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NH2-terminal Deletion of beta -Catenin Results in Stable Colocalization of Mutant beta -Catenin with Adenomatous Polyposis Coli Protein and Altered MDCK Cell Adhesion

Rockefeller University Press
Journal of Cell Biology (JCB)
Authors:
Angela I M Barth at Stanford University
Angela I M Barth
Anne L Pollack at The University of Arizona
Anne L Pollack
Yoram Altschuler at UCSF University of California, San Francisco
Yoram Altschuler
Keith E Mostov at UCSF University of California, San Francisco
Keith E Mostov
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Abstract and Figures

β-Catenin is essential for the function of cadherins, a family of Ca²⁺-dependent cell–cell adhesion molecules, by linking them to α-catenin and the actin cytoskeleton. β-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant β-catenins in MDCK epithelial cells to gain insights into the regulation of β-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length β-catenin, β-catenin mutant proteins with NH2-terminal deletions before (ΔN90) or after (ΔN131, ΔN151) the α-catenin binding site, or a mutant β-catenin with a COOH-terminal deletion (ΔC) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All β-catenin mutant proteins form complexes and colocalize with E-cadherin at cell–cell contacts; ΔN90, but neither ΔN131 nor ΔN151, bind α-catenin. However, β-catenin mutant proteins containing NH2-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal– deleted β-catenin poorly colocalize with APC protein. NH2-terminal deletions result in increased stability of β-catenin bound to APC protein and E-cadherin, compared with full-length β-catenin. At low density, MDCK cells expressing NH2-terminal–deleted β-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH2 terminus, but not the COOH terminus of β-catenin, regulates the dynamics of β-catenin binding to APC protein and E-cadherin. Changes in β-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of β-catenin binding to α-catenin. These results demonstrate that regulation of β-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.
Schematic representation of KT3-tagged full-size and mutant β-catenin proteins. NH2- and COOH-terminal domains and the 13 internal armadillo-like repeats of β-catenin are indicated. A stretch of unrepeated amino acids between repeat 10 and 11 (empty box) is shown. The binding sites for α-catenin, E-cadherin, APC, and the epitopes for rabbit β-catenin antisera β-cat.N, β-cat.C, and mouse mAb KT3 are indicated. The epitope for antiserum β-cat.N is deleted in ΔN90, ΔN131, and ΔN151, and these mutant proteins are not detected by β-cat.N. A mouse mAb raised against the 212 COOH-terminal amino acids of β-catenin was used instead of antiserum β-cat.C, and both β-cat.C antibodies do not detect ΔC. The following amino acids are deleted in the mutants: ΔC, 696–781; ΔN90, 1–90; ΔN131, 1–131; ΔN151, 1–151.
Schematic representation of KT3-tagged full-size and mutant β-catenin proteins. NH2- and COOH-terminal domains and the 13 internal armadillo-like repeats of β-catenin are indicated. A stretch of unrepeated amino acids between repeat 10 and 11 (empty box) is shown. The binding sites for α-catenin, E-cadherin, APC, and the epitopes for rabbit β-catenin antisera β-cat.N, β-cat.C, and mouse mAb KT3 are indicated. The epitope for antiserum β-cat.N is deleted in ΔN90, ΔN131, and ΔN151, and these mutant proteins are not detected by β-cat.N. A mouse mAb raised against the 212 COOH-terminal amino acids of β-catenin was used instead of antiserum β-cat.C, and both β-cat.C antibodies do not detect ΔC. The following amino acids are deleted in the mutants: ΔC, 696–781; ΔN90, 1–90; ΔN131, 1–131; ΔN151, 1–151.
… 
Dox-repressible expression of β-catenin mutant proteins in MDCK cells. MDCK clones were cultured for 4 d without or with Dox (−/+ Dox) and extracted with 1% SDS. 15-μg protein lysates were subjected to SDS-PAGE and immunoblotted with different antibodies (see Fig. 1). β-catenin mutant proteins (stars in a–c) were expressed only in cultures without Dox; endogenous β-catenin was expressed in all cultures (a and b). Expression levels of exogenous full-length β-catenin*, ΔN90, ΔN131, and ΔN151 were compared with that of endogenous β-catenin by immunoblotting with mAb β-cat.C (a). Exogenous β-catenin* and ΔC were compared with endogenous β-catenin by immunoblotting with β-cat.N (b). The expression levels of mutant β-catenin proteins in different clones were compared by immunoblotting with the tag antibody KT3 (c). The β-cat.C blot was reblotted with E-cadherin antiserum (d). The KT3 blot was reblotted with α-catenin antiserum (e). Molecular mass standards are indicated in kD.
