cover slips were pre-coated with 0.2% gelatin for 15 minutes.

The following primary antibodies were used for the immunofluorescent staining and Western blot analyses: mouse anti-p120, mouse anti-β-catenin and mouse anti-plakoglobin mAbs (BD Transduction Laboratories), rat anti-mouse E-cadherin mAb (ECCD-2) (Shirayoshi et al., 1986), rat anti-α-catenin mAb (α18) (Nagafuchi and Tsukita, 1994), mouse anti-HA tag mAb (12C5A, Roche), rat anti-mouse LAMP-1 mAb (1D4B; Southern Biotechnology Associates, Inc.), rabbit anti-E-cadherin pAb (Nagafuchi et al., 1987), anti-GFP polyclonal antibody (pAb) (Molecular Probes), anti-α-tubulin mAb (DM1A; Sigma-Aldrich). FITC- or Cy3-conjugated donkey anti-rabbit, anti-mouse and anti-rat IgGs (Jackson Immunoresearch) were used as secondary antibodies for the immunofluorescent staining. HRP-conjugated secondary antibodies (Amersham Biosciences) were used for Western blot analyses.

Targeting vectors, gene targeting and Southern blot analysis

The 43.8 kb p120 genome clone was isolated as three fragments from a 129/Sv mouse genomic library. One short gap was connected using PCR. For the p120 knock-out (KO) and the p120KI targeting vectors, two cassettes, the PESIBAP trap-selection and P(p120’HA)IBP knock-in (KI) cassettes, were initially constructed. In PESIBAP, an en-2 sequence with a splice acceptor (Mountford et al., 1994), IRES (internal ribosome entry site) and β-geo sequences were connected in tandem and then inserted between two loxP sequences. p(p120’HA)IBP KI cassette was constructed whereby p120’HA, IRES and β-geo sequences were connected in tandem and then inserted between two loxP sequences. In p120’HA, a splice acceptor region of the ATG exon of the p120 genome and one of the internal introns (indicated by black arrow) (Fig. 1C) were added into a full-length p120 cDNA (see below) in which the stop codon was replaced with a HA-tag sequence. Further, a loxP sequence was inserted into the intron. The 27.5 kb EcoRI-ApaI fragment of mouse p120 genomic DNA contains the complete exons for the ORF. For construction of the KO and KI targeting vectors, PESIBAP and P (p120’HA)IBP, respectively, took the place of the 23.4 kb SacI-ApaI fragment of this p120 genomic DNA that contains almost all of the exons except for the latter half of the polyA exon. All targeting vectors were constructed based on the pBluescript SK(–) plasmid (Stratagene).

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Fig. 1. Isolation of p120KI cells. A: The structure of the wild-type allele, KO targeting vector (KO), targeted allele, and the PESIBAP trap-selection cassette are shown together with the pertinent restriction sites. B: The structure of the wild-type allele, the p120 knock-in targeting vector, targeted allele, and P(p120’HA)IBP trap-selection cassette are shown together with the pertinent restriction sites. The actual recombination site in the p120 cDNA sequence is indicated by a black triangle. In panels A-B, the probe is shown as a small closed box. Insertion of each trap cassette resulted in the generation of a new BamHI fragment of 4.9 kb, which hybridizes with the probe in Southern blot. C: Southern blot analysis of DNA derived from each cell clone. Genomic DNA was digested with BamHI and hybridized with the probe indicated in panel A-B. p120SKO, p120DKO: single and double KO cells, respectively. p120KI, p120DKO cells with the third allele of p120 recombined with the p120KI targeting vector.

p120 single KO (p120SKO) cells were then isolated as follows. The p120 KO vector (25 μg) was linearized with NotI and electroporated at 250 V and 960 μF using a gene pulser into 2×10⁷ F9 cells in 0.4 ml of HEPES-buffered saline. Cells were then subjected to G418 (Nacalai Tesque, Inc.) selection at 400 μg/ml. For the isolation of the p120 double KO (p120DKO) cells, p120SKO cells were subjected to 8 mg/ml G418 selection, until colonies emerged from single cells. To isolate p120KI cells, the drug resistance cassettes in the p120DKO cells were initially removed by the transient expression of Cre recombinase, and G418-sensitive cells were subsequently isolated. The G418-sensitive p120DKO cells were re-electroporated with the p120KI targeting vector and then subjected to 400 μg/ml G418 selection. (We also attempted to isolate p120 KO cells. For this purpose, p120DKO cells were subjected to 16 mg/ml G418 selection. Furthermore, the G418-sensitive p120DKO cells were re-electroporated with the p120KO targeting vector and then subjected to 400 μg/ml G418 selection. Although more than 1600 and 600 colonies, respectively, were picked in these experiment, we could not isolate cells lacking p120 expression (data not shown).)

