1 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5426; and 2 Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202-5116
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ABSTRACT |
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The role of E-cadherin, a calcium-dependent adhesion protein, in organizing and maintaining epithelial junctions was examined in detail by expressing a fusion protein (GP2-Cad1) composed of the extracellular domain of a nonadherent glycoprotein (GP2) and the transmembrane and cytoplasmic domains of E-cadherin. All studies shown were also replicated using an analogous cell line that expresses a mutant cadherin construct (T151) under the control of tet repressor. Mutant cadherin was expressed at ~10% of the endogenous E-cadherin level and had no apparent effect on tight junction function or on distributions of adherens junction, tight junction, or desmosomal marker proteins in established Madin-Darby canine kidney cell monolayers. However, GP2-Cad1 accelerated the disassembly of epithelial junctional complexes and delayed their reassembly in calcium switch experiments. Inducing expression of GP2-Cad1 to levels approximately threefold greater than endogenous E-cadherin expression levels in control cells resulted in a decrease in endogenous E-cadherin levels. This was due in part to increased protein turnover, indicating a cellular mechanism for sensing and controlling E-cadherin levels. Cadherin association with catenins is necessary for strong cadherin-mediated cell-cell adhesion. In cells expressing low levels of GP2-Cad1, protein levels and stoichiometry of the endogenous cadherin-catenin complex were unaffected. Thus effects of GP2-Cad1 on epithelial junctional complex assembly and stability were not due to competition with endogenous E-cadherin for catenin binding. Rather, we suggest that GP2-Cad1 interferes with the packing of endogenous cadherin-catenin complexes into higher-order structures in junctional complexes that results in junction destabilization.
E-cadherin; adherens junction; tight junction
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INTRODUCTION |
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EPITHELIAL CELL-TO-CELL junctional complexes include tight junctions (zonula occludens; ZO), adherens junctions (zonula adherens), and desmosomes (macula adherens) (11). These junctions maintain structural integrity and physiological function of epithelial tissues and are a locus of intercellular signaling machinery. Adherens junctions, tight junctions, and desmosomes share a common structural organization; all three types of junctional complexes consist of transmembrane components with adhesive function and cytosolic adaptor proteins that provide a link to the cytoskeleton. In the case of adherens junctions, catenins link the transmembrane E-cadherin protein to the actin-based cytoskeleton (27, 50, 55; reviewed in 42, 63, 65). Tight junctions are also linked to the actin cytoskeleton; the transmembrane protein occludin binds directly to ZO-1, which binds actin and ZO-2 (16, 17, 21, 30, 62; reviewed in 5, 10, 64). Desmosomal cadherins, distantly related to E-cadherin, are linked to intermediate filaments through cytosolic proteins of the desmosomal plaque, including desmoplakin and plakoglobin (reviewed in 9, 20).
During junctional complex assembly, cadherin-catenin complexes are
stabilized at sites of cell-cell contact (2, 4, 37, 58) and
subsequently develop into mature adherens junctions. The cadherin
superfamily of adhesion molecules shares common structural features: an
extracellular domain with calcium-binding sequence repeat motifs, a
single transmembrane domain, and a COOH-terminal cytoplasmic domain
(39, 56; reviewed in 63). Classical cadherins (e.g., E-, P- and
N-cadherin) bind homotypically to identical family members on
neighboring cells (27, 47; reviewed in 63). The conserved cytoplasmic
domain interacts with the cytoskeleton, leading to strengthened
cell-cell adhesion (40, 49, 50; reviewed in 42, 63, 65). Specifically,
cadherin interactions with catenins (-catenin, 102 kDa;
-catenin,
97 kDa;
-catenin, also called plakoglobin, 86 kDa) have been
extensively characterized (1, 12, 25, 31, 36, 41, 49; reviewed in 42, 44). Cadherin-catenin complexes also mediate intracellular signaling
(reviewed in 6, 38, 54).
Coordinate assembly of junctional complexes is dependent on E-cadherin
(22, 23, 52, 53). Evidence placing E-cadherin at the top of the
hierarchy of epithelial junctional complex assembly has come largely
from experiments in which E-cadherin function was completely inhibited.
Incubating epithelial cells in low-calcium medium (LCM), or in medium
containing E-cadherin function-blocking antibodies, prevents assembly
of adherens junctions, tight junctions, and desmosomes (22, 23, 52, 53,
59). Furthermore, cells genetically lacking -catenin also lack
E-cadherin function, and junctional complexes do not assemble (67).
Several studies have employed mutant cadherins with a truncated
extracellular domain to study perturbations of cadherin-based adhesion
(3, 14, 32, 69). Formation of both adherens junctions and desmosomes was inhibited in cells overexpressing mutant cadherin molecules (3, 14,
69). How these mutant cadherin molecules generate an abnormal phenotype
is unclear. Some investigators have suggested that the phenotype is due
to competition between mutant cadherin and endogenous cadherin for
catenin binding, resulting in a decrease in catenin association with
endogenous cadherin that compromises cadherin-catenin complex function
(32, 69).
Here, we analyze the effects of mutant E-cadherin fusion proteins on the assembly and disassembly of cell-cell junctional complexes. Our results show that the mutant cadherin, when expressed at very low levels (10% that of endogenous E-cadherin), allowed assembly of functionally and morphologically normal junctions, but junctional complex stability and assembly rates were affected. Stoichiometry of mutant cadherin and endogenous cadherin and direct analysis of cadherin-catenin complexes in cells expressing low levels of mutant cadherin showed that competition for catenins was not responsible for the observed phenotype. We also found that mutant cadherin overexpression in Madin-Darby canine kidney (MDCK) cells reduced steady-state endogenous E-cadherin levels. Together, these results provide new insight into roles of E-cadherin in junctional complex organization and how mutant cadherin strategies affect cell-cell adhesion.
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MATERIALS AND METHODS |
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Cell culture and transfection. MDCK cells (type II, strain G; Ref. 35) and transfected cell lines were maintained in DMEM (GIBCO-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), penicillin, streptomycin, and glutamine (GIBCO-BRL), and cells were passaged by trypsinization.
MDCK IIG cells were cotransfected with a fusion protein (GP2-Cad1), composed of the extracellular domain of a nonadherent glycoprotein (GP2) and the transmembrane and cytoplasmic domains of E-cadherin, and with pSV2neo (60) using calcium phosphate (19). G418 (GIBCO-BRL, 0.4 mg/ml) was used to select for transfected clones. Drug-resistant colonies were isolated and were screened for GP2-Cad1 expression by immunofluorescence and immunoblotting using 4A9, an anti-GP2 monoclonal antibody (see below). More than 10 clones were isolated that all showed virtually identical immunofluorescence staining patterns and immunoblot profiles. Low-passage-number cells were used in all experiments (passage 15 or less). Two clones were extensively characterized. MDCK cells transfected with wild-type GP2 have been described previously (35).
MDCK T23 cells were cotransfected with pUC1-1 [which encodes glutathione S-transferase-E-cadherin (GST-ECad) fusion protein; see below] and pCB7 (which confers hygromycin resistance; Ref. 7) or cotransfected with pDOX151 (which encodes T151 mutant cadherin) and pCB7 using lipofectamine, according to the recommended protocol (GIBCO-BRL). Cells were selected using hygromycin, and positive clones were identified by immunoblotting with an anti-cadherin cytoplasmic domain polyclonal antibody (see below).