Dox-repressible expression of β-catenin mutant proteins in MDCK cells. MDCK clones were cultured for 4 d without or with Dox (−/+ Dox) and extracted with 1% SDS. 15-μg protein lysates were subjected to SDS-PAGE and immunoblotted with different antibodies (see Fig. 1). β-catenin mutant proteins (stars in a–c) were expressed only in cultures without Dox; endogenous β-catenin was expressed in all cultures (a and b). Expression levels of exogenous full-length β-catenin*, ΔN90, ΔN131, and ΔN151 were compared with that of endogenous β-catenin by immunoblotting with mAb β-cat.C (a). Exogenous β-catenin* and ΔC were compared with endogenous β-catenin by immunoblotting with β-cat.N (b). The expression levels of mutant β-catenin proteins in different clones were compared by immunoblotting with the tag antibody KT3 (c). The β-cat.C blot was reblotted with E-cadherin antiserum (d). The KT3 blot was reblotted with α-catenin antiserum (e). Molecular mass standards are indicated in kD.
… 
β-catenin mutant proteins compete with endogenous β-catenin for binding to E-cadherin and α-catenin. MDCK clones were cultured 4 d without or with Dox (−/+) and extracted with 1% Triton X-100 lysis buffer. Protein lysates were split: 400-μg lysates were immunoprecipitated with E-cadherin antiserum to analyze binding of mutant β-catenin proteins to E-cadherin (a and b). 400 μg protein lysates were immunoprecipitated with α-catenin antiserum to analyze binding of mutant β-catenin proteins to α-catenin (c and d). Equivalent fractions of the immunoprecipitates were subjected to SDS-PAGE and blotted with mAbs KT3 (a and c) or β-cat.C (b and d). β-catenin mutant proteins are indicated (stars).
β-catenin mutant proteins compete with endogenous β-catenin for binding to E-cadherin and α-catenin. MDCK clones were cultured 4 d without or with Dox (−/+) and extracted with 1% Triton X-100 lysis buffer. Protein lysates were split: 400-μg lysates were immunoprecipitated with E-cadherin antiserum to analyze binding of mutant β-catenin proteins to E-cadherin (a and b). 400 μg protein lysates were immunoprecipitated with α-catenin antiserum to analyze binding of mutant β-catenin proteins to α-catenin (c and d). Equivalent fractions of the immunoprecipitates were subjected to SDS-PAGE and blotted with mAbs KT3 (a and c) or β-cat.C (b and d). β-catenin mutant proteins are indicated (stars).
… 
ΔN90, ΔN131, and ΔN151 are enriched in APC protein complexes. Aliquots of the same Triton X-100 lysates as described in Fig. 3 were either subjected to SDS-PAGE or used for immunoprecipitation with APC antiserum (a–d). 9-μg protein lysates were subjected to SDS-PAGE and immunoblotted with the indicated mAbs to compare the ratio of β-catenin mutant proteins to endogenous β-catenin (a), and to each other (b). 1,500-μg protein lysates were immunoprecipitated with APC antiserum. Equivalent fractions of the immunoprecipitates were subjected to SDS-PAGE and blotted with the indicated mAbs to analyze binding of mutant β-catenin proteins to APC (c–d). β-catenin mutant proteins are indicated (stars). A longer exposure of the blot in (d) is shown for lanes 7 and 8. Triton X-100 lysates were prepared from clones β-catenin*–7 and ΔN131-7, and aliquots of the lysates were immunoprecipitated with antiserum β-cat.C or APC antiserum (e). Immunoprecipitates were subjected to SDSPAGE and blotted with antiserum β-cat.C to analyze the ratio of exogenous β-catenin* and ΔN131 to endogenous β-catenin. β-catenin mutant proteins are indicated (stars).
