Institut National de la Santé et de la Recherche Médicale U442, Signalisation Cellulaire et Calcium, Université Paris-Sud, Centre Universitaire, 91405 Orsay Cedex, France
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ABSTRACT |
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WIF-B9 is a suitable
model for in vitro studies of hepatocyte polarity. To better understand
polarity establishment, we have localized key proteins of the adhesion
system, cytoskeleton, and tight junctions soon after plating, when most
cells are isolated or in doublets. In isolated attached cells, only
cytoskeletal proteins (tubulin, cytokeratins) displayed a precise
localization. As soon as two cells formed a doublet, E-cadherin, -,
-, and
-catenins, and p120 protein were present at the doublet
contiguous membrane. Actin, ezrin, and zonula occludens-1 (ZO-1)
colocalized at this membrane, but not in all doublets: ezrin was
present only at contiguous membrane expressing ZO-1, and ZO-1 was
present only at membrane expressing actin. In contrast, occludin was
spread throughout the doublet cytoplasm. With time in culture, these proteins localized transiently, as in cells expressing simple epithelial polarity, and finally, as in hepatocytes. We conclude that
during WIF-B9 early polarization, key proteins are settled according to
a hierarchy, as has been shown for Madin-Darby canine kidney cells.
Cytoplasmic complexes of E-cadherin-catenin were detected during the
whole polarization process; they were more abundant in fully polarized cells.
hepatoma hybrid WIF-B9; polarity establishment; cell-cell adhesion; cytoskeleton; tight junctions; zonula occludens-1
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INTRODUCTION |
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CELL POLARITY is at
the end of a multistage process that establishes and maintains
functionally specialized domains in the plasma membrane (PM) and the
cytoplasm (17). The pathway for polarity development in
epithelial cells starts with cell adhesion to the matrix and to the
contiguous cells. Cell-cell adhesion is mediated by homotypic
Ca2+-dependent binding between E-cadherin molecules
(44, 58, 65); this type of interaction is at the top of a
molecular cascade of protein interactions leading to the development of
epithelial cell structure and function (45). Intracellular
reorganization involves interactions of E-cadherin with the cortical
cytoskeleton components (32, 49), mediated by catenins
(22, 51). Ozawa and Kemler (52) have shown
that -catenin first associates with E-cadherin, while
- and
-catenin join the complex afterward.
-Catenin, by direct
interaction with F-actin (57), links the E-cadherin-catenin complex to the actin cytoskeleton and thereby increases cell adhesion (3). Previous studies have
indicated that the cadherin-mediated adhesion is a prerequisite for the formation of all intercellular junctions in Madin-Darby canine kidney
cells (MDCK): tight junction (TJ), zonula adherens, and desmosomes
(25). Recent studies demonstrate that
-catenin, by direct interaction with vinculin, plays a role in the assembly of
the apical junctional complex in epithelia (67). After the formation of intercellular junctions, structurally and functionally distinct PM domains are settled, leading to the development of cellular polarity.
Moreover, dynamic complexes of E-cadherin and catenins have been
described (27, 60); the steps of assembly and the exchange of proteins in these complexes are potential targets for regulation. Indeed, recent studies have indicated that catenins are at the convergence of several transduction pathways (1, 6);
accumulating evidence suggests that junctional integrity is regulated
by tyrosine phosphorylation, with catenins being target molecules
(7), as well as the p120 protein (p120), a substrate of
the Src tyrosine kinase and classified as a new catenin
(56). For example, -catenin mutations and changes in
level are implicated in colonic tumorigenesis (35, 47) via
regulation by the adenomatous polyposis coli (APC) protein, the product
of a tumor suppressor gene (29, 64). These changes in the
pool size of
-catenin may affect the APC-microtubule interactions
(62).
-Catenin is also a key effector in the pathway of
Wnt/Wingless (26), one of the major families of
developmentally signaling molecules in mouse and Drosophila
(13). By moving to the nucleus,
-catenin can regulate
gene expression by direct interaction with the transcription factor
LEF-1 (7). Thus catenins are key molecules connecting
cell-cell adhesion and signaling pathways with the cytoskeleton;
consequently, the interplay between cytoskeletal components and
signaling pathways may regulate morphogenesis (6).