For experiments, GP2-Cad1 and control cells were plated at a density of 1.5 million per filter on six-well polycarbonate filter inserts (Corning-Costar, Kennebunk, ME) and were used 8 days after plating. Sodium butyrate was utilized to selectively but nonspecifically increase the expression of the stably integrated GP2-Cad1 sequences (see Ref. 18). For sodium butyrate induction, 5 mM sodium butyrate was added to both the apical and basal-lateral culture media 48 h before harvest; control medium and sodium butyrate-containing medium were replaced once ~24 h before harvest. Doxycycline treatment was performed by diluting 20 µg/ml stock solution in culture medium to 0.1 or 20 ng/ml concentration. Cells were maintained in 20 ng/ml doxycycline to prevent phenotypic drift. For experiments, T151 cells were cultured in the different doxycycline concentration for 5 days before experiments were performed.
Fusion protein construction. To create GP2-Cad1, the following oligonucleotides were used as PCR primers: a pair of complementary oligonucleotides with the COOH terminal sequence of rat GP2 [without the glycosyl phosphatidylinositol (GPI) modification signal] joined in frame with canine E-cadherin transmembrane sequences (italic) GP2-Cad1-1S (5' CAG AAT CCT GAT ACC TCC GCG CCT TAC GCC GAA GCA 3') and GP2-Cad1-3A (5' TGC TTC GGC GTA AGG CGC GGA GGT ATC AGG ATT CTG 3'); an oligonucleotide hybridizing with the COOH terminal of canine E-cadherin, including the stop codon, GP2-Cad1-2A (5' CGC GGG GCG GCC GCT TTA GTC GTC CTC GCC ACC TCC); an oligonucleotide hybridizing with the NH2 terminal of GP2, including the start codon, GP2-1S (5' CGC GGG AAG CTT AGG ATG GTG GCT TGT GAC 3'). GP2-1S and GP2-Cad1-3A were used to amplify GP2 using a cDNA clone of rat GP2 (a gift from Dr. Anson Lowe, Stanford University School of Medicine). GP2-Cad1-2A was used with GP2-Cad1-1S to amplify the transmembrane and cytoplasmic domains of canine E-cadherin using a cDNA clone of canine E-cadherin (a gift of Lee Rubin, Eisai London Lab, University College, London, UK) as template. The PCR products from the two reactions were mixed and used as template for amplification using oligonucleotides GP2-1S and GP2-Cad1-2A as primers. The final PCR product was then subcloned into the Hind III/Not I sites of CDM8 (Invitrogen, La Jolla, CA), behind the cytomegalovirus (CMV) promoter. The plasmid was named GP2-Cad1. The construct was confirmed by DNA sequencing (PAN facility, Stanford University).
T151 mutant cadherin construct was derived from mouse E-cadherin cDNA (pSUM, a gift of Rolf Kemler, MPI Freiberg; Ref. 61). This plasmid was digested with Aat II. Two Aat II internal fragments were deleted, removing 1,170 base pairs (390 amino acids) of the cadherin extracellular domain. The remaining E-cadherin sequences were religated. A HA epitope tag was inserted into the deleted cadherin plasmid by digesting with EcoR I, filling in with Klenow, and then ligating a 54-base pair HA sequence (derived from plasmid pSKHAV2, a gift of Drs. Lisa Elferink and Richard Scheller). The tagged cadherin mutant, T151, was directionally cloned into Bgl II/Kpn I sites of the eukaryotic expression vector pCB6+. To express T151 behind a tetracycline-repressible promoter, the entire T151 sequence was amplified with the following primers: 5pCB6 + Xba (5' CTC GTC TAG AGA ACC GTC 3') and 3pCB6 (5' GGC GAG GCA CTG GGG 3'). The PCR product was digested with Xba I and ligated to an Xba I-digested pUHD 10-3 vector. This plasmid was named pDOX151.
GST-ECad fusion protein was subcloned from a bacterial expression plasmid into a eukaryotic expression vector. PCR was used to amplify a previously described fusion protein consisting of GST and the cytoplasmic domain of mouse E-cadherin (34). The following oligonucleotides were used for PCR: 5GEXH3 (5' TCG TAT AAG CTT TGG AAT TGT GAG CGG 3') and 3GEXXba (5' GCG CGA TCT AGA TCG TCA GTC AG). The resulting PCR product was ligated into the Hind III/Xba I sites of pCB6+ (a gift of Dr. Vikas Sukhatme; Ref. 7) downstream of the CMV promoter, and named pUC1-1.
Antibodies and reagents. GP2
monoclonal antiserum, 4A9, was a kind gift from Dr. Anson Lowe and has
been described previously (13). HA monoclonal antibody 12CA5 was
purchased from BABCO (Berkeley, CA). The rr-1 hybridoma, recognizing
the extracellular domain of E-cadherin (22), and the ZO-1 hybridoma
(R26.4C; Ref. 62) were purchased from Developmental Studies Hybridoma
Bank maintained by the Department of Biological Sciences, The
University of Iowa, Iowa City, IA, under contract from the National
Institute of Child Health and Human Development. Conditioned ascites
fluid was used for rr-1 immunoprecipitation experiments, and
conditioned culture medium was used for rr-1 immunoblotting
experiments. Conditioned culture medium was used for all experiments
using the ZO-1 monoclonal antibody. Decma-1 ascites, which also
recognizes the E-cadherin extracellular domain (66), was purchased from
Sigma (St. Louis, MO). An anti-occludin polyclonal antibody was
purchased from Zymed (South San Francisco, CA). Antiserum to the
cadherin cytoplasmic domain (ECAD-B.5) was raised in rabbits against a
GST-ECad cytoplasmic domain fusion as antigen and has been
characterized (34). Anti--catenin and anti-
-catenin polyclonal
antisera were gifts from Dr. Inke Näthke and have been described
previously (26). The desmoplakin polyclonal rabbit antiserum was
described in Pasdar and Nelson (52).
Chemicals were purchased from Sigma or Midwest Scientific (St. Louis, MO) unless otherwise noted.
Calcium switch and immunofluorescence. For calcium removal experiments, 8-day filter-grown cells were washed once in LCM (DMEM with 5 µM CaCl2; Refs. 46, 51) and incubated with LCM plus 2.5% dFBS (FBS extensively dialyzed against Tris-saline; see Refs. 46, 51). At time points from 15 to 240 min after calcium removal, cells were washed and fixed immediately for immunofluorescence staining (see below).
For calcium replacement experiments, 8-day filter-grown cells were washed in LCM and switched to LCM plus 2.5% dFBS as above for 2, 3, or 4 h. LCM was then replaced with complete DMEM [normal (high)-calcium medium (HCM) containing 1.8 mM CaCl2 and 10% FBS], and cells were washed, fixed, and stained at time points from 15 to 120 min after calcium replacement.
For immunofluorescence, cells were washed briefly three times in cold PBS (2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, 8.1 mM Na2HPO4). Cells were fixed for 10 min at room temperature in 3.75% paraformaldehyde, washed three times for 5 min in PBS, and extracted for 5 min in CSK buffer (10 mM PIPES, pH 6.8, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5% Triton X-100). Cells were washed again, as above, and blocked for 30 min in PBS-B (PBS plus 0.2% BSA, 0.45 mM CaCl2, 0.5 mM MgCl2) with 2% goat serum (GIBCO-BRL), 0.05 M NH4Cl, 10 mM glycine, and 10 mM lysine. Filters were cut into quarters, incubated sequentially with primary antibodies diluted in PBS-B containing 2% goat serum for 45 min, and then washed three times in PBS-B. Secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) were diluted 1:200 in PBS-B containing 2% goat serum and incubated on cells for 30 min. Filters were washed as above and mounted in elvanol containing 0.1% paraphenylene diamine. Samples were viewed on a Bio-Rad MRC 1024 confocal microscope (Renal Epithelial Biology Laboratory, Indiana University School of Medicine). Individual planes through the entire cell volume were collected at 0.4-µm intervals and combined in a projection using Metamorph software (Universal Imaging, West Chester, PA).
