* Department of Biology, University of Toledo, Toledo, Ohio 43606; and Department of Dermatology, University of
Pennsylvania, Philadelphia, Pennsylvania 19104
Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these
cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious
why formation of an adherens junction is a prerequisite
for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial
cells) linked to either -catenin or plakoglobin, which is
linked to
-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the
intermediate filament cytoskeleton, presumably
through plakoglobin and desmoplakin. To begin to
study the role of adherens junctions in the assembly of
desmosomes, we produced an epithelial cell line that
does not express classical cadherins and hence is unable
to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however,
overexpression of plakoglobin, along with E-cadherin,
did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens
junction before the cell can begin to assemble desmosomal components at regions of cell-cell contact. Although adherens junctions can form in the absence of
plakoglobin, making use only of
-catenin, such junctions cannot support the formation of desmosomes.
Thus, we speculate that plakoglobin plays a signaling
role in desmosome organization.
Squamous epithelial cells typically contain two prominent types of cell-cell junctions: the adherens junction and the desmosome. The adherens junction is
an intercellular adhesion complex that is composed of a
transmembrane protein (a classical cadherin) and numerous cytoplasmic proteins ( The organization of the proteins within the adherens
junction is well understood (for reviews see Kemler, 1993 The importance of the classical cadherins to the formation of adherens junctions and desmosomes has been demonstrated. Keratinocytes maintained in medium with low
Ca2+ (i.e., 30 µM) grow as a monolayer and do not exhibit
adherens junctions or desmosomes; however, elevation of
Ca2+ concentration induces the rapid formation of adherens junctions followed by the formation of desmosomes
(Hennings et al., 1980 Plakoglobin is found to be associated with the cytoplasmic domains of both the classical cadherins and the desmosomal cadherins. Despite the high degree of identity
between plakoglobin and In the present study, we have tested the hypothesis that
plakoglobin, through its interaction with E- or P-cadherin,
serves as a regulatory molecule for desmosome organization. Even though plakoglobin is not an essential structural component of the adherens junction (Sacco et al.,
1995 Reagents
Unless otherwise stated, all reagents were from Sigma Chemical Co. (St.
Louis, MO).
Cell Culture
The human epidermoid carcinoma cell line A431 was obtained from
American Type Culture Collection (Rockville, MD) and maintained in
DME (Gibco Laboratories, Grand Island, NY), 5% FCS (Hyclone Laboratories, Logan, UT), glutamine at 540 mg/L (Gibco Laboratories), and
antibiotics (Penicillin-Streptomycin) at 50 U penicillin and 50 mg streptomycin/L (Gibco Laboratories). Rat 2 cells and Wnt 1-expressing Rat 2 cells were kindly provided by Dr. Anthony M.C. Brown (Cornell University Medical College, New York) (Jue et al., 1992 Derivation of A431D Cells
A431 cells were placed into DME, 5% FCS containing 10 Antibodies
Rat monoclonal (E9) and rabbit polyclonal antibodies against human
E-cadherin (Wheelock et al., 1987 Molecular Constructs and Transfections
The human P-cadherin cDNA was a gift of Dr. Setsuo Hirohashi (National Cancer Center Research Institute, Tokyo, Japan). The entire P-cadherin cDNA was excised from pBR322 by EcoRI digestion and placed
into the EcoRI site of pLKneo-1 expression vector (Hirt et al., 1992 For cotransfection experiments, the neomycin gene in pLKneo was replaced with the puromycin gene from pBSpac Cell cultures were transfected using a calcium phosphate kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. Colonies
of G-418 or puromycin-resistant cells were isolated and screened for expression of the transfected gene(s) by immunoblot analysis. Positive
clones were further examined by immunofluorescence microscopy, immunoblotting, and immunoprecipitation.
Microscopy
Cells were plated in DME, 5% FCS with or without dexamethasone on
glass coverslips and grown until almost confluent. Cells were fixed in 1%
paraformaldehyde buffered with Hanks' balanced salt solution (Gibco
Laboratories) and 10 mM Hepes followed by permeabilization in 100%
cold methanol for 4 min at Electron Microscopy
2 × 105 cells were plated in DME, 5% FCS onto Falcon cyclopore membrane filters that inserted into 24-well plates (Becton-Dickinson Labware,
Franklin Lakes, NJ). After incubation for 24-72 h, cultures were washed
in protein-free DME, fixed overnight in 2% glutaraldehyde in 0.13 M sodium cacodylate buffer, pH 7.2, fixed in 2% OsO4 in 0.13 M cacodylate
buffer, pH 7.2, dehydrated, infiltrated, and embedded in Epon. Thin sections were cut, stained with uranyl acetate and lead citrate, and examined
on an electron microscope (model H7000; Hitachi Ltd., Tokyo, Japan).
Protein Assays
For the purpose of loading equal amounts of protein onto SDS-PAGE or
for immunoprecipitation experiments, quantification was done using the
BioRad Protein Assay reagent (Richmond, CA) according to the manufacturer's protocol.
Immunoprecipitations
A431 cells were grown to confluence and either immediately extracted or
metabolically labeled with [35S][Methionine/Cysteine] (Translabel; ICN
Biomedicals, Inc., Costa Mesa, CA) for 1 h followed by extraction as described (Lewis et al., 1994 Gels containing labeled extracts were enhanced (EN3HANCE, New
England Nuclear, Boston, MA), dried, and autoradiographed with Kodak
X-Omat AR film (Rochester, NY) at Cell Fractionation
For some experiments, cells were fractionated into an aqueous-soluble
fraction, an NP-40-soluble fraction, and an NP-40-insoluble fraction. Confluent 225 cm2 flasks of cells were washed with PBS, scraped into 5 ml of
10 mM Tris acetate, pH 8.0, 1 mM EDTA (TE) saturated with PMSF at 0°C, and Dounce homogenized until all cells were broken but nuclei remained intact, as determined by phase microscopy. The extract was centrifuged at 15,000 g for 45 min. The pellet was washed once with TE, extracted with 5 ml 0.5% NP-40 in TE, and centrifuged again at 15,000 g for
45 min. The resulting pellet was solubilized in 5 ml of boiling Laemmli
sample buffer (Laemmli, 1970 Quantitative Analysis of Gels
Quantitative analysis of protein bands from immunoblots or labeled immunoprecipitation reactions was performed on a Macintosh Quadra 800 using the Plotting Macros of the NIH Image program (developed at the
U.S. National Institutes of Health, Bethesda, MD, and acquired from the
Internet by anonymous FTP from zippy.nimh.nih.gov).