ΔN90, ΔN131, and ΔN151 are enriched in APC protein complexes. Aliquots of the same Triton X-100 lysates as described in Fig. 3 were either subjected to SDS-PAGE or used for immunoprecipitation with APC antiserum (a–d). 9-μg protein lysates were subjected to SDS-PAGE and immunoblotted with the indicated mAbs to compare the ratio of β-catenin mutant proteins to endogenous β-catenin (a), and to each other (b). 1,500-μg protein lysates were immunoprecipitated with APC antiserum. Equivalent fractions of the immunoprecipitates were subjected to SDS-PAGE and blotted with the indicated mAbs to analyze binding of mutant β-catenin proteins to APC (c–d). β-catenin mutant proteins are indicated (stars). A longer exposure of the blot in (d) is shown for lanes 7 and 8. Triton X-100 lysates were prepared from clones β-catenin*–7 and ΔN131-7, and aliquots of the lysates were immunoprecipitated with antiserum β-cat.C or APC antiserum (e). Immunoprecipitates were subjected to SDSPAGE and blotted with antiserum β-cat.C to analyze the ratio of exogenous β-catenin* and ΔN131 to endogenous β-catenin. β-catenin mutant proteins are indicated (stars).
… 
Increased stability of ΔN90, ΔN131, and ΔN151 in the E-cadherin– and APC protein–bound pools. MDCK clones were cultured 0, 6, 12, or 18 h with Dox and extracted with 1% Triton X-100 lysis buffer. Protein lysates were split: 1,500-μg protein lysates were immunoprecipitated with APC antiserum (first column), and 500-μg lysates were immunoprecipitated with E-cadherin antiserum (second column). Equivalent fractions of the immunoprecipitates and 50-μg (β-cat.*-10,-7, ΔC-9) or 25-μg (ΔN90-A, ΔN131-D, ΔN151-D) protein lysates (third column) were subjected to SDS-PAGE and immunoblotted with mAb KT3. For β-catenin*–10, –7, and ΔC-9 blots, three times more of the APC immunoprecipitations were used than for the ΔN90-A, ΔN131-D, and ΔN151-D blots, and the blots were exposed longer.
+3
Increased stability of ΔN90, ΔN131, and ΔN151 in the E-cadherin– and APC protein–bound pools. MDCK clones were cultured 0, 6, 12, or 18 h with Dox and extracted with 1% Triton X-100 lysis buffer. Protein lysates were split: 1,500-μg protein lysates were immunoprecipitated with APC antiserum (first column), and 500-μg lysates were immunoprecipitated with E-cadherin antiserum (second column). Equivalent fractions of the immunoprecipitates and 50-μg (β-cat.*-10,-7, ΔC-9) or 25-μg (ΔN90-A, ΔN131-D, ΔN151-D) protein lysates (third column) were subjected to SDS-PAGE and immunoblotted with mAb KT3. For β-catenin*–10, –7, and ΔC-9 blots, three times more of the APC immunoprecipitations were used than for the ΔN90-A, ΔN131-D, and ΔN151-D blots, and the blots were exposed longer.
… 
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The Journal of Cell Biology, Volume 136, Number 3, February 10, 1997 693–706 693
NH
2
-terminal Deletion of
b
-Catenin Results in
Stable Colocalization of Mutant
b
-Catenin with
Adenomatous Polyposis Coli Protein and
Altered MDCK Cell Adhesion
Angela I.M. Barth,* Anne L. Pollack,
‡
Yoram Altschuler,
‡
Keith E. Mostov,
‡
and W. James Nelson*
*Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305-5426;
and
‡
Department of Anatomy, Department of Biochemistry and Cardiovascular Research Institute, University of California at
San Francisco, San Francisco, California 94143-0452
Abstract.
b
-Catenin is essential for the function of cad-
herins, a family of Ca
2
1
-dependent cell–cell adhesion
molecules, by linking them to
a
-catenin and the actin
cytoskeleton.
b
-Catenin also binds to adenomatous
polyposis coli (APC) protein, a cytosolic protein that is
the product of a tumor suppressor gene mutated in
colorectal adenomas. We have expressed mutant
b
-catenins in MDCK epithelial cells to gain insights
into the regulation of
b
-catenin distribution between
cadherin and APC protein complexes and the functions
of these complexes. Full-length
b
-catenin,
b
-catenin
mutant proteins with NH
2
-terminal deletions before
(
D
N90) or after (
D
N131,
D
N151) the
a
-catenin binding
site, or a mutant
b
-catenin with a COOH-terminal dele-
tion (
D
C) were expressed in MDCK cells under the
control of the tetracycline-repressible transactivator.