Cell extraction, SDS-PAGE, and
immunoblotting. For total cell protein extracts, cells
were washed twice with cold PBS, extracted in 200 µl hot SDS sample
buffer (4% SDS, 80 mM Tris, pH 6.8, 100 mM DTT, 15% glycerol, 0.005%
bromophenol blue), and scraped from the filter with a rubber policeman.
Extracts were boiled for 10 min. For pulse and pulse-chase experiments,
cells were washed with ice cold PBS. Then, cells were extracted on ice
with R buffer [20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100
(Bio-Rad, Hercules, CA), 0.5% deoxycholate, 0.1% SDS, and 0.9 mM
CaCl2 plus 10 mM phenylmethylsulfonyl fluoride (PMSF); modified from Ref. 22] for
10 min on ice. Cells were scraped and centrifuged for 15 min at 13,000 rpm. Supernatants were collected, aliquoted as equal halves, and frozen
at 80°C. For catenin coimmunoprecipitation, cells were
washed and extracted in K buffer consisting of 1 mM CaCl2, 1% Nonidet P-40, and 1%
Triton X-100 in PBS (61) plus 10 mM PMSF for 10 min on ice, scraped,
and centrifuged as above. Supernatants were collected, aliquoted as
equal halves, and frozen at
80°C.
Extracts or immune complexes were separated by SDS-PAGE (33), in 6.25% or 7.5% polyacrylamide gels made from a stock of 30% acrylamide-0.6% bis-acrylamide. For immunoblotting, proteins were transferred to nitrocellulose membrane (Bio-Rad) in a buffer containing 25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol. Membranes were stained with Ponceau S and blocked overnight at 4°C in TBS-T (10 mM Tris, pH 7.4, 100 mM NaCl, 0.1% Tween 20) plus 5% Carnation nonfat powdered milk and 3% BSA (Sigma). Primary antibodies were diluted (1:5,000-1:10,000) in blocking solution and incubated with the membrane for 1 h at room temperature with constant rotation. Membranes were washed 5 times for 10 min each wash with TBS-T and then incubated as above with a species-matched horseradish peroxidase-conjugated secondary antibody (Amersham, Arlington Heights, IL) diluted 1:10,000 in blocking solution. Membranes were again washed five times for 10 min each wash with TBS-T, developed by electrochemiluminescence (Amersham), and exposed to film (Kodak Bio-Max ML, Eastman Kodak, Rochester, NY). Bands were scanned with a Silverscanner III (LaCie, Beaverton, OR) and quantified with BioImage IQ software (Ann Arbor, MI).
Metabolic labeling and immunoprecipitation. For pulse labeling experiments, cells were washed once and then starved for 20 min in methionine- and cysteine-free DMEM (46, 51) supplemented with 2.5% dFBS, then pulse labeled with 0.250 mCi [35S]methionine/cysteine (Pro-mix, Amersham) per filter in the same media for 5, 10, or 15 min. Cells were washed three times in cold PBS and extracted for 10 min on ice with R buffer plus PMSF, as above.
For pulse-chase labeling experiments, cells were washed, starved, and pulse labeled for 20 min with 0.125 mCi [35S]methionine/cysteine, as above. Filters were then washed twice with DMEM containing 10,000-fold excess of cold methionine and cysteine with 5 mM sodium butyrate and chased in the same media for up to 24 h. At the end of the chase period, cells were washed with PBS and extracted in R buffer as above.
For steady-state labeling, cells were washed once in methionine and cysteine-free media, then incubated for 18-24 h in DMEM supplemented with 2.5% dFBS, 10 µM methionine, 10 µM cysteine, and 0.250 mCi [35S]methionine/cysteine. At the end of the labeling period, cells were washed in PBS and extracted in K buffer as above.
Prepared extracts were precleared on ice for 45 min with 5 µl of
nonimmune serum and 30 µl of Pansorbin cells (Calbiochem, San Diego,
CA). Pansorbin cells were removed by centrifugation for 5 min at 13,000 rpm. Primary antibody (4-16 µl) was added to the extracts for at
least 1 h on ice; for mouse monoclonal antibodies (rr-1, 4A9), a rabbit
anti-mouse bridging antibody (DAKO, Carpinteria, CA) was added
(10-40 µl). Sixty microliters of protein A Sepharose CL-4B beads
(Pharmacia, Uppsala, Sweden) were added and incubated for at least 1 h
at 4°C with rotation. Alternatively, rr-1 immune complexes were
incubated with 50-100 µl of packed anti-mouse-coupled Sepharose
beads (ICN, Costa Mesa, CA), for 60 min at 4°C with rotation. The
protein A or anti-mouse beads were washed three times with extraction
buffer (R buffer or K buffer), and 60 µl of SDS sample buffer were
added before boiling at 100°C for 10 min. The immunoprecipitates
were separated by 6.25 or 7.5% SDS-PAGE as above. Gels were processed
for immunoblotting, or, for
35S-labeled samples, gels were
processed for fluorography, as described (26). Gels were dried and
exposed to Kodak X-OMAT AR film at 80°C. Fluorographs were
scanned with a Silverscanner III (LaCie) and bands were quantified with
BioImage IQ software. For T151 pulse-chase experiments, gels were
quantified using a Molecular Dynamics STORM 820 phosphoimager
(Sunnyvale, CA) and ImageQuant software (Molecular Dynamics).
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RESULTS |
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Mutant cadherin fusion protein construction and
expression. We constructed chimeric proteins containing
transmembrane and cytoplasmic domain sequences of E-cadherin (Fig.
1A).
For most experiments, GP2-Cad1 was stably transfected in MDCK cells,
and two independent clones were characterized. To engineer the GP2-Cad1 fusion protein, the extracellular domain of canine E-cadherin was
replaced by that of a GPI-linked protein, GP2 (a membrane protein of
pancreatic acinar cells; 15, 28). Cells expressing GP2 (13, 35) and
parental, untransfected MDCK cells (type II) were used as controls.
GP2-Cad1 and GP2 were detected using a monoclonal antibody that
recognizes GP2 extracellular domain (4A9; Ref. 13). Immunoblot analysis
of stable clones showed that mature GP2-Cad1 fusion protein migrated
with an apparent molecular weight of ~130 kDa. This protein was
derived from a GP2-Cad1 precursor of ~100 kDa, which was processed to
an ~115-kDa form and then to the ~130-kDa form, as shown by
pulse-chase metabolic labeling experiments (Fig. 1,
B and
D). These changes in apparent molecular weight reflect complex glycosylation of GP2 extracellular domain (28). In the absence of sodium butyrate (see below), GP2-Cad1
was expressed at ~10% the level of endogenous E-cadherin (Fig.
1B), and the level of endogenous
E-cadherin was unchanged relative to control cells (Fig.
1C). Transfected gene expression (GP2-Cad1 or GP2) was induced to higher levels by treating cells with 5 mM sodium butyrate (Fig. 1D; Ref. 18).
Unlike wild-type GP2, GP2-Cad1 fusion protein was localized to the
basal-lateral plasma membrane domain (Fig.