Treatment of A431 Cells with Dexamethasone Induces
Dramatic Morphological Changes
Confluent monolayers of A431 cells had the cobblestone
appearance that is typical of squamous epithelial cells.
Treatment of A431 cultures with 10
Both the morphological appearance as well as the unusual growth properties of the A431D cultures suggested
to us that the adhesive properties of the cells were altered.
We therefore compared the expression of adherens junction and desmosome components in the parent and derived cell lines.
Stable Expression of Most Adherens Junction
Components Is Depressed in A431D Cells
In the first series of experiments, we immunocytochemically stained A431 cultures that had been treated with dexamethasone for ~14 d and therefore were comprised of areas
with normal-appearing cells as well as foci with altered morphology. The areas of normal morphology consistently
and intensely stained with antibodies to E-cadherin, P-cadherin,
Several clones of A431D cells were then analyzed by immunoblotting and immunofluorescence for their levels of
adherens junctional and desmosomal proteins. Since there
was minimal variation, the results from one clone are
presented. As shown in Fig. 4, there was no detectable
E- or P-cadherin in the A431D cells, although comparable
amounts of cell lysate protein from the A431 parent cells
contained readily detectable amounts of these cadherins. When A431D cell extracts were immunoblotted with a
monoclonal antibody against human N-cadherin or with a
polyclonal anti-pan-cadherin antibody, the results were
totally negative, indicating that the A431D cells did not
express any classical cadherins (data not shown). Immunoblotting and immunofluorescence with antibodies to the
catenins revealed that greatly decreased levels of both
To compare the synthetic capacity of A431D and A431
cells for cadherins and catenins, both types of cultures were
biosynthetically labeled with 35S[Methionine/Cysteine] for
1 h. Extracts were prepared from equal numbers of cells,
subjected to immunoprecipitation with antibodies against
each of these proteins, resolved with SDS-PAGE, and visualized with autoradiography. When immunoprecipitated
with antibodies to either E- or P-cadherin, A431D extracts
revealed no bands, although intense bands were present in
the A431 parent extracts (data not shown). These data are
consistent with the immunofluorescence and immunoblotting results, all of which demonstrate that A431D cells do
not make detectable levels of E- or P-cadherin.
In contrast, when immunoprecipitation was performed
with antibodies to the catenins, it became clear that A431D
cells synthesized close to normal levels of both
Expression of Desmosomal Components Is Not Severely
Affected in A431D Cells
Extracts of A431D or A431 cells were compared by immunoblot analysis for expression of the desmosomal cadherins (desmoglein and desmocollin). A431D cells showed
approximately equal levels of desmocollin (data not shown)
and desmoglein when compared with the parent A431
cells (Fig. 4).
Immunofluorescence staining with antibodies against
the desmosomal proteins similarly revealed the presence
of these proteins in A4341D cells (Figs. 5 and 8). However,
the pattern of staining was distinct in the A431D and A431
cells. When A431 cells were examined at a higher magnification, it was clear that both desmoglein and desmoplakin
were present in a punctate pattern along the cell-cell borders, indicative of desmosome organization (Figs. 8, A
and C). In contrast, staining in the A431D cells was much
more diffuse; desmoplakin was completely cytosolic (Fig.
8 D), and desmoglein appeared to be diffuse but with
some indication of membrane staining. However, it was
not present in a punctate pattern (Fig. 8 B). To rule out
the possibility that A431D cells had lost the ability to either synthesize or properly organize keratin filaments, we compared staining patterns of A431 cells and A431D cells
(Fig. 9). Cells were plated sparsely so that the A431 cells
would have minimal cell-cell contact. Keratin filaments were
expressed in A431D cells and were organized in a pattern
similar, but not identical, to that seen in A431 cells. Thus,
the data we have presented indicate that, although A431D
cells synthesize the proteins necessary for desmosome formation, they are not able to organize these constituents into a functional structure (Figs. 2, 4, 5, and 8).
Transfection of E- or P-Cadherin into A431D Cells
Restores Some but Not All Cadherin-related Functions
In previous studies using human keratinocytes and other
epithelial cells, we and others have demonstrated that either E- or P-cadherin function is required for normal desmosome organization, as determined by redistribution of
the desmosomal components to a punctate pattern along
cell-cell borders and by ultrastructural analysis (Hodivala
and Watt, 1994
Stable expression of both Immunofluorescence localization of either E-cadherin
in the A431DE cells (Fig. 11 A) or P-cadherin in the
A431DP cells (data not shown) showed that the cadherin
was found at cell-cell borders and was not diffusely distributed in the cytoplasm, suggesting that the cadherin was
organized into junctional structures. The desmosomal
protein desmoglein was also localized at cell-cell borders,
but it did not exhibit the punctate pattern that is indicative of desmosome formation (compare Fig. 11 B with 8 A).
Furthermore, desmoplakin remained in a diffuse cytoplasmic pattern (Fig. 11 C) similar to that of A431D cells (Fig.
8 D). These data indicate that, despite the expression and
cell-cell border localization of the classical cadherin in
these cells, the desmosomal proteins were not being organized into punctate structures indicative of desmosomes.
Throughout the remainder of this paper, only the A431DE cells will be discussed, although identical experiments
were carried out for the A431DP cells, and similar results
were obtained.
To evaluate in another way the functionality of the
transfected cadherin, we tested its ability to form complexes with the cellular catenins. Whole cell extracts of
the A431DE cells were immunoprecipitated with anti-Ecadherin antibodies, followed by immunoblot analysis to
look for the presence of coimmunoprecipitated
To test the possibility that plakoglobin expressed by the
A431DE cells was altered such that it could not associate
with any cadherins, we immunoprecipitated desmoglein
from A431, A431D, and A431DE cells and then compared
the levels of plakoglobin coimmunoprecipitated with this
cadherin in each cell line (Fig. 12 B). In all three cell lines,
plakoglobin was found to be complexed with desmoglein to a similar (although not identical) extent. Similarly, plakoglobin was also associated with desmocollin in all the
tested cell lines (data not shown). The domain of plakoglobin that associates with the classical cadherins overlaps the
domain that associates with the desmosomal cadherins
(Chitaev et al., 1996 Plakoglobin Association with E-Cadherin Is Necessary
for Desmosome Organization
As described in the previous section, transfected E-cadherin in A431DE cells appeared in many ways to be functional in that it associated with To determine whether restoration of the plakoglobin
level led to restoration of the ability to form desmosomes,
we performed immunofluorescence and electron microscopic analyses. Staining of A431DEpg cells with antibody
to desmoplakin revealed a punctate pattern along the cell-
cell borders (Fig. 13 B), strongly suggesting formation of
desmosomes. This was confirmed by ultrastructural analysis which demonstrated the presence of desmosomes in
A431DEpg cells (Fig. 13, C and D).