All
b
-catenin mutant proteins form complexes and
colocalize with E-cadherin at cell–cell contacts;
D
N90,
but neither
D
N131 nor
D
N151, bind
a
-catenin. How-
ever,
b
-catenin mutant proteins containing NH
2
-termi-
nal deletions also colocalize prominently with APC
protein in clusters at the tips of plasma membrane pro-
trusions; in contrast, full-length and COOH-terminal–
deleted
b
-catenin poorly colocalize with APC protein.
NH
2
-terminal deletions result in increased stability of
b
-catenin bound to APC protein and E-cadherin, com-
pared with full-length
b
-catenin. At low density,
MDCK cells expressing NH
2
-terminal–deleted
b
-cate-
nin mutants are dispersed, more fibroblastic in mor-
phology, and less efficient in forming colonies than pa-
rental MDCK cells. These results show that the NH
2
terminus, but not the COOH terminus of
b
-catenin,
regulates the dynamics of
b
-catenin binding to APC
protein and E-cadherin. Changes in
b
-catenin binding
to cadherin or APC protein, and the ensuing effects
on cell morphology and adhesion, are independent of
b
-catenin binding to
a
-catenin. These results demon-
strate that regulation of
b
-catenin binding to E-cad-
herin and APC protein is important in controlling epi-
thelial cell adhesion.
b
-
Catenin
is a ubiquitous protein in multicellular organ-
isms and was originally identified through its association
with the cytoplasmic domain of cadherins, a family of
Ca
2
1
-dependent cell adhesion proteins (McCrea and Gum-
biner, 1991; McCrea et al., 1991; Nagafuchi and Takeichi,
1989; Ozawa et al., 1989). Cadherin-mediated intercellular
adhesion initiates structural and functional changes in cells
and is important for maintaining tissue integrity during
embryonic development and in adult organisms (Nelson et
al., 1992; Takeichi, 1990, 1991). These functions of cadherin
require intracellular attachment of cadherin to the actin
cytoskeleton that is dependent on binding of cadherin to
catenins (Hirano et al., 1987; Nagafuchi and Takeichi,
1988; Ozawa et al., 1990);
b
-catenin mediates the linkage
of cadherins to
a
-catenin, which in turn interacts with the
actin cytoskeleton (Aberle et al., 1994; Hülsken et al.,
1994; Jou et al., 1995; Rimm et al., 1995).
Coordination of intercellular adhesion and cell migra-
tion is important during embryonic development. For ex-
ample, during gastrulation, neurulation, and organogene-
sis, sheets of cells move past neighboring cells without
losing cell–cell contact (Gumbiner, 1992). Significantly,
embryonic development of mice lacking
b
-catenin is dis-
rupted at gastrulation (Haegel et al., 1995). These embryos
form a trophectoderm and develop through preimplanta-
tion stages presumably because of the contribution of ma-
ternal
b
-catenin or replacement of
b
-catenin in adherens
A.I.M. Barth and A.L. Pollack contributed equally to this work.
Address all correspondence to Angela I.M. Barth, Department of Mo-
lecular and Cellular Physiology, Stanford University School of Medicine,
Stanford, CA 94305-5426. Tel.: (415) 723-9788. Fax: (415) 725-8021. e-mail:
angelab@leland.stanford.edu
... Growth conditions for Madin-Darby canine kidney (MDCK) type II/G cells have been described previously [43]. MDCK cells have been transfected with DNA constructs using lipofectamine 3000 reagent as described by the manufacturer (Gibco BRL, Gaithersburg, MD). ...
... SDS PAGE and western blotting was done as described previously [43] with the following variation: protein samples were separated in 4-20% Mini-Protean precast protein gels (Bio-Rad, CA). The following antibodies and dilutions were used dilution 1:500: mouse monoclonal anti-EpCAM antibody UMAB131 (OriGene Technology, MD); mouse anti-ERK and rabbit anti-phospho-ERK (Cell Signaling Technology, MA); rabbit anti-Claudin-7 34-9100, anti-Claudin-1 51-9000, and anti-Claudin-3 34-1700 (Thermo Fisher Scientific, MA); mouse anti-GFP (Roche Diagnostics GmbH, Mannheim, Germany); rabbit E-cadherin 24E10 (Cell Signaling Technology, MA) and mouse anti-GAPDH at 1:1000 (Abcam, MA). ...
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  • Sep 2018
Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro. We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1-deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1-mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.