1E) and colocalized with endogenous E-cadherin by double-label immunofluorescence microscopy (data not
shown).
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All experiments described in this paper were replicated using cells expressing a mutant cadherin (T151) under the control of the tet repressor. T151 mutant cadherin protein consists of an HA epitope tag engineered into a nonfunctional, truncated extracellular domain, the transmembrane domain and complete cytoplasmic domain of E-cadherin (Fig. 1A and data not shown). Using these cells, mutant cadherin overexpression was achieved without using sodium butyrate treatment, and levels of mutant cadherin expression could be titrated by incubating cells in various concentrations of doxycycline (which binds the tet repressor protein and blocks its association with the promoter driving mutant cadherin expression). Incubating T151 cells with 0.1 ng/ml doxycycline in culture medium repressed mutant cadherin protein levels to amounts that were similar to GP2-Cad1 protein expression (in cells that were not treated with sodium butyrate).
Effects of mutant cadherin expression on junctional
complex stability. To determine whether expression of
GP2-Cad1 fusion protein affected cell-cell junction integrity, the
distribution of epithelial junctional complex proteins in established
cell monolayers of GP2-Cad1 and control cells was analyzed by
immunofluorescence. Proteins of the adherens junction (E-cadherin,
-catenin), tight junction (ZO-1, occludin), and desmosome
(desmoplakin) were localized at sites of cell-cell contact in
GP2-Cad1-expressing cells, in patterns indistinguishable from those in
control cells (Fig.
2A). Thus low level expression of GP2-Cad1 had no apparent effect on junctional complex morphology at steady state. Similar results were
obtained using T151 cells expressing low levels of mutant cadherin
(treated with 0.1 ng/ml doxycycline; data not shown). Significantly,
measurements of transepithelial resistance and inulin diffusion in
GP2-Cad1 cells were also indistinguishable from those in control cells,
indicating that tight junctions were functionally intact in MDCK
monolayers expressing mutant cadherin (data not shown).
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A survey of junctional and cytoskeletal proteins (including E-cadherin,
-catenin,
-catenin, ZO-1, occludin, desmoplakin, desmoglein,
ankyrin, fodrin, and
Na+-K+-ATPase)
showed that steady-state levels of these proteins were not altered by
expressing low levels of GP2-Cad1. However, we found that expression of
ZO-1, ankyrin, and
Na+-K+-ATPase
was decreased in the presence of 5 mM sodium butyrate, both in control
and GP2-Cad1-expressing cells (data not shown). The expression level of
endogenous E-cadherin was also decreased, and was shown to be a
specific consequence of increased GP2-Cad1 expression and independent
of sodium butyrate (Figs. 2B and
8A; see below). Furthermore, 48 h of 5 mM sodium butyrate treatment resulted in significant morphological
changes in established monolayers of both control and
GP2-Cad1-expressing cells (Fig. 2B).
Cell-cell junction stability and assembly were examined in sodium
butyrate-treated cells expressing high levels of GP2-Cad1 and using
T151 cells (without sodium butyrate treatment). These data showed that
effects were consistent with those seen for low expression (see below). Interpretation of results using GP2-Cad1 cells treated with sodium butyrate was problematic due to the independent sodium butyrate effects, and therefore only data for low-expression cells were shown.
We examined effects of low-level GP2-Cad1 and T151 expression on the stability of cell-cell junctions. Fully polarized MDCK cell monolayers were switched from HCM (1.8 mM Ca2+) to LCM (5 µM Ca2+) to induce junctional complex disassembly. Cells were processed at different times after the switch for double-label immunofluorescence to follow the distribution of adherens junction, tight junction, and desmosome (data not shown) proteins during junctional complex disassembly. To analyze protein distributions, a z-series of x-y sections through the entire volume of the monolayer were collected with a confocal microscope and combined to visualize any alterations in junction organization in all focal planes simultaneously.
Following a 15-min switch to LCM, we observed significant
redistribution of E-cadherin and -catenin (Fig.
3, A and
B) in GP2-Cad1 cells, but very minor
changes in the distributions of these proteins in control cells. After
30 min in LCM, GP2-Cad1 cells mostly lacked cell contact staining for
E-cadherin and
-catenin. However, at this time, the majority of
control cells still showed only minor alteration to characteristic
adherens junction staining of E-cadherin and
-catenin. By 45 min of
LCM treatment, cell-cell contact staining of E-cadherin and
-catenin
was completely disrupted in GP2-Cad1 cells, whereas control cultures
still had significant cell-cell contact staining for these proteins. It
was noted that the distribution of GP2-Cad1 coincided with that of
E-cadherin and
-catenin throughout this time course (data not
shown).
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Cell monolayers expressing GP2-Cad1 showed significant redistribution
of tight junction proteins by 15 min after switching cells to LCM (Fig.
4, A and
B); large breaks in the continuity of both ZO-1 and occludin cell contact staining were apparent, unlike
the staining patterns in control monolayers. By 30 min in LCM, GP2-Cad1
cells showed nearly complete disruption of ZO-1 and occludin staining
at cell-cell contacts. In contrast, tight junctions appeared relatively
intact in control cells. At 45 min of LCM treatment, GP2-Cad1 cells
showed nearly complete redistribution of ZO-1 and occludin from
cell-cell contacts. In control cell monolayers, many cells maintained
ZO-1 and faint occludin staining in apical belts between cells that
were reminiscent of normal tight junction morphology.
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Again, equivalent results for adherens junction and tight junction disassembly were obtained using T151 cells expressing low levels of mutant cadherin (0.1 ng/ml doxycycline treated) and completely repressed T151 cells (20 ng/ml doxycycline treated) for controls (data not shown).
Effects of mutant cadherin expression on junctional complex reassembly. The results described above demonstrate that junctional complexes in MDCK cells expressing low levels of GP2-Cad1 or T151 were more rapidly disrupted than in control cells. We investigated whether low levels of mutant cadherin protein expression affected junctional complex reassembly. Confluent MDCK monolayers were incubated in LCM for 2 or 4 h, which is sufficient for complete disassembly of junctional complexes (no junctional complex component staining was observed at sites of cell-cell contact). Results of reassembly experiments using cells incubated in LCM for 2 or 4 h were equivalent. This shows that, operationally, the junctions were completely disassembled after 2 h, because additional low-calcium treatment did not change the outcome of the reassembly experiments. Cultures were switched back to normal medium containing 1.8 mM Ca2+ for various times, and processed for double-label immunofluorescence localization of adherens junction, tight junction, and desmosomal (data not shown) proteins. Optical sections through the whole cell volume were combined to analyze the entire junctional complex.
In control cells, accumulation of E-cadherin at sites of cell-cell
contact was evident by 30 min after returning cells to HCM (Fig.
5A). In
contrast, cells expressing GP2-Cad1 showed little or no accumulation of
E-cadherin staining at cell-cell contact sites at this time.
After 60 min in HCM, E-cadherin was present at cell-cell contact sites
in nearly all the cells of the control monolayer. However at 60 min,
cells expressing GP2-Cad1 exhibited weak, discontinuous E-cadherin
staining at cell-cell contacts within a small subset of cells. After 90 min in HCM, the pattern of E-cadherin staining in control cells was
approaching that in an established monolayer. In GP2-Cad1 expressing
cells, E-cadherin was detected at many cell-cell contact sites, but
significantly less than in control cells.
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-Catenin accumulated at sites of cell-cell contact in control cells
after 30 min in HCM, although significant cytoplasmic
-catenin
staining was also observed (Fig.