To demonstrate that we had in fact increased the level
of classical cadherin-plakoglobin complex in the double
transfectants, we semiquantified the relative levels of plakoglobin in different compartments of the parent and
transfected cell lines using immunoblots. We prepared
three sequential cell fractions: (a) an aqueous soluble fraction prepared by homogenizing the cells in the absence of detergent, (b) a nonionic detergent-soluble fraction prepared by NP-40 extraction of the pellet recovered from the
first step, and (c) an insoluble or cytoskeletal fraction prepared by solubilizing the pellet from the second step in
SDS. Fraction b was further fractionated into plakoglobin
that was associated with classical cadherins and plakoglobin that was associated with desmosomal cadherins by exhaustive immunoprecipitation. As shown in Fig. 14, the
amount of plakoglobin in each fraction was very similar in
A431 and A431DEpg cells. Furthermore, the amount of
plakoglobin complexed to cadherins (in the NP-40 soluble
fraction) was also very similar in the two cell lines. All of
these data thus demonstrate that the amount and distribution of plakoglobin in the A431DEpg transfectant is very
close to that observed in the parent A431 cells.
An important control for this experiment was the demonstration that transfection of plakoglobin alone into A431D
cells was not sufficient for desmosomal organization. This
is demonstrated in Fig. 15, which shows the staining pattern for desmoplakin in A431D cells that were overexpressing plakoglobin in the absence of a classical cadherin.
Desmoplakin was localized throughout the cytoplasm in a
diffuse pattern; no desmoplakin was detected at the cell-
cell borders. Even though these clones were expressing as
much plakoglobin as the parent A431 cells (as demonstrated by immunoblotting, not shown), they could not organize desmoplakin in the absence of a classical cadherin.
The most straightforward interpretation of all these data
is that desmosome organization requires a sufficient level
of plakoglobin to permit complex formation both with desmosomal cadherins and with classical cadherins. Hence, we
hypothesize that the complex between classical cadherins
and plakoglobin has signal-transducing capacity that initiates desmosome organization.
A Plakoglobin-E-Cadherin Chimera Can Restore
Desmosome Organization
To test further our hypothesis that a plakoglobin-classical
cadherin complex is required to initiate desmosome organization, we constructed cDNA for an E-cadherin-plakoglobin chimeric molecule (Fig. 16 B) consisting of: (a) the
entire extracellular and transmembrane domains of E-cadherin as well as the first 61 amino acids of its cytoplasmic domain, excluding the region shown to associate with
Thus, our data with the chimeric transfectants verify and
extend our findings with the cotransfected lines expressing
both E-cadherin and plakoglobin. An association between
a classical cadherin and plakoglobin appears to be essential for the formation of desmosomes. Plakoglobin appears
to be a central molecule in desmosomal organization, not
only because of its presumed structural role but also because of a regulatory function in conjunction with the classical cadherins.
The goal of these studies was to further our understanding
of how classical cadherins (e.g., E- and P-cadherins) regulate the organization of desmosomes. In previous studies using
normal human keratinocytes incubated with function-perturbing antibodies against the cadherins, we and others
have shown that either E- or P-cadherin must be functional
in order for these cells to form desmosomes normally
(Hodivala and Watt, 1994 In the present study, we have not addressed the mechanism by which A431 cells ceased to express the cadherins
when treated with dexamethasone. The switch-off of the
cadherins appeared to take place over several weeks and
did not involve every cell. However, the cadherin-negative
cells could be easily recovered from the culture because they
floated in the medium when the culture became confluent.
These floating, cadherin-negative cells could be readily propagated since they would replate and divide when added to
a fresh culture dish. We are still attempting to determine the mechanism by which cadherins are turned off; we suspect it involves alterations in the activity of the cadherin
promoter. We also believe that the mechanism has specificity for A431 cells, as we attempted, without success, to
replicate the dexamethasone effect in other squamous epithelial cell lines.
Regardless of the mechanism by which the A431D cells
were generated, they are a useful experimental tool for
evaluation of the molecular interactions that are required
for intercellular junction organization. Our present studies
with these cells suggest that plakoglobin is a central regulatory molecule. A431D cells express approximately half
the plakoglobin of the parent A431 cell line. When A431D
cells are transfected with E-cadherin, the plakoglobin level
remains approximately the same, and most remains associated with desmosomal cadherins. Very little plakoglobin is
associated with the transfected E-cadherin, even though
Our findings with the A431D cells strongly indicate that
expression of desmosomal components and interaction of
plakoglobin with desmosomal cadherins are not sufficient
for desmosome organization. At least two additional conditions must be met if desmosome formation is to proceed:
(a) The cells must express a classical cadherin; and (b)
there must be sufficient plakoglobin to permit association not only with desmosomal cadherins, but also with the
classical cadherins. It has previously been shown that plakoglobin is not required for organization of an adherens
junction (Sacco et al., 1995 Ruiz et al. (1996) The assembly of structures like the desmosome and the
adherens junction is indeed complex. It is interesting that
one protein, plakoglobin, is found in both structures, while
the remaining components are restricted to one structure
or the other. Bornslaeger et al. (1996) Plakoglobin and Armadillo family members are components of the wingless or wnt-signaling pathway (for review see Orsulic and
Peifer, 1996-catenin,
-catenin and plakoglobin, vinculin and
-actinin; for reviews see Takeichi,
1990
; Geiger and Ayalon, 1992
). The cadherins are directly
responsible for adhesive interactions via a Ca2+-dependent,
homotypic mechanism, i.e., in the presence of sufficient Ca2+, cadherin on one cell binds to an identical molecule
on an adjacent cell. The desmosome, also an intercellular
adhesion complex, is composed of at least two different
transmembrane proteins (desmoglein and desmocollin) as
well as several cytoplasmic proteins, including desmoplakins
and plakoglobin (Koch and Franke, 1994
). The transmembrane components of the desmosome are members of the
broadly defined cadherin family and also require Ca2+ for
adhesive activity. However, decisive experimental evidence for homophilic or heterophilic interactions between desmosomal cadherins via their extracellular domains has not yet
been presented (Koch and Franke, 1994
; Kowalczyk et al.,
1996
). While members of the cadherin family constitute
the transmembrane portion of both adherens junctions
and desmosomes, the different classes of cadherins are
linked to different cytoskeletal elements by the cytoplasmic components of each junction. Specifically, the classical
cadherins are linked to actin filaments and the desmosomal cadherins to intermediate filaments.