Established cell lines such as renal MDCK (38) or intestinal HT29 and Caco-2 (53) have been widely used to study cell polarity. However, no polarized line derived from hepatocytes was available until we isolated and characterized WIF 12-1 cells (14) and their improved derivatives, WIF-B and WIF-B9 (16, 59). These rat hepatoma-human fibroblast hybrid cells are differentiated and polarized (30); in particular, they form bile canalicular-like structures (BC) that are equivalent to the apical pole of a hepatocyte. This polarity is not only structural but also functional, because efficient vectorial transport of a bile acid analog was demonstrated (12).
We have previously shown that the establishment of WIF-B9 polarity is a
biphasic phenomenon going from a simple epithelial polarized phenotype
to a hepatic polarized one, with the expression of these two phenotypes
being mutually exclusive (16). Here we have studied how
the cell surface domains, previously evidenced in WIF-B9 cells, are
generated. Because the generation and regulation of these domains in
polarized epithelial cells were shown to be mediated by the adhesion
system components, the cytoskeleton, and the TJ proteins, we have
analyzed WIF-B9 early polarity establishment according to the
expression of these key components. Three questions were addressed. Are
the proteins considered to be key proteins in polarity establishment
for MDCK cells also present in WIF-B9 cells? What is their
localization, and do they exhibit a hierarchy in their settlement at
the very beginning of polarity establishment? Are soluble cytoplasmic
complexes of E-cadherin and catenins present, and does their amount
vary when cells polarize? To answer these questions, we have
1) studied the expression and localization of components of
the adhesion system (-,
-, and
-catenins and p120) in relation
to E-cadherin; 2) performed experiments to detect complexes
of E-cadherin with each of the four catenins; and 3) analyzed in parallel the localization of cytoskeletal components (actin, actin-associated ezrin,
-tubulin, and cytokeratins) and the
TJ-associated complex (occludin and ZO-1). The study was done on WIF-B9 cells at 24 h of culture by using a plating cell density such that most cells were isolated and in doublets. The results obtained at that time were compared with those found after several days
in culture, when WIF-B9 cells transiently expressed a simple polarized
phenotype, and later, when cells expressed the typical hepatic
polarity. We have shown here that, at the very beginning of polarity
establishment, there is a hierarchy in the settlement of the key
proteins examined. Those of the adhesion system are localized very
early, whereas those of the TJ, in particular, occludin, are the last
to settle. Cytoplasmic complexes of E-cadherin with each of the
catenins and with p120 were detected all over the polarization process;
they were more abundant when cells expressed a fully polarized phenotype.
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MATERIALS AND METHODS |
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Antibodies and Materials
Ascitic mouse monoclonal antibodies raised against E-cadherin,Cell Culture
Cells were grown as described previously (14) on plastic petri dishes (Falcon Plastics, Cockeysville, MD) or on glass coverslips for immunofluorescence. Cells were regarded as confluent at 1.1-1.5 × 105 cells/cm2. Cells of polarized subconfluent cultures were detached with trypsin-EDTA and plated at 2.5 × 103 or 1.5 × 104 cells/cm2. At different times, from day 1 to days 8-10, the total cell number and the number of cells engaged in BC formation were counted on phase-contrast micrographs (Leitz inverted microscope with a ×20 lens).Cell Examination and Counting at Day 1
After trypsin-EDTA treatment, well-isolated WIF-B9 cells were plated at densities between 2.5 × 103 (low density) and 1.5 × 104 cells/cm2 (high density). To determine the best conditions from which to obtain isolated cells and cells in doublets, we used three different methods. The first method comprised counting on CELLocate coverslips (5 experiments) that permit easy relocalization of individual cells with time. Five hours after cells were plated, as soon as they were fixed on the support, four areas per CELLocate coverslip with adherent cells were mapped on two or three different coverslips, and cells were counted on phase-contrast micrographs; the same areas were analyzed 24 h later. Each area contained 7-30 cells. The second method entailed counting in culture flasks (1 experiment) of 27 areas of 5 × 5 mm, automatically scanned in Hoffman ×20 on an inverted Nikon Diaphot-300 microscope with a ×20 lens by using a computerized stage controller system (Alliance-Vision, Mirmande, France) based on LabView 4.0VI (National Instruments, Austin, TX), operating a Märzhauser scanning stage, with a resolution digital camera. The positions of adherent cells (5-14 cells/area) were mapped 4 h after they were plated and then were stored in memory to allow subsequent analysis at 24 h of culture. Images were processed with Photoshop (Adobe Systems). Finally, the third method consisted of videomicroscopy experiments in which observation and counting occurred in culture flasks maintained at 37°C in a heated Plexiglas chamber and was recorded during the 24 h following plating on a phase-contrast micrograph with a Zeiss inverted microscope equipped with a video camera connected to a television monitor and a time-lapse recorder, with a ×10 lens.At the low plating density, the distribution of the 1,000 counted cells was as follows: 44% were isolated, 41% were in doublets, and 15% were in small islets (3-6 cells). At the high plating density, the distribution of the 600 counted cells was as follows: 23% were isolated, 24% were in doublets, and 53% were in small islets (3-10 cells). As determined in two videomicroscopy experiments, cell doublets were the result of either mitosis (30% of isolated cells underwent mitosis in 24 h) or a union between two isolated cells. These cell unions occurred more frequently at the higher plating density, as can be inferred from the higher percentage of cells in small islets.