5B). By comparison,
-catenin
localization in GP2-Cad1 cells was primarily cytoplasmic, with little
or no staining at cell-cell contacts. Sixty minutes after the switch to
HCM in control cells,
-catenin was enriched at sites of cell-cell
contact, like that seen for E-cadherin. In contrast,
-catenin
staining was still diffuse in the cytoplasm with little staining at
cell-cell contact sites after 60 min of reassembly in
GP2-Cad1-expressing cells. After 90 min in HCM,
-catenin staining
in control cells was prominent at cell-cell contacts and cytoplasmic
staining was largely lost. At the same time, GP2-Cad1 cells
showed only weak
-catenin staining at sites of cell-cell contacts
and prominent cytoplasmic staining.
Tight junctions also assembled more rapidly in control cells than in
cells expressing GP2-Cad1 (Fig. 6,
A and
B). After 30 min in HCM, large
patches of control cells showed significant ZO-1 and occludin staining
at cell contact sites. In contrast, there was little or no accumulation
of either ZO-1 or occludin at sites of cell-cell contact after 30 min
in HCM in GP2-Cad1-expressing cells. Control cells showed nearly
complete redistribution of ZO-1 and occludin to sites of cell-cell
contact after 45 min in HCM. GP2-Cad1-expressing cells showed some ZO-1
and occludin accumulation at sites of cell-cell contact after 45 min in
HCM, but the staining rarely formed a complete belt around cells. By 60 min in HCM, control monolayers showed a continuous network of ZO-1 and
occludin staining in an apical belt at contacts between cells
throughout the monolayer. By comparison, GP2-Cad1 cells at 60 min in
HCM showed discontinuous ZO-1 and occludin staining at cell-cell
contacts.
|
Equivalent results for adherens junction and tight junction reassembly were again obtained using T151 cells expressing low levels of mutant cadherin (0.1 ng/ml doxycycline treated) and completely repressed T151 cells (20 ng/ml doxycycline treated) for controls (data not shown).
Mutant cadherin expression does not alter the
composition of the endogenous cadherin-catenin complex.
The results described above show that low-level expression of GP2-Cad1
or T151 in MDCK cells significantly accelerated disassembly and
retarded reassembly of junctional complexes. Previous studies using
overexpression of analogous mutant cadherin molecules (32, 69)
suggested that mutant phenotypes were generated by competition for
endogenous catenin binding. Because the level of mutant cadherin
expression was ~10% of endogenous cadherin in our experiments,
catenin competition mechanisms are not responsible for generating
effects on junctional complex stability and reassembly. Total cellular
levels of -catenin and
-catenin were similar in control cells and
in cells expressing low levels of GP2-Cad1, and
-catenin,
-catenin, and plakoglobin were detected in GP2-Cad1
immunoprecipitates (data not shown).
To directly examine the possibility that low-level expression of
GP2-Cad1 somehow alters the stoichiometry of E-cadherin-catenin complexes, immunoprecipitation was performed on GP2-Cad1 and control cells using the monoclonal antibody rr-1, which specifically recognizes the E-cadherin extracellular domain, and thus only endogenous E-cadherin (22). Immune complexes were separated by SDS-PAGE. Immunoblotting for -catenin,
-catenin, and plakoglobin revealed that catenin binding to endogenous E-cadherin was similar in
GP2-Cad1-expressing cells and control cells (Fig.
7). Similar results were obtained from
cells metabolically labeled to steady state with
[35S]methionine/cysteine
(data not shown).
|
Overexpression of mutant cadherin reduces the amount
and metabolic stability of endogenous E-cadherin. The
expression level of GP2-Cad1 fusion protein increased by incubating
cells in 5 mM sodium butyrate (Figs.
1D and
8A).
Under these conditions, the GP2-Cad1 level was approximately threefold
higher than that of endogenous E-cadherin in control cells.
Overexpression of GP2-Cad1 decreased the amount of endogenous
E-cadherin at steady state by ~10-fold relative to control cells
(Figs. 8A and
2B). However, the levels of - and
-catenin (Fig. 8B) and
stoichiometry of the cadherin-catenin complex were comparable under
control and high-level GP2-Cad1 expression conditions (data not shown).
This effect on endogenous E-cadherin expression levels was not due to
sodium butyrate treatment alone or to overexpression of any exogenous
protein from a transfected gene. Both untransfected MDCK cells and MDCK
cells transfected with wild-type GP2 had high levels of endogenous
E-cadherin in the presence of 5 mM sodium butyrate (Figs.
8A and
2B). Additionally, overexpression of
T151 mutant cadherin protein resulted in downregulation of endogenous E-cadherin (Fig. 8C). Repressing
mutant cadherin expression levels to low levels (0.1 ng/ml doxycycline
treatment) or completely repressing T151 expression (20 ng/ml
doxycycline treatment; Fig. 8C)
reversed this effect on T151 cells. Thus the decrease in endogenous E-cadherin was a consequence of mutant cadherin expression and not
sodium butyrate per se.
|
To determine whether the decrease in the level of endogenous cadherin was related to the observed effect of mutant cadherin on the plasma membrane junctional complex assembly events, we expressed another mutant cadherin fusion protein that consists of GST fused to the complete E-cadherin cytoplasmic domain (named GST-ECad; Fig. 1A). This fusion protein is a cytoplasmic protein that contains neither a signal sequence nor transmembrane sequence. Overexpressing GST-ECad at very high levels did not affect endogenous E-cadherin levels (Fig. 8C), suggesting that the cellular system for sensing cadherin levels acts at the plasma membrane.
To determine whether the rate of synthesis or the metabolic stability
of endogenous E-cadherin was affected by overexpression of mutant
cadherin, pulse labeling and pulse-chase labeling experiments were
performed to follow synthesis and catabolism of E-cadherin, respectively. Cells were pulse labeled with
[35S]methionine/cysteine
for 5, 10, and 15 min followed by immunoprecipitation using the
monoclonal antibody rr-1, specific for endogenous E-cadherin. Analysis
of the immunoprecipitates showed that the rate of E-cadherin synthesis
was similar in control and GP2-Cad1 cell lines with or without sodium
butyrate induction of GP2-Cad1 expression (Fig. 9A).
|
To examine the stability of endogenous E-cadherin in GP2-Cad1-expressing cells and control cells, cells induced with sodium butyrate were pulse labeled for 20 min with [35S]methionine/cysteine and chased with medium containing excess methionine and cysteine over a 24-h time course (Fig. 9B). E-cadherin was immunoprecipitated from cell extracts, and the immunoprecipitates were separated by SDS-PAGE. Quantitation of fluorographs showed that the half-life of endogenous E-cadherin was reduced from 6.1 h (±0.88 h SD, n = 4) in control cells to 3.4 h (±0.68 h SD, n = 4) in GP2-Cad1-expressing cells. Decreased stability of endogenous E-cadherin is consistent with the reduction in steady-state E-cadherin in cells expressing high levels of mutant cadherin.
These metabolic labeling experiments were also replicated using the T151 cell system. T151 overexpression did not affect the rate of endogenous E-cadherin synthesis (Fig. 9A). However, T151 overexpression reduced the half-life of endogenous E-cadherin from 8.8 h (±0.13 h SD, n = 2) in the presence of 20 ng/ml doxycycline to 2.5 h (±0.71 h SD, n = 2) without doxycycline present (Fig. 9B), confirming the data collected using GP2-Cad1 cells.