Genomic DNA preparation and Southern blot analysis were performed as described previously (Maeno et al., 1999; Saitou et al., 1997). The probe for Southern blot analysis was a 400 bp fragment located immediately downstream from the region of homology of the targeting vector (Fig. 1). The primers for this probe fragment were; P1: 5'-ACCTGTAGTCTTGGAGCTGA-3', and P2: 5'-GCGCTTATAGGTCAGTCCAA-3'.

Expression vectors and transfection

pCAG-NGFP (Matsuda et al., 2004) was used for the construction of GFP fusion protein expression vectors. The p120 cDNA used in this paper was isolated from the cDNA library of F9 cells. The cDNA is 98% identical to mouse p120 cDNA (Z17804). Since the cDNA sequence contains exon C, F9 cells may express p120-1C type polypeptide as a major p120 protein. To construct an expression vector (pCAG-NGFP-wp120) for the GFP-p120 fusion, the XhoI-EcoRI fragment of the p120 cDNA sequence, which contains the whole ORF, was inserted into XhoI-EcoRI sites of pCAG-NGFP. The XhoI site in p120 cDNA was artificially inserted in place of an initiation ATG codon. Expression vectors for all GFP-mutant p120 fusions were generated based on pCAG-NGFP-wp120 using PCR. The nucleotide sequence of all PCR products was checked by direct sequencing. Deletion mutants were denoted with missing amino acid sequences presented in parentheses: ΔN1 (1–267); ΔN (1–346); ΔA1 (352–645); ΔC (832–911). For general understanding, the amino acid number was based on the amino acid numbering system for mouse p120-1 composed of 911 amino acids (Anastasiadis and Reynolds, 2000). In the p120-1C type polypeptide, 6 amino acids were inserted between residues 625 and 626 of the p120-1 polypeptide.

The expression vectors were transfected into p120KI cells using the Lipofectamine 2000 system (GIBCO BRL). Stable colonies were isolated as follows. p120KI cells were co-transfected with an expression vector and the pGK-puromycin vector, and then subjected to puromycin selection at 1 μM for two weeks. At least 5 positive clones were isolated for each transfectant and one clone, which showed a common phenotype, was chosen as the representative clone.

Western blot analysis

SDS-PAGE and immunoblotting was performed as previously described (Nagafuchi and Tsukita, 1994). Samples were solubilized in SDS sample buffer and then separated by SDS-PAGE. Proteins were electrophoretically transferred onto nitrocellulose sheets for the immunoblotting. Nitrocellulose membranes were then incubated with primary antibodies. Antibody detection was performed using ECL (Amersham Biosciences). The signal was collected by LumiVision (TAITEC) and analyzed by LumiViewer 130 (TAITEC).

Immunofluorescent staining

Immunofluorescent staining was performed as follows. Cells cultured on 15-mm gelatin-coated cover slips were fixed with 3.7% formaldehyde for 15 minutes, washed with 0.2% Triton X-100 for 5 minutes, and then incubated with the primary and secondary antibodies as previously described (Imamura et al., 1999). Samples were then embedded using a SlowFade Light Antifade Kit (Molecular Probes). Images were acquired using the Acquavision 3.0.6 equipped with a microscope (model Axiovert 200; Carl Zeiss MicroImaging, Inc.) and AxioCAM-cooled CCD camera fitted with a 40/0.75 NA plan-Neofluar objective. Adjustment of brightness, contrast, color balance and final image size was achieved using Adobe ImageReady 7.0.1. An Olympus confocal microscope (model FLUOVIEW FV500; OLYMPUS) fitted with a 60/1.25 oil lris UPlanF1 objective was used to acquire the confocal images.