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Nonmuscle myosin IIA is involved in recruitment of apical junction components through activation of α-catenin
Article
Full-text available
  • Apr 2018
  • Biol Open
MDCK dog kidney epithelial cells express two isoforms of nonmuscle myosin heavy chain II, IIA and IIB. Using the CRISPR/Cas9 system, we established cells in which the IIA gene was ablated. These cells were then transfected with a vector that expresses GFP-IIA chimeric molecule under the control of tetracycline-responsible element. In the absence of Dox (doxycyclin), when GFP-IIA is expressed (GFP-IIA+), the cells exhibit epithelial cell morphology, but in the presence of Dox, when expression of GFP-IIA is repressed (GFP-IIA-), the cells lose epithelial morphology and strong cell-cell adhesion. Consistent with these observations, GFP-IIA- cells failed to assemble junction components such as E-cadherin, desmoplakin, and occludin at cell-cell contact sites. Therefore, IIA is required for assembly of junction complexes. MDCK cells with an ablation of the α-catenin gene also exhibited the same phenotype. However, when in GFP-IIA- cells expressed α-catenin lacking the inhibitory region or E-cadherin/α-catenin chimeras, the cells acquired the ability to establish the junction complex. These experiments reveal that IIA acts as an activator of α-catenin in junction assembly.
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Purification of a 92-kDa cytoplasmic protein tightly associated with the cell-cell adhesion molecule E-cadherin (uvomorulin)
Article
Full-text available
  • Mar 1991
The transmembrane epithelial cell-cell adhesion protein E-cadherin (uvomorulin) associates via its cytoplasmic domain with three or more proteins whose structure and function are not yet established. The associated proteins, also termed catenins (Ozawa, M., Baribault, H., and Kemler, R. (1989) EMBO J. 8, 1711-1717), are of interest because they may form a link to the cytoskeleton, and/or regulate E-cadherin function. In this report immunoprecipitates of E-cadherin complexes, isolated from Xenopus laevis A6 and Madin-Darby canine kidney cell monolayers, were stringently washed to leave a single very tightly associated protein of ∼ 92 kDa. We report on the 92-kDa protein's association with E-cadherin in the presence of various perturbants, its preferential dissociation from the immune complex upon exposure to mixed detergent micelles containing sodium dodecyl sulfate, and its preparative purification to homogeneity. We have additionally found that E-cadherin and its associated proteins may be easily and quantitatively extracted from both subconfluent and fully confluent cells by a variety of mild nonionic detergents made up in isotonic buffers. In contrast, such extractions at short times left most of the cytoskeletal protein fodrin in the insoluble pellet fraction. Western blots of immunoprecipitated E-cadherin complexes failed to detect the presence of fodrin, or that of the cytoskeletal proteins adducin, α-actinin, and vinculin. If the E-cadherin-associated protein complex interacts with known proteins of the cell cytoskeleton, such interactions are labile and/or transient.
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Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly
Article
Full-text available
  • Jun 1994
Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed alpha-, beta-, and gamma-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/alpha-catenin, and either beta-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, beta-catenin, and plakoglobin cosedimented as complexes. alpha-Catenin was not associated with these complexes after synthesis, but a subpopulation of alpha-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of E-cadherin at the plasma membrane coincided with an increase in its insolubility in TX-100, but extraction of this insoluble pool with 1% SDS disrupted the cadherin/catenin complex. Therefore, to examine protein complex assembly in both the TX-100-soluble and -insoluble fractions, we used [35S]methionine labeling followed by chemical cross-linking before cell extraction. Analysis of cross-linked complexes from cells labeled to steady state indicates that, in addition to cadherin/catenin complexes, there were cadherin-independent pools of catenins present in both the TX-100-soluble and -insoluble fractions. Metabolic labeling followed by chase showed that immediately after synthesis, cadherin/beta-catenin, and cadherin/plakoglobin complexes were present in the TX-100-soluble fraction. Approximately 50% of complexes were titrated into the TX-100-insoluble fraction coincident with the arrival of the complexes at the plasma membrane and the assembly of alpha-catenin. Subsequently, > 90% of labeled cadherin, but no additional labeled catenin complexes, entered the TX-100-insoluble fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
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Armadillo is required for adherens junction assembly, cell polarity, and morphogenesis during Drosophila embryogenesis
Article
  • Jul 1996
Morphological and biochemical analyses have identified a set of proteins which together form a structure known as the adherens junction. Elegant experiments in tissue culture support the idea that adherens junctions play a key role in cell-cell adhesion and in organizing cells into epithelia. During normal embryonic development, cells quickly organize epithelia; these epithelial cells participate in many of the key morphogenetic movements of gastrulation. This prompted the hypothesis that adherens junctions ought to be critical for normal embryonic development. Drosophila Armadillo, the homologue of vertebrate beta-catenin, is a core component of the adherens junction protein complex and has been hypothesized to be essential for adherens junction function in vivo. We have used an intermediate mutant allele of armadillo, armadilloXP33, to test these hypotheses in Drosophila embryos. Adherens junctions cannot assemble in the absence of Armadillo, leading to dramatic defects in cell-cell adhesion. The epithelial cells of the embryo lose adhesion to each other, round up, and apparently become mesenchymal. Mutant cells also lose their normal cell polarity. These disruptions in the integrity of epithelia block the appropriate morphogenetic movements of gastrulation. These results provide the first demonstration of the effect of loss of adherens junctions on Drosophila embryonic development.