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DISCUSSION |
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We have expressed mutant cadherin proteins to examine the consequence on the formation and stability of tight junctions and adherens junctions. Low-level mutant cadherin disrupted dynamic processes of cell-cell junction disassembly and reassembly. Despite a normal level of endogenous E-cadherin, disassembly of adherens junctions and tight junctions in cells expressing mutant cadherin exposed to LCM (5 µm Ca2+) was more rapid than in control cells, indicating that established junctional complexes were inherently less stable in the presence of the low level of mutant cadherin. Furthermore, low level of mutant cadherin retarded the reassembly of tight junctions and adherens junctions compared with control cells. These observations extend previous studies demonstrating that junctional complex formation was inhibited under conditions of large-scale blockage of cadherin function (3, 22, 23, 67, 69).
We have conducted detailed analysis of the distribution of the desmosomal marker protein desmoplakin during the process of junctional disassembly and reassembly (data not shown) like that performed for adherens junction and tight junction components. We observed that the time courses of adherens junction and tight junction disassembly and reassembly closely corresponded to one another, but desmosome disassembly and reassembly lagged behind adherens junction and tight junction disassembly and reassembly in control, GP2-Cad1, and T151 cells. This suggests that adherens junction and tight junction assembly processes are somehow coupled (30).
Despite the effects on junctional complex stability and assembly,
low-level expression of mutant cadherin allows establishment of
polarized MDCK cell monolayers. With time, cell-cell junctions formed
that were morphologically indistinguishable from those in control
cells, when distributions of tight junction (ZO-1 and occludin),
adherens junction (E-cadherin and -catenin), and desmosome (desmoplakin) marker proteins were examined. Cells expressing a low
level of mutant cadherin also developed a measurable transepithelial resistance and restricted inulin passage similar to control cell monolayers.
How does mutant cadherin expression alter junctional complex integrity? Previous notions were that overexpressed mutant ("dominant negative") cadherin proteins sequester catenins from endogenous cadherins (32, 69). However, this mechanism is unlikely in cells expressing low levels of mutant cadherin. Mutant cadherins expressed at approximately one-tenth the level of endogenous cadherin are too low to support a catenin competition mechanism. In immunoprecipitation experiments, low-level mutant cadherin expression did not alter the extent or stoichiometry of catenin binding to endogenous E-cadherin. The possibility remains that the observed effects on junctional complexes are due to competition for some other unidentified component of the cadherin complex, present in limiting quantities, as suggested by Fujimori and Takeichi (14).
Because catenin binding to endogenous E-cadherin was shown to be unaltered in the presence of low levels of mutant cadherin, we suggest that GP2-Cad1 and T151 proteins interfered structurally with endogenous E-cadherin packing in forming cell-cell adhesion junctions. GP2-Cad1 and endogenous E-cadherin colocalized at sites of cell-cell contact (Fig. 1E and data not shown). Studies suggest that cadherin extracellular domain dimers form lateral associations and intercalate with dimers on adjacent cells to build a lattice of cadherin-adhesive interactions (8, 43, 48, 57, 68). We suggest that the juxtaposition of mutant cadherin molecules and endogenous E-cadherin at adherens junctions, even at low expression levels, disrupts cadherin spacing and substitutes nonfunctional binding partners within the extracellular cadherin lattice, producing the observed structural weakness of the junctions. Our data indicate that GP2-Cad1 interferes with cell junction dynamics via a structural effect on cadherin packing, instead of altering cadherin-catenin complex composition as suggested in previous studies (32, 69).
At high levels of GP2-Cad1 or T151 expression (approximately threefold greater than endogenous E-cadherin), steady-state endogenous E-cadherin levels were downregulated. This could be attributed in part to decreased metabolic stability of endogenous E-cadherin. Previous studies have shown little change in E-cadherin half-life even under low calcium growth conditions (E. Shore, L. Hinck, and W. J. Nelson, unpublished results). However, it has been demonstrated that cytoskeletal association may prevent turnover of other membrane-cytoskeletal proteins (24, 45). Mutant cadherin could occupy cytoskeletal-binding sites normally available to endogenous E-cadherin, forcing more rapid turnover of endogenous E-cadherin molecules. Zhu and Watt (69) observed that a mutant cadherin construct lacking the catenin binding domain no longer caused downregulation of endogenous cadherin in keratinocytes. Taken together, these data indicate that the reduced half-life of endogenous E-cadherin, contributing to decreased steady-state E-cadherin levels, is dependent on catenin binding. However, overexpressing a cytoplasmic fusion protein that contains the entire cadherin cytoplasmic domain but no transmembrane sequence (GST-ECad) had no effect on endogenous E-cadherin levels (Fig. 8C), suggesting that catenin binding and plasma membrane localization are necessary for regulating cadherin levels. In squamous carcinoma cells, levels of E- and N-cadherin were reciprocally regulated by an unknown mechanism (29), and our results may help explain the observed phenomenon.
The observed downregulation of endogenous E-cadherin in conditions of mutant cadherin overexpression is in contrast to observations of Fujimori and Takeichi (14) and Amagai et al. (3), who found no decrease in endogenous cadherin expression in the presence of high levels of a mutant cadherin protein. It is interesting to note that in these other two studies the cytoplasmic domain of the mutant protein did not match that of the endogenous cadherin family member (mutant N-cadherin was overexpressed, whereas keratinocytes express E- and P-cadherin). Like our results, Zhu and Watt (69) found that endogenous cadherin was downregulated in the presence of a mutant cadherin molecule with an identical cytoplasmic domain. These data point to the existence of a posttranslational, cellular mechanism for regulating cadherin protein levels.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Inke Näthke (The University of Dundee, Scotland) for providing catenin antisera and Dr. Anson Lowe (Stanford University Medical Center) for providing both GP2 antibody and rat GP2 cDNA. We thank Dr. Vikas Sukhatme (Harvard University) for the gift of the expression vector pCB6+, Dr. Michael Roth (University of Texas Southwestern) for the gift of pCB7, and Dr. Michael Kinch (Purdue University) for assistance with ascites production. Dr. Lee Rubin (Eisai London Lab, University College, London) generously provided the canine E-cadherin cDNA. We also thank Dr. Rolf Kemler (Max Plank Institut fur Immunbiology, Freiburg) for providing the mouse E-cadherin cDNA. Drs. Lisa Elferink and Richard Scheller (Stanford University Medical Center) are gratefully acknowledged for providing plasmid containing HA tag sequences. Dr. Kenneth Dunn and Paul Brown (Indiana University Medical Center) provided expert assistance with confocal microscopy and image processing.
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FOOTNOTES |
---|
M. L. Troxell is supported by a Medical Scientist Training Program Grant GM-07365 from the National Institutes of Health (NIH). Y.-T. Chen is a recipient of a postdoctoral fellowship from the National Kidney Foundation. NIH awards to W. J. Nelson and J. A. Marrs (EY-11365 and DK-54518) supported this work.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J. A. Marrs, Dept. of Medicine, Indiana Univ. School of Medicine, Fesler Hall 115, 1120 South Dr., Indianapolis, IN 46202-5116.
Received 6 May 1998; accepted in final form 10 November 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aberle, H.,
S. Butz,
J. Stappert,
H. Weissig,
R. Kemler,
and
H. Hoschuetzky.
Assembly of the cadherin-catenin complex in vitro with recombinant proteins.
J. Cell Sci.
107:
3655-3663,
1994
2.
Adams, C. L.,
W. J. Nelson,
and
S. J. Smith.
Quantitaive analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion.
J. Cell Biol.
135:
1899-1911,
1996[Abstract].
3.