E-cadherin internalization assay

The E-cadherin internalization assay was performed using procedures adapted from Paterson et al. (Paterson et al., 2003). Cells were passaged 12 hours prior to analysis. On the day of the assay, the cell medium was changed to a balanced salt solution (Hepes-buffered saline (HCMF) (Takeichi, 1977) supplemented with 5 mM CaCl₂ and 50 mg/ml bovine serum albumin). Cells were then allowed to equilibrate for 1 hour at 37°C. Samples were then incubated for 1 hour at 4°C with anti-E-cadherin mAb ECCD-2 diluted in balanced salt solution. Coverslips were washed with ice-cold HMF (HCMF with 1 mM CaCl₂) to remove unbound antibody and the medium was replaced with a balanced salt solution pre-warmed to 37°C. Following incubation at 37°C for 10 minutes, cells were then washed with HMF and returned to 4°C. The residual surface-bound antibody was removed by washing three times for 5 minutes each in PBS (pH 2.7) containing 25 mM glycine and 3% BSA. Following fixation, cells were incubated with anti-β-catenin antibody. Anti-β-catenin antibodies and internalized ECCD-2 were detected with anti-mouse and anti-rat second antibodies, respectively.

Results

Isolation of F9 cells which express only a trace amount of HA-tagged p120

For the detailed analysis of p120 function, we first attempted to isolate p120KO F9 cells using a conventional p120KO target vector (Fig. 1A). In the course of targeting, it was found that there were three p120 alleles within the genome of F9 cells. Following introduction of the KO vector into F9 cells we isolated 1 recombinant clone, referred to as p120 single KO (p120SKO) cells, from approximately 600 clones picked. p120 single KO cells were then treated with a high concentration of G418 (8 mg/ml). From 100 colonies picked, we isolated 35 clones in which the second p120 allele was disrupted (p120 double KO cells (p120DKO)) (Fig. 1C). Although two alleles were successfully disrupted with the p120 KO vector, we never isolated cells in which all three p120 alleles had been disrupted after extensive screening of approximately 2000 clones (see Materials and Methods). Considering the possibility that complete loss of p120 was lethal to F9 cells, a p120 conditional KO vector (p120KI vector) was then constructed. In this p120KI vector, p120 cDNA sequence, which contains the splicing acceptor of ATG exon, an internal intron with loxP sequence and C-terminal HA tag, was replaced with SA sequence of p120 KO vector (Fig. 1B). The p120KI vector was then introduced into p120DKO cells and from 20 colonies picked, 2 clones were isolated in which the final allele had recombined (Fig. 1C). The recombination, however, had occurred in the latter part of the p120 cDNA in this KI vector and the first two loxP sequences were not included in the recombinant allele (Fig. 1B; the black triangle indicates the recombination site). Consequently, we were unable to isolate complete KO and proper KI cells in which p120 cDNA could be excised. We, however, isolated KI cells that expressed HA-tagged p120 protein but not endogenous p120 (Fig. 2A); this line was referred to as p120KI cells. Western blot analysis demonstrated that the expression level of p120 was seriously affected in the p120KI cells, probably due to genetic manipulation of the p120 genomes. The expression level of HA-tagged p120 protein in the p120KI cells was 5% compared to that of endogenous p120 in the parental F9 cells (Fig. 2A). We used these clones to investigate the role of p120 in cadherin transport inside cells.

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Fig. 2. Expression of cadherin-catenin complex components in p120KI cells. A: Western blot analysis of parental F9, p120SKO, p120DKO and p120KI cells with anti-p120 and anti-HA tag mAbs. α-tubulin levels were used as an internal control. B: Western blot analysis of parental F9, p120SKO, p120DKO and p120KI cells with anti-E-cadherin, anti-β-catenin, anti-plakoglobin and anti-α-catenin mAbs. α-tubulin levels were used as an internal control. Note that the expression level of E-cadherin, β-catenin, plakoglobin and α-catenin was also reduced in p120KI cells compared to that in other cells. C: F9 and p120KI cells were co-cultured and stained with anti-p120 and anti-E-cadherin mAbs. Note that the expression level of E-cadherin corresponded to that of p120. E-cad: E-cadherin. Phase: The phase contrast image. Bar: 20 μm.