;
Cowin, 1994
; Wheelock et al., 1996
). Specifically, the intracellular domain of cadherin interacts directly with either
plakoglobin or
-catenin, which in turns binds to
-catenin
(Jou et al., 1995
; Sacco et al., 1995
).
-Catenin interacts with
-actinin and actin filaments, thereby linking the cadherin/
catenin complex to the cytoskeleton (Knudsen et al., 1995
;
Rimm et al., 1995
). Cadherin/catenin complexes include
either plakoglobin or
-catenin but not both (Näthke et al.,
1994
).
; Tsao et al., 1982
; Boyce and Ham,
1983
; Hennings and Holbrook, 1983
; O'Keefe et al., 1987
;
Wheelock and Jensen, 1992
; Hodivala and Watt, 1994
;
Lewis et al., 1994
). Simultaneous blocking with functionperturbing antibodies against the two classical cadherins (E- and P-cadherin) found in keratinocytes inhibits not
only Ca2+-induced adherens junction formation but also
severely limits desmosome formation (Lewis et al., 1994
;
Jensen et al., 1996). Consistent with these findings, expression of a dominant-negative cadherin by keratinocytes
results in decreased E-cadherin expression and delayed
assembly of desmosomes (Fujimori and Takeicki, 1993; Amagai, et al., 1995). These data suggest some form of
cross-talk between the proteins of the adherens junction
and those of the desmosome. One candidate protein that
might mediate such cross-talk is plakoglobin, since it is the
only known common component of both junctions.
-catenin (65% at the amino acid
level; Fouquet et al., 1992
),
-catenin only associates with
the classical cadherins and not with the desmosomal cadherins. In the adherens junction, plakoglobin and
-catenin have at least one common function, i.e., the linking of
cadherin to
-catenin and thus to actin. However, there is emerging evidence that other functions of these two proteins are not identical. For example, in a study by Navarro
et al. (1993)
, E-cadherin transfected into a spindle cell carcinoma was shown to associate with
- and
-catenin, but
not with the low levels of endogenous plakoglobin. The
transfected cells did not revert to a more epithelial morphology in spite of the presence of functional E-cadherin,
and the authors suggested that the lack of plakoglobin may have prevented such morphological reversion.
), our data indicate that plakoglobin can function as a
regulator of desmosome formation only when it is associated with a classical cadherin. Thus, we propose that plakoglobin has at least two functions: (a) as a structural component of the adherens junction and the desmosome and
(b) as a signaling molecule that regulates communication
between the adherens junction and the desmosome.
Materials and Methods
) and maintained in
DME, 5% FCS.
7 M dexamethasone and grown until confluent. Cells were then lightly trypsinized to recover approximately half the cell population; trypsin was neutralized
with FCS, and the cells were centrifuged and resuspended in fresh medium, still containing dexamethasone. This procedure was continued for
several weeks until the population consisted mainly of fibroblastic cells.
Clones were obtained from this population by limiting dilution and characterized for cadherin expression, morphology, and clonal integrity. Cadherin-negative clones were selected and renamed A431D cells to distinguish them from the parent A431 cells.
) and rabbit pan-cadherin (Knudsen et al.,
1995
) and mouse monoclonal antibodies against
-catenin (1G5) and
-catenin (5H10; Johnson et al., 1993
), plakoglobin (15F11; Sacco et al.,
1995
), P-cadherin (6A9; Lewis et al., 1994
), N-cadherin (13A9; Sacco et al.,
1995
), and desmoglein 2 (6D8; Wahl et al., 1996
) have been previously described. The mouse monoclonal antidesmoplakin multi-epitope cocktail
(dp) was purchased from American Research Products (Belmont, MA).
The mouse monoclonal anticytokeratin antibody (CY90), which was made
against A431 material, was purchased from Sigma Chemical Co.
),
which was a gift of Dr. Nicholas Fasel (University of Lausanne). Genes inserted into the pLK series of vectors are under control of the mouse mammary tumor virus promoter and are inducible by dexamethasone. The
human E-cadherin cDNA was isolated from a JAR PR497 human gestational choriocarcinoma (Pattillo et al., 1971
) cDNA library. The entire
E-cadherin open reading frame was excised from pBluescript using HindIII and inserted into the HindIII site of pLKneo.
p (de la Luna and Ortín,
1992), a gift of Dr. Juan Ortín (Universidad Autónoma de Madrid,
Madrid, Spain). This vector is referred to as pLKpac. The full-length human plakoglobin clone in pBluescript (HPG Ca2.1; Franke et al., 1989
), a gift
from Dr. Werner W. Franke (German Cancer Research Center, Heidelberg, Germany), was inserted into pLKpac. The chimera between E-cadherin and plakoglobin was constructed of (a) the entire extracellular and
transmembrane domains of E-cadherin as well as the first 61 amino acids
of its cytoplasmic domain, excluding the region shown to associate with
-catenin, (b) a nine-amino acid spacer, and (c) amino acids 19-745 of
plakoglobin, which includes the domain that is necessary for association
with
-catenin (Sacco et al., 1995
). The chimeric E-cadherin-plakoglobin cDNA was inserted into pLKpac.
20°C. Coverslips were blocked for 1 h in PBS
containing 10% goat serum and 0.1 M glycine and stained with primary
antibodies for 1 h followed by FITC-conjugated anti-IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Fluorescence was detected
with a microscope (model Axiophot; Carl Zeiss, Inc., Thornwood, NY)
equipped with epifluorescence and filters appropriate for visualizing
FITC, and cells were photographed using T-Max 3200 film.
). Briefly, monolayers of cells were washed three
times with PBS at room temperature, extracted at 4°C with 2 ml/75 cm2
flask of 10 mM Tris acetate, pH 8.0, 0.5% NP-40 (BDH Chemicals Ltd.,
Poole, England), and 1 mM EDTA saturated with PMSF. The cells were
scraped into this buffer, followed by vigorous agitation for 30 min on ice.
Insoluble material was removed by centrifugation at 15,000 g for 45 min.