Amagai, M.,
T. Fujimori,
T. Masunaga,
H. Shimizu,
T. Nishikawa,
N. Shimizu,
M. Takeichi,
and
T. Hashimoto.
Delayed assembly of desmosomes in keratinocytes with disrupted classic-cadherin-mediated cell adhesion by a dominant negative mutant.
J. Invest. Dermatol.
104:
27-32,
1995[Abstract].
4.
Angres, B.,
A. Barth,
and
W. J. Nelson.
Mechanism for transition from initial to stable cell-cell adhesion: kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay.
J. Cell Biol.
134:
549-557,
1996[Abstract].
5.
Balda, M. S.,
and
K. Matter.
Tight junctions.
J. Cell Sci.
111:
541-547,
1998
6.
Barth, A. I. M.,
I. S. Näthke,
and
W. J. Nelson.
Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways.
Curr. Opin. Cell Biol.
9:
683-690,
1997[Medline].
7.
Brewer, C.
Cytomegalovirus plasmid vectors for permanent lines of polarized epithelial cells.
Methods Cell Biol.
43:
233-245,
1994[Medline].
8.
Brieher, W. M.,
A. S. Yap,
and
B. M. Gumbiner.
Lateral dimerization is required for the homophilic binding activity of C-cadherin.
J. Cell Biol.
135:
487-496,
1996[Abstract].
9.
Buxton, R. S.,
and
A. I. Magee.
Structure and interactions of desmosomal and other cadherins.
Semin. Cell Biol.
3:
157-167,
1992[Medline].
10.
Fanning, A. S.,
L. A. Lapierre,
A. R. Brecher,
C. M. Van Itallie,
and
J. M. Anderson.
Protein interactions in the tight junction: the role of MAGUK proteins in regulating tight junction organization and function.
Curr. Top. Membr. Transp.
43:
211-235,
1996.
11.
Farquhar, M. G.,
and
G. E. Palade.
Junctional complexes in various epithelia.
J. Cell Biol.
17:
375-412,
1963
12.
Franke, W. W.,
M. D. Goldschmidt,
R. Zimbelmann,
H. M. Mueller,
D. L. Schiller,
and
P. Cowin.
Molecular cloning and amino acid sequence of human plakoglobin, the common junctional plaque protein.
Proc. Natl. Acad. Sci. USA
86:
4027-4031,
1989[Abstract].
13.
Fritz, B. A.,
and
A. W. Lowe.
Polarized GP2 secretion in MDCK cells via GPI targeting and apical membrane-restricted proteolysis.
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33):
G176-G183,
1996
14.
Fujimori, T.,
and
M. Takeichi.
Disruption of epithelial cell-cell adhesion by exogenous expression of a mutated nonfunctional N-cadherin.
Mol. Biol. Cell
4:
37-47,
1993[Abstract].
15.
Fukuoka, S.-I.,
and
G. A. Scheele.
Nucleotide sequence encoding the major glycoprotein (GP2) of rat pancreatic secretory (zymogen) granule membranes.
Nucleic Acids Res.
18:
5900,
1990[Medline].
16.
Furuse, M.,
T. Hirase,
M. Itoh,
A. Nagafuchi,
S. Yonemura,
S. Tsukita,
and
S. Tsukita.
Occludin: a novel integral membrane protein localizing at tight junctions.
J. Cell Biol.
123:
1777-1788,
1993[Abstract].
17.
Furuse, M.,
M. Itoh,
T. Hirase,
A. Nagafuchi,
S. Yonemura,
S. Tsukita,
and
S. Tsukita.
Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.
J. Cell Biol.
127:
1617-1626,
1994[Abstract].
18.
Gorman, C. M.,
and
B. H. Howard.
Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate.
Nucleic Acids Res.
11:
7631-7648,
1983[Abstract].
19.
Graeve, L.,
A. Patzak,
K. Drickamer,
and
E. Rodriguez-Boulan.
Polarized expression of functional rat liver asialoglycoprotein receptor in transfected Madin-Darby canine kidney cells.
J. Biol. Chem.
265:
1216-1224,
1990
20.
Green, K. J.,
and
J. C. R. Jones.
Desmosomes and hemidesmosomes: structure and function of molecular components.
FASEB J.
10:
871-881,
1996
21.
Gumbiner, B.,
T. Lowenkopf,
and
D. Apatira.
Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1.
Proc. Natl. Acad. Sci. USA
88:
3460-3464,
1991[Abstract].
22.
Gumbiner, B.,
and
K. Simons.
A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide.
J. Cell Biol.
102:
457-468,
1986[Abstract].
23.
Gumbiner, B.,
B. Stevenson,
and
A. Grimaldi.
The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex.
J. Cell Biol.
107:
1575-1587,
1988[Abstract].
24.
Hammerton, R. W.,
K. A. Krzeminski,
R. W. Mays,
T. A. Ryan,
D. A. Wollner,
and
W. J. Nelson.
Mechanism for regulating cell surface distribution of Na+-K+-ATPase in polarized epithelial cells.
Science
254:
847-850,
1991[Medline].
25.
Herrenknecht, K.,
M. Ozawa,
C. Eckerskorn,
F. Lottspeich,
M. Lenter,
and
R. Kemler.
The uvomorulin-anchorage protein alpha catenin is a vinculin homologue.
Proc. Natl. Acad. Sci. USA
88:
9156-9165,
1991[Abstract].
26.
Hinck, L.,
I. S. Näthke,
J. Papkoff,
and
W. J. Nelson.
Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly.
J. Cell Biol.
125:
1327-1340,
1994[Abstract].
27.
Hirano, S.,
A. Nose,
K. Hatta,
A. Kawakami,
and
M. Takeichi.
Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles.
J. Cell Biol.
105:
2501-2510,
1987[Abstract].
28.
Hoops, T. C.,
and
M. J. Rindler.
Isolation of the cDNA endcoding glycoprotein-2 (GP-2), the major zymogen granule membrane protein.
J. Biol. Chem.
266:
4257-4263,
1991
29.
Islam, S.,
T. E. Carey,
G. T. Wolf,
M. J. Wheelock,
and
K. R. Johnson.
Expression of N-cadherin by human squamous carcinoma cells induces a scattered fibroblastic phenotype with disrupted cell-cell adhesion.
J. Cell Biol.
135:
1643-1654,
1996[Abstract].
30.
Itoh, M.,
A. Nagafuchi,
S. Moroi,
and
S. Tsukita.
Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to -catenin and actin filaments.
J. Cell Biol.
138:
181-192,
1997
31.
Jou, T.-S.,
D. B. Stewart,
J. Stappert,
W. J. Nelson,
and
J. A. Marrs.
Genetic and biochemical dissection of protein linkages in the cadherin-catenin complex.
Proc. Natl. Acad. Sci. USA
92:
5067-5071,
1995[Abstract].
32.
Kintner, C.
Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain.
Cell
69:
225-236,
1992[Medline].
33.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
34.
Marrs, J. A.,
E. W. Napolitano,
C. Murphy-Erdosh,
R. W. Mays,
L. F. Reichardt,
and
W. J. Nelson.
Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K+-ATPase distributions in polarized epithelia.
J. Cell Biol.
123:
149-164,
1993[Abstract].
35.
Mays, R. W.,
K. A. Siemers,
B. A. Fritz,
A. W. Lowe,
G. van Meer,
and
W. J. Nelson.
Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells.
J. Cell Biol.
130:
1105-1115,
1995[Abstract].
36.
McCrea, P. D.,
C. W. Turck,
and
B. Gumbiner.
A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin.