The expression of E-cadherin, β-catenin, plakoglobin and α-catenin was also reduced in p120KI cells

It was reported that a deficiency in p120 could reduce the expression level of cadherins in epithelial and endothelial cells (Davis et al., 2003; Xiao et al., 2003). We then set out to determine whether a low level of p120 has the same effect on the expression level of the E-cadherin complex in nonepithelial F9 cells. Protein samples isolated from confluent cultured F9 parental cells, p120SKO cells, p120DKO cells and the p120KI cells were subjected to Western blot analysis. The Western blot results were enhanced by ECL methods and the amount of each protein was quantified by densitometry. The expression level of E-cadherin in p120KI cells was reduced to about 25%, compared to that of the parental F9 cells (Fig. 2B), whereas E-cadherin mRNA expression is not affected in p120KI cells (data not shown). On the other hand, the expression level of E-cadherin was not affected in p120SKO and p120DKO cells, and was similar to the normal expression of p120 in those cells. These data are consistent with the results obtained from p120 knock-down epithelial or endothelial cells expressing E-cadherin, N-cadherin, VE-cadherin and P-cadherin (Davis et al., 2003; Xiao et al., 2003). The expression level of β-catenin, plakoglobin and α-catenin was also reduced in p120KI cells (Fig. 2B). The immunostaining results for the mixed-culture of p120KI cells and parental F9 cells also demonstrated that the cells displaying weak p120 staining also showed weak E-cadherin staining (Fig. 2C). Unlike the results from the p120 knock-down epithelial cells, however, the components of the E-cadherin complex could still be detected at cell-cell boundaries, although these were weaker than those of parental F9 cells. (Fig. 2C), When HA-tagged p120 was stably expressed in p120KI cells, expression of E-cadherin, β-catenin and α-catenin was restored (data not shown). This indicated that decrease of p120 expression mainly caused low level expression of these molecules and that HA-tagged p120 did not function as a dominant-negative molecule.

Expression of the Arm-repeat domain of p120 is sufficient for normal expression of E-cadherin

p120 is composed of three domains: a central Arm-repeat domain, and N- and C- terminal tails. In an effort to delineate the potential function of these different domains on the restoration of E-cadherin expression, wild-type, partial N-terminal (ΔN1), full N-terminal (ΔN), Arm 1–7 (ΔA1) and C-terminal (ΔC) p120 deletions were introduced into p120KI cells and stable transfectants were isolated. The structures of these various p120 molecules are shown in Fig. 3A. The N-terminus of all these molecules was linked to GFP (green fluorescent protein) molecules, so that the expression of exogenous p120 variant molecules could be achieved using anti-GFP antibody. The expression level of exogenous p120 variant molecules and endogenous E-cadherin was examined by Western blot analysis. The expression of E-cadherin could be restored by wild-type p120, ΔN1p120, ΔNp120 and ΔCp120, but not by ΔA1p120 which lacks most of the Arm-repeat sequences of p120 (Fig. 3B). Since the p120 N- and C-terminal deletions could restore the expression of E-cadherin, we postulated that the p120 Arm-repeat domain was sufficient for maintaining the stability of E-cadherin. To confirm this hypothesis, we set out to determine whether expression of the p120 Arm-repeat domain affected the expression level of E-cadherin. As expected, the expression of the Arm-repeat domain alone was able to restore the expression of E-cadherin (Fig. 3B, Arm).

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Fig. 3. Restoration of E-cadherin expression level in p120KI cells is dependent on expression of the Arm-repeat domain of p120. A: Schematic structure of GFP fusion proteins with wild-type and p120 deletion mutants. The deleted amino acids in each mutant are described in Materials and Methods. B: Western blot analysis of p120KI cells expressing various GFP-p120 fusion proteins with anti-E-cadherin, anti-GFP Abs. The expression vectors for the GFP fusions were introduced into p120KI cells and stable transfectants were isolated. α-tubulin levels were used as a control. Note that the expression of wild-type (WT) p120 or its mutants containing the full Arm-repeat domain, ΔN1, ΔN, ΔC and Arm restored E-cadherin expression.