[35S][Methionine/Cysteine]-labeled cell extracts were adjusted to 0.25 M
NaCl and 0.3% BSA; 100 µl extract was precleared by mixing with 50 µl
Sepharose 4B (Pharmacia LKB Biotechnology, Piscataway, NJ) at 4°C for
30 min. The supernatant was separated from the Sepharose by brief centrifugation. Unlabeled extracts were not precleared. Primary antibody was
added to unlabeled or labeled extract and mixed at 4°C for 1 h. Antimouse or anti-rabbit IgG-Sepharose (Organon-Technica, Durham, NC) was
added, and mixing continued for an additional 30 min. The Sepharosebound immune complexes were washed five times with 50 mM Tris-HCl,
pH 7.5, 0.5% NP-40, and 1 mM EDTA. Pellets of Sepharose-bound immune complex were boiled in Laemmli sample buffer (Laemmli, 1970
)
and resolved on 7% SDS-PAGE as described (Lewis et al., 1994
). For gels
with labeled extracts 14C-labeled molecular mass markers (Gibco Laboratories) included myosin (205 kD), phosphorylase b (97 kD), BSA (66 kD),
ovalbumin (43 kD), and carbonic anhydrase (30 kD). For gels with unlabeled extracts, molecular mass markers (Sigma Chemical Co.) included myosin (205 kD),
-galactosidase (116 kD), phosphorylase b (97 kD),
BSA (66 kD), ovalbumin (43 kD), and carbonic anhydrase (30 kD).
70°C. Gels containing unlabeled
extracts were transferred electrophoretically to nitrocellulose and immunoblotted as described (Wheelock et al., 1987
) using primary antibodies
followed by alkaline phosphatase-conjugated anti-IgG (Promega Corp.,
Madison, WI) and nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate substrates.
).
Results
7 M dexamethasone
resulted in the development of small islands of cells that
showed distinct morphological changes from the parent cells, beginning at ~14 d after addition of steroid. Specifically, the affected cells displayed a more fibroblastic morphology along with marked plasma membrane blebbing
(Fig. 1). We found that we could enrich for the affected
subpopulation by mild trypsinization, since these cells
were detached much more readily by trypsin than the normal-appearing ones. Several rounds of such selective
trypsinization, followed by further incubation and growth,
led to the development of cultures that consisted almost
entirely of the abnormal-appearing cells, which we renamed A431D cells to distinguish them from the parent
A431 cell line. To characterize the A431D cells, several clones, all of which exhibited a similar phenotype, were
isolated by limiting dilution. Aside from their fibroblastic
morphology and membrane blebbing, another characteristic that distinguished the A431D cells from the parent
A431 line was their growth pattern. Confluent A431 cultures exhibited several vertical layers with desmosomal attachments between layers; however, the A431D grew as a
monolayer of cells without desmosomal attachments to
one another (Fig. 2). Although both types of cultures,
when confluent, contained numerous floating cells, they
exhibited very different properties. The floating cells of
the parent A431 cultures were not viable, as evidenced by
their inability to attach and proliferate when transferred to
a new tissue culture plate; however, the floating cells of
the A431D cultures rapidly reattached to a fresh culture plate and continued to grow (data not shown).
Fig. 1.
A431D cells have fibroblast-like morphology. Living A431 cells (A and C) and A431D cells (B and D) were photographed using the 40× objective (A and B) or the 100× objective (C and D). Note the fibroblast-like morphology and the minimal cell-cell contact in the A431D cells. Note also the blebbing of the plasma membrane in the A431D cells. Bar, 30 µm.
[View Larger Version of this Image (143K GIF file)]
Fig. 2.
A431D cells grow as a monolayer without desmosomal contacts. A431 cells (A and B) and A431D cells (C) were plated on filters and processed for electron microscopy. A431 cells formed several layers with extensive desmosomal contacts. A431D cells grew as a
monolayer and did not form desmosomes. Bars: (A and C) 2.5 µm; (B) 0.4 µm.
[View Larger Version of this Image (76K GIF file)]
-catenin, and
-catenin, with concentration along
the cell-cell borders, as expected for an epithelial cell line.
In marked contrast, the foci of altered morphology did not stain with antibodies to constituents of the adherens junction (Fig. 3). Cells that lacked expression of E- and P-cadherin initially appeared very flat and developed noticeable
surface blebs with even fewer cell-cell contacts, as can be
noted in Fig. 3, A and C. To analyze this population biochemically, we cloned out cadherin-negative cells from the
population; all clones exhibited the more fibroblast-like appearance seen in Fig. 1, which appears to represent the
final stage of the morphological changes induced by longterm culture in dexamethasone.
Fig. 3.
A431 cells show loss of E-cadherin staining as they convert to A431D cells. A431 cells were grown in 10-7 M dexamethasone
for 2 wk, transferred to glass coverslips, and processed for immunofluorescence. Phase microscopy (A and C) depicts the altered morphology of the cells as they are converted to A431D cells. Normal-looking A431 cells surround the islands of A431D cells in A and in
the bottom right hand corner of C. Arrows in A and B point to islands of A431D cells. Arrows in C and D point to borders between the
A431 parent cells and the A431D cells. Immunofluorescence microscopy shows cell border staining for E-cadherin (B and D). Note the
absence of staining in the cells that have converted to A431D cells and the decreased staining in cells that appear to be converting to
A431D cells (arrowheads). Bar, 30 µm.
[View Larger Version of this Image (125K GIF file)]
- and
-catenins were present in the A431D cells compared to the A431 parent line. To accurately compare
A431D cells with A431 cells in immunofluorescence,
timed exposures were taken (Fig. 5). Under conditions in
which intense staining was found in A431 cells,
-catenin,
-catenin, and plakoglobin were barely visible in A431D
cells. When longer exposures were taken, however, all three catenins were detectable (Fig. 6 and not shown). In
contrast, neither E-cadherin nor P-cadherin was detectable even after prolonged exposures (data not shown).
Fig. 4.
A431D cells have decreased levels of cadherins and
catenins. (A) A431 cells (odd numbered lanes) and A431D cells
(even numbered lanes) were extracted with NP-40, and equal
amounts of protein from each extract were resolved by 7% SDSPAGE, transblotted to nitrocellulose, and probed with various
monoclonal antibodies. Lanes 1 and 2: anti--catenin (
-cat);
lanes 3 and 4: anti-
-catenin (
-cat); lanes 5 and 6: anti-plakoglobin (pg); lanes 7 and 8: anti-E-cadherin (E-cad); lanes 9 and 10:
anti-P-cadherin (P-cad); lanes 11 and 12: antidesmoglein (dg).