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Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles
Article
  • Dec 1987
Cadherins are a family of cell-cell adhesion molecules and are divided into subclasses with distinct adhesive specificities and tissue distribution. Here we examined the distribution of cadherins at contact sites between cells expressing the same or different cadherin subclasses. Each cadherin was concentrated at the boundary between cells expressing an identical cadherin subclass, irrespective of the cell types connected. However, such localization decreased or disappeared at the boundary between cells containing different cadherin subclasses. We also found that the localization of cadherins precisely coincided with that of actin bundles; both were detected at the apical region of cell sheets. This co-localization was retained even after cells were either treated with cytochalasin D or extracted with the detergent NP40. These results suggest that each cadherin subclass preferentially interacts with its own molecular type at intercellular boundaries, and that cadherin molecules may be associated with actin-based cytoskeletal elements.
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Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and E-cadherin and increased cellular adhesion
Article
  • Dec 1993
The Wnt-1 gene plays an essential role in fetal brain development and encodes a secreted protein whose signaling mechanism is presently unknown. In this report we have investigated intracellular mechanisms by which the Wnt-1 gene induces morphological changes in PC12 pheochromocytoma cells. PC12 cells expressing Wnt-1 show increased steady-state levels of the adhesive junction protein plakoglobin, and an altered distribution of this protein within the cell. This effect appears similar to a modulation of the plakoglobin homolog, Armadillo, that occurs in Drosophila embryos in response to the Wnt-1 homolog, wingless (Riggleman, B., P. Schedl, and E. Wieschaus. 1990. Cell. 63:549-560). In addition, PC12/Wnt-1 cells show elevated expression of E-cadherin and increased calcium-dependent cell-cell adhesion. These results imply evolutionary conservation of cellular responses to Wnt-1/wingless and indicate that in certain cell types Wnt-1 may act to modulate cell adhesion mechanisms.
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Spatial and temporal dissection of immediate and early events following cadherin-mediated epithelial cell adhesion
Article
  • Mar 1993
Cell-cell adhesion is at the top of a molecular cascade of protein interactions that leads to the remodeling of epithelial cell structure and function. The earliest events that initiate this cascade are poorly understood. Using high resolution differential interference contrast microscopy and retrospective immunohistochemistry, we observed that cell-cell contact in MDCK epithelial cells consists of distinct stages that correlate with specific changes in the interaction of E-cadherin with the cytoskeleton. We show that formation of a stable contact is preceded by numerous, transient contacts. During this time and immediately following formation of a stable contact, there are no detectable changes in the distribution, relative amount, or Triton X-100 insolubility of E-cadherin at the contact. After a lag period of approximately 10 min, there is a rapid acquisition of Triton X-100 insolubility of E-cadherin localized to the stable contact. Significantly, the total amount of E-cadherin at the contact remains unchanged during this time. The increase in the Triton X-100 insoluble pool of E-cadherin does not correlate with changes in the distribution of actin or fodrin, suggesting that the acquisition of the Triton X-100 insolubility is due to changes in E-cadherin itself, or closely associated proteins such as the catenins. The 10 minute lag period, and subsequent prompt and localized nature of E-cadherin reorganization indicate a form of signaling is occurring.
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Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules
Article
  • Apr 1995
A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E-cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.
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