Large cytoplasmic E-cadherin complex aggregates were detected in p120KI cells

Contrary to the weak junctional staining of E-cadherin, cytoplasmic large aggregates of E-cadherin were detected in p120KI cells. Staining with anti-E-cadherin and anti-β-catenin antibodies (Fig. 4A) or anti-E-cadherin and anti-plakoglobin antibodies (Fig. 4B) clearly showed that β-catenin and plakoglobin were colocalized with the cytoplasmic pool of E-cadherin. The frequency of cytoplasmic E-cadherin aggregates in p120KI cells ranged from about 10% to 50% depending on unidentified condition. Considering that the expression level of E-cadherin was reduced, we suspected that cytoplasmic E-cadherin was actively degraded in p120KI cells. To address whether lysosomes are involved in E-cadherin aggregate formation, we compared localization of LAMP-1, a type of late endosome/lysosome marker with that of E-cadherin in parental F9 cells and p120KI cells. Immunostaining showed that the localization of the LAMP-1-positive vesicles was related to the cytoplasmic E-cadherin aggregates. Many LAMP-1-positive vesicles underwent a change in that they became concentrated in the vicinity of cytoplasmic E-cadherin aggregates, and enveloped these aggregates in p120KI cells (Fig. 5). The vesicles showed random distribution in parental F9 cells (Fig. 5), implying that LAMP-1 positive lysosomes may be involved in determining the fate of the cytoplasmic E-cadherin complexes.

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Fig. 4. Cytoplasmic E-cadherin complex aggregates frequently detected in p120KI cells. p120KI cells were double-labeled with E-cadherin and β-catenin (A) or E-cadherin and plakoglobin (B). E-cad: E-cadherin; β-cat: β-catenin; plako: plakoglobin. Note that, in addition to weak junctional E-cadherin signals, cytoplasmic E-cadherin aggregate signals were frequently detected in p120KI cells. β-catenin and plakoglobin colocalized with both junctional and cytoplasmic E-cadherin. Bar: 20 μm.

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Fig. 5. Altered distribution of LAMP-1 in p120KI cells. F9 cells and p120KI cells were double-labeled with E-cadherin and LAMP-1. Immunofluorescent staining shows that large aggregates of LAMP-1 localized close to cytoplasmic E-cadherin vesicles in p120KI cells, whereas in parental F9 cells, small aggregates of LAMP-1 were randomly distributed in the cytoplasm. Bar: 10 μm.

Internalization of cell surface E-cadherin in p120KI cells

It was reported that loss of p120 did not affect the arrival of E-cadherin to the cell surface but affected the stability of surface E-cadherin (Davis et al., 2003; Ireton et al., 2002; Miranda et al., 2003). An internalization assay was performed in an effort to determine whether surface E-cadherin was internalized into cytoplasm and incorporated into these cytoplasmic large aggregates. Cells were initially labeled with anti-E-cadherin antibody ECCD-2 at 4°C for 1 hour. Cells were then rinsed and placed at 37°C for 10 minutes to facilitate the internalization of E-cadherin. Antibody bound to cell surface E-cadherin was then removed using a low pH wash. Following fixation, cells were incubated with anti-β-catenin antibody. The anti-β-catenin antibodies and internalized ECCD-2 were detected using anti-mouse and anti-rat secondary antibodies, respectively. In p120KI cells, clear cytoplasmic E-cadherin signals were detected, which colocalized with cytoplasmic β-catenin (Fig. 6), indicating that the cytoplasmic E-cadherin in p120KI cells included internalized E-cadherin. In parental F9 cells, cytoplasmic E-cadherin and β-catenin signals were negligible.

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Fig. 6. Internalized E-cadherin retained in the cytoplasmic E-cadherin aggregates. In an effort to determine whether surface E-cadherin was internalized into the cytoplasm, cell surface E-cadherin of F9 and p120KI cells was labeled in living cells using anti-E-cadherin mAb ECCD-2 at 4°C. Cells were rinsed and then placed at 37°C for 10 minutes to facilitate E-cadherin internalization. After internalization, the residual surface-bound antibody was removed. Following fixation, cells were incubated with anti-β-catenin antibody. Anti-β-catenin antibodies and internalized ECCD-2 were detected with anti-mouse and anti-rat secondary antibodies, respectively. Note that E-cadherin was actively internalized in p120KI cells, and that internalized E-cadherin colocalized with cytoplasmic β-catenin. E-cad: E-cadherin; β-cat: β-catenin. Bar: 20 μm.