Molecular weight markers are indicated. (B) Each band in A was
quantified. The level of expression in A431 cells was assigned the
value 1, and the level of expression in A431D is presented as a
fraction of the level in A431.
[View Larger Version of this Image (46K GIF file)]
Fig. 5.
Expression of adherens junction and desmosome proteins is abnormal in A431D cells. A431 cells (A, C, E, G, and I) or
A431D cells (B, D, F, H, and J) were grown on glass coverslips and processed for immunofluorescence. (A and B) The localization of
E-cadherin (E-cad); (C and D) -catenin (
-cat); (E and F) plakoglobin (pg); (G and H) desmoglein (dg); (I and J) desmoplakin (dp).
Timed exposures were taken to compare the levels of expression in A431D cells with that in A431 cells. Bar, 30 µm.
[View Larger Version of this Image (98K GIF file)]
Fig. 6.
-Catenin and plakoglobin are expressed at low levels by A431D cells. When automatic exposures were made of cells stained for
-catenin (A;
-cat) or plakoglobin (B; pg), each protein was detectable. Bar, 30 µm.
[View Larger Version of this Image (59K GIF file)]
- and
-catenin (Fig. 7 A). However, anticatenin antibodies did not
coimmunoprecipitate other catenins or cadherins from the
A431D extracts, although catenins, cadherins, and plakoglobin did coimmunoprecipitate in the A431 parent cell extracts. Hence, although A431D cells synthesize
- and
-catenins, they do not form a complex with other cellular
proteins. To examine the stability of the catenins, we performed pulse chase labeling experiments (Fig. 7 B). After
a 1-h chase,
-catenin had almost completely disappeared
in A431D cells. By contrast, in the parent A431 cells after
a 2-h chase,
-catenin was still present at almost the same
level as in the 0-h control. Interestingly,
-catenin was stably expressed by A431D cells, at least for a 2-h chase period. The catenin biosynthetic labeling results, coupled with the evidence for reduced total levels of these proteins as
observed in immunoblots and via immunofluorescence, suggest that
-catenin is expressed but then rapidly degraded
in A431D cells. Given the total lack of E- and P-cadherins
in the A431D cells, the instability of
-catenin is not surprising since in L cells, stable catenin expression has been
shown to be dependent upon cadherin expression (Nagafuchi and Takeichi 1988
; Ozawa et al., 1989
). It was surprising, however, that
-catenin was stably expressed. The
level of expression, as shown by immunoblot analysis (see
Fig. 4), was lower than in A431 cells, but the turnover rate
was not as rapid as that for
-catenin. Consistent with
- and
-catenins, plakoglobin was expressed at a lower level in
A431D cells; its turnover rate in A431D cells and A431
cells appeared to be similar, presumably due to its association with desmosomal cadherins. Thus, our data concerning adherens junction proteins demonstrate that A431D
cells have no detectable classical cadherins and decreased
levels of
-catenin,
-catenin, and plakoglobin.
Fig. 7.
Catenins do not form normal complexes in A431D
cells. (A) A431 cells and A431D cells were labeled with [35S][Methionine/Cysteine] for 1 h and extracted with NP-40. Cell extracts
were immunoprecipitated with monoclonal anti--catenin (lane 1),
monoclonal anti-
-catenin (lane 2), or monoclonal anti-plakoglobin (lane 3). E-cadherin, P-cadherin,
-catenin,
-catenin, and
plakoglobin coimmunoprecipitated with antibodies against
-catenin (lane 1),
-catenin (lane 2), or plakoglobin (lane 3) from
A431 cells. In contrast, only the catenin for which the immunoprecipitating antibody was specific was seen in the A431D reactions. (B) Cells were labeled for 1 h and chased for 1 or 2 h. They
were then extracted with NP-40 and immunoprecipitated as
above.
-Catenin and plakoglobin were stable in both A431 cells
and A431D cells, whereas
-catenin was stable for 2 h in A431
cells but had almost completely disappeared by 1 h in A431D
cells. The level of each catenin was quantified; expression at time
0 was set equal to 100%, and the remaining levels at each time
point are expressed as a percentage of 0 time. The numbers are
presented above each band. Cad, cadherin;
-cat,
-catenin;
-cat,
-catenin; pg, plakoglobin.
[View Larger Version of this Image (74K GIF file)]
Fig. 8.
A431D cells show abnormal localization of desmosomal proteins. A431 cells (A and C) and A431D cells (B and D) were
grown on glass coverslips, processed for immunofluorescence with antibodies against desmoglein (A and B; dg) or desmoplakin (C and
D; dp), and photographed using a 100× objective. Bar, 30 µm.
[View Larger Version of this Image (101K GIF file)]
Fig. 9.
The pattern of keratin staining is similar in
A431 cells and A431D cells.
A431 cells (A) or A431D
cells (B) were grown on glass
coverslips and processed for
immunofluorescence with
antibodies against keratin.
Bar, 30 µm.
[View Larger Version of this Image (48K GIF file)]
; Lewis et al., 1994
; Amagai et al., 1995
;
Jensen et al., 1996). We therefore hypothesized that the
lack of desmosomes in A431D cells was secondary to the loss of E- and P-cadherin expression. To test this hypothesis, we reexpressed a cadherin in the A431D cells by transfecting the entire cDNA for either E- or P-cadherin, using
a plasmid that also conferred G418 resistance. Resistant
colonies were screened for the expression of E- or P-cadherin by immunofluorescence and positive clones were selected. Clones of A431D cells transfected with E-cadherin
(A431DE cells) or P-cadherin (A431DP) that expressed levels of cadherin nearly equivalent to that of the parent
A431 cells were selected for further analysis (Fig. 10). As
anticipated, A431DE cells expressed only E-cadherin and not
P-cadherin, while A431DP cells expressed only P-cadherin
and not E-cadherin. Thus, transfection of one cadherin cDNA
did not lead to expression of the other cadherin by activation of the endogenous genes.
Fig. 10.