Science
254:
1359-1361,
1991[Medline].
37.
McNeill, H.,
T. A. Ryan,
S. J. Smith,
and
W. J. Nelson.
Spatial and temporal dissection of immediate and early events following cadherin-mediated epithelial cell adhesion.
J. Cell Biol.
120:
1217-1226,
1993[Abstract].
38.
Miller, J. R.,
and
R. T. Moon.
Signal transduction through -catenin and specification of cell fate during embryogenesis.
Genes Dev.
10:
2527-2539,
1996[Medline].
39.
Nagafuchi, A.,
Y. Shirayoshi,
K. Okazaki,
K. Yasuda,
and
M. Takeichi.
Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA.
Nature
329:
341-343,
1987[Medline].
40.
Nagafuchi, A.,
and
M. Takeichi.
Cell binding function of E-cadherin is regulated by the cytoplasmic domain.
EMBO J.
7:
3679-3684,
1988[Abstract].
41.
Nagafuchi, A.,
M. Takeichi,
and
S. Tsukita.
The 102-Kd cadherin-associated protein: similarity to vinculin and posttranscriptional regulation of expression.
Cell
65:
849-857,
1991[Medline].
42.
Nagafuchi, A.,
S. Tsukita,
and
M. Takeichi.
Transmembrane control of cadherin-mediated cell-cell adhesion.
Semin. Cell Biol.
4:
175-181,
1993[Medline].
43.
Nagar, B.,
M. Overduin,
M. Ikura,
and
J. M. Rini.
Structural basis of calcium-induced E-cadherin rigidification and dimerization.
Nature
380:
360-364,
1996[Medline].
44.
Näthke, I. S.,
L. E. Hinck,
and
W. J. Nelson.
Epithelial cell adhesion and development of cell surface polarity: possible mechanisms for modulation of cadherin function, organization and distribution.
J. Cell Sci. Suppl.
17:
139-145,
1993[Medline].
45.
Nelson, W. J.,
and
P. J. Veshnock.
Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells.
J. Cell Biol.
104:
1527-1537,
1987[Abstract].
46.
Nelson, W. J.,
R. Wilson,
and
R. W. Mays.
Biochemical methods for studying supramolecular complexes involving cell adhesion molecules, integral membrane proteins, and the cytoskeleton.
In: Cell-Cell Interactions: A Practical Approach, edited by B. R. Stevenson,
W. J. Gallin,
and D. L. Paul. Oxford, UK: Oxford Univ. Press, 1992, p. 227-255.
47.
Nose, A.,
A. Nagafuchi,
and
M. Takeichi.
Expressed recombinant cadherins mediate cell sorting in model systems.
Cell
54:
993-1001,
1988[Medline].
48.
Overduin, M.,
T. S. Harvey,
S. Bagby,
K. I. Tong,
P. Yau,
M. Takeichi,
and
M. Ikura.
Solution structure of the epithelial cadherin domain responsible for selective cell adhesion.
Science
267:
386-389,
1995[Medline].
49.
Ozawa, M.,
H. Baribault,
and
R. Kemler.
The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.
EMBO J.
8:
1711-1717,
1989[Abstract].
50.
Ozawa, M.,
M. Ringwald,
and
R. Kemler.
Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule.
Proc. Natl. Acad. Sci. USA
87:
4246-4250,
1990[Abstract].
51.
Pasdar, M.
Biochemical approaches for analysing de novo assembly of epithelial junctional components.
In: Cell-Cell Interactions: A Practical Approach, edited by B. R. Stevenson,
W. J. Gallin,
and D. L. Paul. Oxford, UK: Oxford Univ. Press, 1992, p. 203-226.
52.
Pasdar, M.,
and
W. J. Nelson.
Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis.
J. Cell Biol.
106:
677-685,
1988[Abstract].
53.
Pasdar, M.,
and
W. J. Nelson.
Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. II. Morphological analysis.
J. Cell Biol.
106:
687-695,
1988[Abstract].
54.
Peifer, M.
-Catenin as oncogene: the smoking gun.
Science
275:
1752-1753,
1997
55.
Rimm, D. L.,
E. R. Koslov,
P. Kebriaei,
C. D. Cianci,
and
J. S. Morrow.
Alpha 1(E)-catenin is an actin-binding and -bundling protein mediating the attachment of F-actin to the membrane adhesion complex.
Proc. Natl. Acad. Sci. USA
92:
8813-8817,
1995[Abstract].
56.
Ringwald, M.,
R. Schuh,
D. Vestweber,
H. Eistetter,
F. Lottspeich,
J. Engel,
R. Dolz,
F. Jahnig,
J. Epplen,
S. Mayer,
C. Muller,
and
R. Kemler.
The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.
EMBO J.
6:
3647-3653,
1987[Abstract].
57.
Shapiro, L.,
A. M. Fannon,
P. D. Kwong,
A. Thompson,
M. S. Lehmann,
G. Grübel,
J.-F. Legrand,
J. Als-Neilsen,
D. R. Colman,
and
W. A. Hendrickson.
Structural basis of cell-cell adhesion by cadherins.
Nature
374:
327-337,
1995[Medline].
58.
Shore, E. M.,
and
W. J. Nelson.
Biosynthesis of the cell adhesion molecule uvomorulin (E-cadherin) in Madin-Darby canine kidney epithelial cells.
J. Biol. Chem.
266:
19672-19680,
1991
59.
Siliciano, J. D.,
and
D. A. Goodenough.
Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells.
J. Cell Biol.
107:
2389-2399,
1988[Abstract].
60.
Southern, P. J.,
and
P. Berg.
Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter.
J. Mol. Appl. Genet.
1:
327-341,
1982[Medline].
61.
Stappert, J.,
and
R. Kemler.
A short core region of E-cadherin is essential for catenin binding and is highly phosphorylated.
Cell Adhes. Commun.
2:
319-327,
1994[Medline].
62.
Stevenson, B. R.,
J. D. Siliciano,
M. S. Mooseker,
and
D. A. Goodenough.
Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia.
J. Cell Biol.
103:
755-766,
1986[Abstract].
63.
Takeichi, M. A.
Cadherins: a molecular family important in selective cell-cell adhesion.
Annu. Rev. Biochem.
59:
237-252,
1990[Medline].
64.
Tsukita, S.,
M. Furuse,
and
M. Itoh.
Molecular dissection of tight junctions.
Cell Struct. Funct.
21:
381-385,
1996[Medline].
65.
Tsukita, S.,
S. Tsukita,
A. Nagafuchi,
and
S. Yonemura.
Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions.
Curr. Opin. Cell Biol.
4:
834-839,
1992[Medline].
66.
Vestweber, D.,
and
R. Kemler.
Identification of a putative cell adhesion domain of uvomorulin.
EMBO J.
4:
3393-3398,
1985[Abstract].
67.
Watabe, M.,
A. Nagafuchi,
S. Tsukita,
and
M. Takeichi.
Induction of polarized cell-cell association and retardation of growth by activation of the E-cadherin-catenin adhesion system in a dispersed carcinoma line.
J. Cell Biol.
127:
247-256,
1994[Abstract].
68.
Yap, A. S.,
W. M. Brieher,
M. Pruschy,
and
B. M. Gumbiner.
Lateral clustering of the adhesive ectodomain: a fundamental determinant of cadherin function.
Curr. Biol.
7:
308-315,
1997[Medline].
69.
Zhu, A. J.,
and
F. M. Watt.
Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes.
J. Cell Sci.
109:
3013-3023,
1996