The potential regulatory role of p120 on cytoplasmic E-cadherin trafficking

Internalized E-cadherin has many fates, including the recycling of E-cadherin back to the cell surface or its transient sequestration inside the cell (by sorting or recycling endosomes) and/or routing for eventual degradation (Bryant and Stow, 2004). In an effort to investigate whether p120 is involved in regulating the trafficking of internalized E-cadherin, wild-type, N-terminal (ΔNp120) and C-terminal (ΔCp120) p120 deletion mutants were transiently introduced into p120KI cells. Forty-eight hours following transfection, the localization of E-cadherin and all p120 variants was visualized by immunofluorescent staining. A significant increase in E-cadherin signals at cell-cell boundaries was observed in cells transiently expressing wild-type p120 and ΔNp120. Furthermore, the obvious cytoplasmic aggregates of E-cadherin also disappeared in those cells (Fig. 7, a, a', b, b'). In cells transiently expressing ΔCp120, however, the distribution of E-cadherin differed from that found in cells expressing wild-type and ΔNp120. Only a small increase in E-cadherin signals at the cell-cell boundaries was detected. In a part of the cells transiently expressing ΔCp120, the cytoplasmic accumulation of E-cadherin was observed (Fig. 7, c'). The ΔCp120 colocalized with cytoplasmic E-cadherin and surface E-cadherin (Fig. 7, c). The cytoplasm staining of E-cadherin in p120KI cells expressing ΔCp120 was much stronger than in the surrounding cells. Further staining showed that ΔCp120 also colocalized with β-catenin and plakoglobin in the cytoplasm (data not shown). The retention of these cytoplasmic E-cadherin complex aggregates in p120KI cells transiently expressing ΔCp120 indicated that the C-terminus of p120 is involved in the regulation of internalized E-cadherin trafficking in the cytoplasm.

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Fig. 7. Transient expression of p120, ΔNp120 and ΔCp120 in p120KI cells. p120KI cells were transfected with wild-type, ΔNp120 and ΔCp120. After 48 hours, cells were fixed and immunofluorescent staining was performed using anti-E-cadherin and anti-GFP antibodies. In cells transiently expressing wild-type and ΔNp120 (a, a', b, b'), a significant increase in E-cadherin staining at the cell-cell boundary was observed. The cytoplasmic aggregates of E-cadherin became unclear. In cells transiently expressing ΔCp120, E-cadherin signals at the cytoplasmic aggregates increased significantly (c, c'), concomitant with a small increase in staining of E-cadherin at the surface. E-cad: E-cadherin. Bar: 20 μm.

Discussion

Isolation of p120KI cells expressing trace levels of p120 molecules

p120, also known as p120 catenin, is a substrate of src tyrosine kinase and recently classified as a member of the cadherin-binding proteins. Its role in the cadherin-mediated cell adhesion system, however, remains largely unknown. Here, a stable cell line was isolated that completely lost the expression of endogenous p120 molecules but expressed trace levels of exogenous HA-tagged p120 molecules. Although our first aim was to acquire p120 KO cells, this was not achieved, even after extensive screening with the use of two different targeting vectors.

The KO vector was not a promoter-enhancer trap-type but a promoter trap-type construct, in which an enhancer sequence was included. Using this vector, we were able to isolate cells with the appropriate recombination. We were unable, however, to isolate complete p120KO cells after extensive screening. One possible reason is that the complete loss of p120 expression could be lethal to F9 cells. It was reported that p120 is essential for Xenopus embryogenesis (Ciesiolka et al., 2004; Fang et al., 2004). Davis et al. (Davis et al., 2003) also discussed that murine p120KO is embryonic lethal. On the other hand, experiments in C. elegans and Drosophila indicated that p120 is not essential in these organisms (Myster et al., 2003; Pettitt et al., 2003). Moreover, recent reports indicated that conditional KO of p120 in mouse epidermis and salivary gland caused severe defects in inflammatory response and cell differentiation, respectively, but not cellular lethality (Davis and Reynolds, 2006; Perez-Moreno et al., 2006). These data suggested that if p120 is required for the survival of cells, the requirement is depending on cellular context. p120 protein is involved in not only cadherin-mediated cell adhesion but also other various cellular events including actin-based cytoskeletal organization. Another possibility of our failure to isolate p120KO cells is that abrupt loss of p120 expression might affect cell division through disorganization of actin-based cytoskeleton. In fact, growth of colonies of p120KI cells was significantly slower than that of normal F9 cells, during initial isolation of knock-in cells (our unpublished observation). Once stable p120KI cells were isolated, however, the growth rate of p120KI cells is similar to that of parental F9 cells, even during cloning of cells. Further efforts to isolate p120 conditional KO F9 cells will help us to understand the role of p120 in terms of the survival or maintenance of cells.