Transfection of E- or P-cadherin into A431D cells stabilizes the expression of - and
-catenin. (A) A431 cells (lane 1), A431D cells (lane 2), A431D cells transfected with E-cadherin (A431DE; lane 3), and A431D cells transfected with P-cadherin (A431DP; lane 4) were extracted with NP-40 and equal amounts
of protein from each extract were resolved by 7% SDS-PAGE,
transblotted to nitrocellulose, and probed with monoclonal antibodies against E-cadherin (E-Cad), P-cadherin (P-cad),
-catenin (
-cat), or
-catenin (
-cat). (B) Each band in A was quantified. The level of expression in A431 cells was assigned the value 1, and the levels of expression in the other cell lines are presented
as fractions of the level in A431.
[View Larger Version of this Image (36K GIF file)]
- and
-catenin increased in
the A431D cells transfected with either E- or P-cadherin
(Fig. 10). These results confirm our hypothesis that the
lower levels of
- and
-catenin in A431D cells were due
to instability because of a lack of association with cadherin
rather than to a direct effect of dexamethasone treatment.
Fig. 11.
A431D cells transfected with E-cadherin do not regain the ability to organize desmosomal proteins at cell-cell borders.
A431D cells transfected with E-cadherin (A431DE) were grown on glass coverslips and processed for immunofluorescence. (A) The localization of E-cadherin (E-cad); (B) the localization of desmoglein (dg); and (C) the localization of desmoplakin (dp). Bar, 30 µm.
[View Larger Version of this Image (89K GIF file)]
-catenin
or plakoglobin. As shown in Fig. 12 A,
-catenin coimmunoprecipitated with E-cadherin to an equivalent extent in
A431 and A431DE cells. In contrast, only very small
amounts of plakoglobin coimmunoprecipitated with the
transfected E-cadherin when compared with that normally seen in A431 cells (Fig. 12 A).
Fig. 12.
Plakoglobin is not associated with E-cadherin in
A431DE cells. (A) A431 cells (lane 1) or A431D cells transfected
with E-cadherin (A431DE; lane 2) were extracted with NP-40 and
equal amounts of protein from each extract were immunoprecipitated (IP) with antibodies against E-cadherin (anti-E-cad). The
immunoprecipitated proteins were resolved by 7% SDS-PAGE,
transblotted to nitrocellulose, and probed with monoclonal antibodies against -catenin (
-cat) or plakoglobin (pg).
-Catenin
coimmunoprecipitated with the transfected E-cadherin, whereas
plakoglobin did not. (B) A431 cells (lane 1), A431D cells (lane 2),
or A431DE (lane 3) were extracted with NP-40, and equal amounts
of protein from each extract were immunoprecipitated with antibodies against desmoglein (dg). The immunoprecipitation reactions
were resolved by 7% SDS-PAGE, transblotted to nitrocellulose, and probed with monoclonal antibodies against plakoglobin. (C) Each band in A was quantified. The amount of
-catenin or plakoglobin that was associated with E-cadherin in A431 cells was
assigned the value 1 and the amount in A431DE cells is presented as a fraction of that in A431. (D) Each band in B was
quantified. The amount of plakoglobin that was associated with
desmoglein in A431 cells was assigned the value 1, and the
amounts in the other cell lines are presented as fractions of that
in A431.
[View Larger Version of this Image (35K GIF file)]
; Wahl et al., 1996
; Witcher et al., 1996
).
Thus, the inherent ability of plakoglobin to associate with
desmosomal (and presumably classical) cadherins did not
seem to be completely disrupted in A431D cells.
- and
-catenins and was
localized along cell-cell borders. Surprisingly, it did not
complex with cellular plakoglobin, and it apparently could
not mediate desmosome organization. Based on these observations, we hypothesized that the inability of the A431DE cells to form desmosomes might be related to a
lack of E-cadherin-plakoglobin interaction. To address
this hypothesis, we attempted to increase the amount of
E-cadherin-plakoglobin complex within the A431DE cells
by overexpressing plakoglobin. A431D cells were therefore cotransfected with full-length plakoglobin cDNA as
well as E-cadherin cDNA, and coexpressing clones were isolated (A431DEpg cells).
Fig. 13.
Cotransfection of
A431D cells with E-cadherin
and plakoglobin results in formation of desmosomes.
A431D cells transfected with
E-cadherin (A431DE; A) or
cotransfected with E-cadherin
and plakoglobin (A431DEpg;
B) were grown on glass coverslips and processed for immunofluorescence with antibodies against desmoplakin. A punctate pattern of desmoplakin staining indicative
of desmosome organization
was observed in the A431DEpg cells but not in the A431DE cells. A431DEpg
cells were plated on filters
and processed for electron
microscopy (C and D). The
cells grew in several layers and formed desmosomal
contacts. The arrow in C
points to a desmosome shown
at higher magnification in D. Bars: (A) 30 µm; (C) 2 µm; (D) 0.2 µm.
[View Larger Version of this Image (126K GIF file)]
Fig. 14.
Relative amount and distribution of plakoglobin are
similar in A431 and A431DEpg cells. Cells were fractionated into
soluble (no detergent), NP-40-soluble, and NP-40-insoluble fractions. Equal volumes of each fraction was resolved by SDS-PAGE,
immunoblotted for plakoglobin, and quantified. The NP-40-soluble
fraction was further fractionated into E- and P-cadherin-associated plakoglobin and into desmoglein- and desmocollin-associated
plakoglobin by exhaustive immunoprecipitation. Each fraction is
represented graphically. The white insets in the NP-40-soluble
graph represent the plakoglobin that was associated with E-cadherin + P-cadherin. The remainder was associated with desmoglein + desmocollin.
[View Larger Version of this Image (40K GIF file)]
Fig. 15.
Overexpression of
plakoglobin alone does not
result in organization of desmoplakin at cell-cell borders.
A431D cells (A and C) and
A431D cells transfected with
plakoglobin (pg; B and D) were grown on glass coverslips and processed for immunofluorescence with antibodies against plakoglobin
(A and C) or desmoplakin
(dp; B and D). Bar, 30 µm.
[View Larger Version of this Image (139K GIF file)]
-catenin (Stappert and Kemler, 1994
), (b) a 9-amino acid
spacer, and (c) amino acids 19-745 of plakoglobin, which
includes the domain that is necessary for association with
-catenin (Sacco et al., 1995
). This chimeric cDNA was transfected into A431D cells. Clones expressing the chimeric
protein (A431DchiE/pg) were verified by immunoblotting
with anti-E-cadherin and antiplakoglobin antibodies, both
of which reacted with a protein at 150 kD, the expected
molecular mass (not shown). Immunoprecipitations of the
chimeric protein with anti-E-cadherin antibodies showed
that it was capable of associating with
-catenin, as expected (data not shown). Localization of desmoplakin in
the A431DchiE/pg cells revealed a punctate pattern concentrated along the cell-cell borders, highly indicative of desmosome organization and indistinguishable from the
parent A431 cells (Fig. 16).