Immunofluorescence
Cells were fixed with 3% paraformaldehyde-phosphate-buffered saline (PBS) and permeabilized in cold methanol or in acetone (16). Cells were then incubated in primary antibody(ies) (1:100-500 in PBS, undiluted culture supernatant for monoclonal antibodies against ZO-1) or in TRITC-conjugated phalloidin (100 ng/coverslip) for 1 h at room temperature. After PBS rinses, cells were incubated for 30 min at room temperature with fluorescent secondary antibodies (1:100-500). In double-labeling experiments, the cells were incubated with both primary and then secondary antibodies at the same time. Cell nuclei were then stained with Hoechst 33258 and counted in parallel to cell counting in phase contrast. After being mounted in buffered glycerin or the SlowFade Antifade kit, cells were viewed on a Zeiss Axioskop fluorescence microscope. Confocal images were collected on a Bio-Rad MRC 600 confocal microscope with a ×63 objective in z series at 0.5- to 1-µm increments. Optical sections were collected by Kalman averaging of eight images. The images were processed with the use of SOM and CoMOS software (Service d'Imagerie, Orsay, France).Cell Extraction and Immunoprecipitation
In three different experiments, WIF-B9 cells were isolated by trypsinization and plated at 1.3 × 104 cells/cm2 on 10-cm petri dishes and cultured. Cell extraction was done at days 1, 4, and 8 after cells were plated by using six to eight, three, and one petri dish, respectively. After three PBS washes, cells were extracted by scraping on ice with 500 µl per petri dish of immunoprecipitation buffer [1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.5% Nonidet P-40 in 20 mM Tris · HCl (pH 7.4) with a cocktail of protease inhibitors]. The crude extracts were centrifuged, and the supernatants (soluble fractions) and pellets (insoluble fractions) were separated. Protein amount was estimated in the soluble fractions by DC protein assay.For immunoprecipitation, 300- to 400-µg proteins of soluble
fractions were rotated for 4 h at 4°C with 5 µg of
anti-E-cadherin in 1 ml of immunoprecipitation buffer, followed by
1 h of incubation with 40 µl of a 50% protein A-Sepharose
solution; after four washes, immunoprecipitates (IP) were eluted from
protein A beads by being boiled for 7 min in 2× SDS-electrophoresis
buffer with 10% -mercaptoethanol.
Western Blotting
Cell lysates (soluble and insoluble parts), after being boiled for 8 min in 5% ![]() |
RESULTS |
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Key Proteins Involved in Cell Polarity Exhibit a Hierarchy in Their Localization at Early Stages of Polarity Establishment
We wanted to know which components were present as soon as cells were fixed on the support and as soon as two cells associated. We have determined the conditions in which most cells were either single or in doublets at the very beginning of the culture. Comparing the low and high plating densities, using different methods (see MATERIALS AND METHODS for details), we observed that, at day 1, 85% of cells at the low density vs. 50% of those at the high density were either isolated or in doublets.According to these observations, we decided to analyze the expression
and localization of E-cadherin, catenins, and cytoskeletal and
TJ-associated proteins at day 1 after cells were plated, at the low density, when 44% cells were isolated, 41% were in doublets, and 15% were in small cell islets. The results are summarized in Table
1.