Cytoplasmic E-cadherin aggregates

Immunofluorescence-based internalization assay showed that cytoplasmic E-cadherin aggregates contained E-cadherin internalized from the cell surface. To quantify the internalization, we attempted biotinylation-based internalization assay. However, biotinylated E-cadherin in cytoplasm was under detection sensitivity. In p120KI cells, weak cell surface E-cadherin was detected. This E-cadherin, however, might form complex with HA-tagged p120 expressed in these cells. When E-cadherin failed to interact with p120, they might be quickly internalized and barely present on plasma membrane in p120KI cells. As a result, internalization of E-cadherin from cell surface would be detected only by sensitive immunofluorescence-based assay but not by biotinylation-based one.

It was reported that internalized E-cadherin possessed many goals that depended on the cell lines used and the signals that induced internalization. For example, the E-cadherin complex can be internalized into a unique storage compartment by calcium depletion in intestinal epithelial T84 cells (Ivanov et al., 2004), whereas Src activation induced internalization of E-cadherin into lysosomes and prevented its recycling to the basolateral plasma membrane (Palacios et al., 2005). In p120KI cells, E-cadherin seems to be degraded in lysosome. The E-cadherin aggregates in p120KI cells possess a relationship with LAMP-1-positive vesicles. In p120KI cells, LAMP-1-positive vesicles formed large aggregates localized in the vicinity of cytoplasmic E-cadherin aggregates. It has been reported that the degradation of E-cadherin could be inhibited by the lysosome inhibitor chloroquine in cells expressing low levels of p120 (Davis et al., 2003; Xiao et al., 2003). Since chloroquine is severely toxic to F9 cells, we could not examine the effect of chloroquine treatment on E-cadherin expression by Western blot analysis. However, we observed an increase in cytoplasmic E-cadherin aggregates in p120KI cells just following treatment with chloroquine (data not shown). Since LAMP-1 is a marker of late endosome/lysosomes, it was likely that lysosomes were mobilized in p120KI cells in terms of facilitating the degradation of internalized E-cadherin. It remains to be determined exactly how the degradation system is activated following a change in the expression level of p120. Although decrease of cadherin expression in p120-deficient cells was reported previously, formation of cytoplasmic aggregates of cadherins was not. Even in our p120KI cells, cytoplasmic aggregates of cadherin were not constantly observed. These data suggested that internalized cadherins were actively destructed in lysosomes or recycled to plasma membrane. In p120KI F9 cells these processes might be slower than in endothelial or epithelial cells used in previous works.

In p120KI cells, β-catenin and plakoglobin were colocalized with cytoplasmic cadherin aggregates, suggesting that these catenins could not prevent internalization of cadherin in the absence of p120. Consistent with these results, it was reported that cadherin is associated with β-catenin when it was internalized for recycling (Le et al., 1999). On the other hand, it was also reported that internalized VE-cadherin was not colocalized with β-catenin and α-catenin, when cells were treated with chloroquine, a lysosome inhibitor (Xiao et al., 2003). Degradation mechanism of cadherin thus might be different from that of α- and β-catenins.

Possible involvement of the C-terminus of p120 in trafficking of E-cadherin from the cytoplasm to the plasma membrane or lysosomes

It was recently reported that p120 prevented the consecutive internalization and degradation of VE-cadherins (Xiao et al., 2003). The stable expression of p120 or its mutants in p120KI cells confirmed this hypothesis. The stable expression of molecules including the Arm-repeat domain of p120 restored the expression of E-cadherin. The Arm-repeat domain was reported to be involved in the binding to E-cadherin JMD (Thoreson et al., 2000). This leads us to speculate that the direct binding of the Arm-repeat domain to E-cadherin JMD might be necessary for maintaining the stability of E-cadherin on plasma membrane.