Fig. 16.
Transfection of an E-cadherin-plakoglobin chimeric protein restores the ability of A431D cells to organize desmoplakin at cell-cell borders. (A) A431 cells (A) and A431D cells transfected with the chimera (A431DchiE/pg; B) were grown on glass coverslips and processed for immunofluorescence with antibodies against desmoplakin. (B) The chimeric molecule consisted of the entire extracellular and transmembrane (TM) domains of E-cadherin as well as the first 61 amino acids of its cytoplasmic domain, excluding the region shown to associate with -catenin, a nine-amino acid spacer, and amino acids 19-745 of plakoglobin, which includes the domain that is
necessary for association with
-catenin. Bar, 15 µm.
[View Larger Versions of these Images (68 + 12K GIF file)]
Discussion
; Lewis et al., 1994
; Amagai et al.,
1995
; Jensen et al., 1996). Similar observations have been
made with other epithelial cells, but the mechanism involved in cadherin-regulated formation of desmosomes
has been difficult to address. Our approach has been to
generate a cell line that expresses the components of the
desmosome but does not express a classical cadherin. We
created this cell line by treating A431 cells with dexamethasone for an extended period of time. Upon treatment with 10
7 M dexamethasone for about 2 wk, A431
cultures formed foci with a distinct morphology; examination of these foci revealed a lack of both E- and P-cadherin. Upon further analysis, it became clear that the affected cells, named A431D cells, were still synthesizing the
catenins, although they were not stable in the cell, probably because of lack of interaction with a cadherin. Interestingly, the A431D cells expressed all of the desmosomal
components examined at close to normal levels, but they
did not form desmosomes.
- and
-catenins readily form complexes with the transfected cadherin. Recently, Chitaev et al. (1996)
showed that
the binding affinity of plakoglobin for the desmosomal
cadherins is five times stronger than for the classical cadherins, indicating that the preferential association we observe could be explained on the basis of stronger interactions at the protein level.
), since
-catenin can be used in
its stead. However, the present results indicate that adherens junctions containing only
-catenin cannot direct subsequent desmosome organization; rather, plakoglobin interaction with a classical cadherin, probably in the context
of an adherens junction, is necessary to organize the desmosome components.
recently showed that plakoglobin
knock-out mice could assemble desmosomes in the squamous epithelial cells of the skin but not in the muscle cells
of the heart. These authors postulated that other armadillo
family members, in particular plakophilin 1, which is expressed in the skin but not in the heart, may be able to
substitute for plakoglobin. Consistent with this idea, our
line of A431 cells does not express plakophilin 1.
have presented data
suggesting that desmoplakin plays a role in segregating adherens junction and desmosomal proteins. In their studies,
cells expressing a mutant, dominant-negative desmoplakin
were not able to restrict adherens junction components from regions where desmosomal cadherins were localized.
These authors suggested that an interaction between plakoglobin and desmoplakin (although no direct interaction
has been demonstrated) may play a role in organizing the
desmosomal and adherens junctional components into
their separate structures.
-catenin are members of the armadillo family of proteins. Recent studies (for review see Peifer, 1995
) have suggested that armadillo family members
can act as signaling molecules in addition to their structural role in the adherens junction. Our results suggest that
one signaling function for plakoglobin is to direct desmosome organization. It is interesting that plakoglobin (which
is a component of both adherens junctions and desmosomes) can perform this function, but
-catenin (which is
restricted to adherens junctions) cannot. One theoretical
explanation for this divergence of function between plakoglobin and
-catenin is that formation of a desmosome involves exchange of plakoglobin molecules between the
classical cadherin and the desmosomal cadherin, an interaction not possible for
-catenin. However, our studies using A431D cells transfected with a cadherin-plakoglobin
chimera make this explanation highly unlikely since these
cells could organize desmosomes even though E-cadherin
was associated permanently with plakoglobin. With ongoing studies in our laboratory using chimeric molecules containing
-catenin and plakoglobin domains, we are attempting to understand the differences between these two
proteins.
). Karnovsky and Klymkowsky (1995)
showed
that overexpression of plakoglobin in Xenopus embryos
mimicked the effect of overexpression of wnt. Expression
of exogenous wnt 1 by some tissue culture cells results in
increased cell-cell adhesion (Bradley et al., 1993
; Hinck
et al., 1994
). It is possible that overexpression of plakoglobin in the A431D cells mimics a wnt signal, which results in
increased cell-cell adhesion. We have cocultured A431DE
cells with wnt 1-expressing Rat 2 cells (kindly provided by
Dr. Anthony Brown; Jue et al., 1992
) and have not observed any effect on the ability of the cells to organize desmosomal components. This does not, however, rule out a
role for the wnt family in desmosomal organization because our cells may not express the appropriate receptor
for wnt 1. The wnt family is large (Nusse and Varmus,
1992
), and the recently identified family of wnt receptors
(frizzled; Bhanot et al., 1996
) is also very large (Wang et
al., 1996
). Further studies along these lines require identification of the specific wnt receptors expressed by A431D
cells as well as the functional pairing of specific wnt family
members with the appropriate frizzled receptors.
Jani Lewis' current address is Department of Anatomy and Cell Biology, University of Michigan Medical School, Ann Arbor, MI.
Received for publication 23 May 1996 and in revised form 9 December 1996.
Address all correspondence to Margaret J. Wheelock, Department of Biology, University of Toledo, Toledo, OH 43606. Tel.: (419) 530-4918. Fax: (419) 530-7737. E-mail: mwheelo{at}uoft02.utoledo.eduThe authors wish to thank Drs. Werner W. Franke, Nicolas Fasel, Setsuo Hirohashi, Juan Ortín, and Anthony Brown for reagents. The authors thank Tammy Sadler and Laura Sauppé (Department of Biology, University of Toledo) for excellent technical help and Dr. Robert Lavker (Department of Dermatology, University of Pennsylvania) for assistance with electron microscopy.
This work was supported by National Institutes of Health (NIH) GM51188 to M.J. Wheelock and K.R. Johnson, by NIH AR42682 to P.J. Jensen and M.J. Wheelock, by grants from the Ohio Chapters of The American Cancer Society and The American Heart Association, and by the Ohio Board of Regents.