The N- and C-terminal regions of p120 were not required for the stable expression of E-cadherin. Although it was reported that the N-terminal region of p120 is involved in the regulation of cadherin adhesion activity (Aono et al., 1999), the role of the C-terminal region in the cell adhesion system remains unclear. Using transient expression analysis in p120KI cells, we found that the C-terminal region of p120 was probably involved in the recruitment of cytoplasmic E-cadherin to the plasma membrane. The transient expression of wild-type or N-terminal deleted p120 could increase the level of surface E-cadherin while diminishing the level of cytoplasmic E-cadherin in p120KI cells. It is plausible that the expression of exogenous p120 could decrease the internalization of cell surface E-cadherin, leading to a reduction in cytoplasmic E-cadherin vesicles. This is consistent with the results reported by Xiao and colleagues using VE-cadherin positive cells (Xiao et al., 2003). The results achieved from the transient expression of ΔCp120, however, are quite different from those observed for wild-type and ΔN p120. Beside the small increase in E-cadherin levels at the cell-cell contacts, cytoplasmic E-cadherin also increased in p120KI cells transiently expressing ΔCp120. As discussed above, the ΔCp120 molecules showed similar function to wild-type p120 molecules in terms of stabilizing surface E-cadherin molecules. Just as in p120KI cells stably expressing wild-type p120, E-cadherin was also predominantly localized at cell-cell boundaries in p120KI cells stably expressing ΔCp120 (data not shown). This means that a decrease in internalization alone is not sufficient for the clearance of cytoplasmic E-cadherin and substantial increase of cell surface E-cadherin in transiently transfected p120KI cells. The apparent discrepancy could be explained as follows. Transiently expressed p120 and its mutants might preferentially interact with cytoplasmic E-cadherin and prevent degradation in lysosomes. Wild-type and N-terminal-deleted p120 could then efficiently recycle E-cadherin onto cell surface, but C-terminal-deleted p120 could not. As a result, cytoplasmic E-cadherin aggregates are disappeared in cells expressing wild-type and N-terminal deleted p120. In contrast, cytoplasmic E-cadherin would increase in C-terminal deleted p120 expressing cells until excess amount of mutant molecules prevent internalization of surface E-cadherin. In stable transfectants, constitutive suppression of internalization might prevent formation of cytoplasmic E-cadherin aggregates. It was previously reported that cadherin is constantly trafficked through an endocytic, recycling pathway (Bryant and Stow, 2004). The C-terminal region lacking in ΔCp120 might be involved in this recycling machinery. Thus p120 might possess two functions. Firstly, p120 stabilizes surface E-cadherin at cell-cell contacts, protecting surface E-cadherin from being internalized. Secondly, p120 clears internalized E-cadherin in the cytoplasm and sorts it to the plasma membrane.

In conclusion, this work showed that the armadillo repeat domain and the C-terminal region of p120 possesses critical functionality in terms of E-cadherin stabilization on plasma membrane and trafficking at the cytoplasm, respectively. Defining the mechanism underlying these processes will provide further insight into our understanding of the mechanisms regulating cadherin-mediated cell adhesion.

Acknowledgements

We would like to thank Masatoshi Takeichi for kindly providing the ECCD-2 monoclonal antibody, Miho Matsuda for pCAG-NGFP, and Osamu Chisaka for 129/Sv mouse genomic library. We would also like to thank Chigusa Fujiwara and Erika Morikawa for their excellent technical assistance.

Part of this work at the Division of Cellular Interactions of the Institute of Molecular Embryology and Genetics of Kumamoto University was supported by a grant to A.N. from the Core Research for Evolutional Science and Technology (CREST); Grants-in-Aid for Scientific Research and Cancer Research and a Grant-in-Aid for 21st Century COE Research “Cell Fate Regulation Research and Education Unit” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. H.L. was a postdoctoral fellow of Japan Society for the Promotion of Science (JSPS).