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Isolated cells.
In isolated cells, -tubulin (Fig.
1A) and cytokeratins (Fig.
1B) formed cytoplasmic networks. p58 Golgi was located near the nucleus (Fig. 1C). The other proteins were spread in the
cytoplasm as shown for p120 (Fig. 1D), except for ezrin,
which also was located in a cap at the cell apex (Fig. 1E);
when present, these caps might be surrounded by apical ZO-1 and
-catenin.
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Doublets.
In doublets, -tubulin and cytokeratin formed cytoplasmic networks as
in isolated cells, and p58 Golgi was located near the nucleus.
E-cadherin,
-,
-, and
-catenins and p120 were always localized
at the contiguous membranes between the two cells of the doublet (Fig.
2, A-E). Actin, ZO-1, and
ezrin were present in the cytoplasm but also were located at the
contiguous membranes in a fraction of the doublets: 90% of doublet
contiguous membranes for actin (Fig. 2F), 9% for ezrin
(Fig. 2G), and 63% for ZO-1 (Fig. 2H).
Ezrin was also sometimes present in a cap at the cell apex (Fig.
2I'), surrounded by apical ZO-1 (Fig. 2I), and in
focal contacts at the cell periphery (Fig. 2G). Occludin was
spread in the cytoplasm (Fig. 2J). Using double-labeling
experiments, we have established that ezrin was present only at
contiguous membranes expressing ZO-1 and that ZO-1 was present at
contiguous membranes expressing actin, thereby showing an order in
expression (see Fig. 2, middle).
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Small cell islets. In small cell islets, the adhesion system proteins and actin were always located at the contiguous membranes (not shown). ZO-1 was expressed at apical networks (Fig. 2L), often as dotted lines (Fig. 2K). Occludin was still mostly spread in the cytoplasm (Fig. 2L') and rarely colocalized with ZO-1 at the apical networks.
In summary, 1) as soon as WIF-B9 cells were fixed on the support, some cytoskeletal organization was observed, with the adhesion and TJ-associated proteins still spread in the cytoplasm; and 2) as soon as two cells associated, the cell-cell adhesion proteins were localized at the contiguous membranes between them. Actin, ZO-1, and ezrin located later at the contiguous membranes according to a hierarchy. Occludin, never located at these contiguous membranes, was the last to settle. The diagram in Fig. 2 summarizes these results. 3) In doublets forming BC, proteins of the cytoskeleton, of the adhesion system, and associated with the TJ are localized according to a hepatic phenotype. 4) In small cell islets, the localization of these proteins indicates that the simple epithelial phenotype had not yet been achieved, because ZO-1 apical networks were sometimes absent or discontinuous in some cells of the islets, even if these islets contained 10-20 cells, and because occludin was still cytoplasmic.With Time in Culture, Key Proteins in Cell Polarity Localize Transiently in a Simple Epithelial Cell-Like Pattern and Finally in a Hepatocyte-Like Pattern
Previously, we have defined apical, lateral, and basal poles in WIF-B9 cells, according to the localization of membrane domain protein markers, by immunofluorescence studies and by analysis of freeze-fracture replicas of TJ. We also have observed that WIF-B9 cells express a simple epithelial phenotype before they express the final hepatic phenotype (16, 30). This study was enlarged to determine the localization of the adhesion system proteins, in particular, that of catenins, and of an intrinsic protein of the TJ, occludin.WIF-B9 cells with simple epithelial phenotype.
A few days after they were plated, WIF-B9 cells formed islets wherein
70-80% of the cells displayed a simple epithelial phenotype with
the apical pole at the cell apex, that is, at the zone opposite to the
substratum attachment and the lateral pole at the contiguous membranes
between cells. The localization of cell-cell adhesion proteins and of
occludin in these cells, determined with the use of specific
antibodies, is summarized in Table 2 and
shown in Fig. 4. All the catenins, -,
-, and
-catenin (Fig. 4A) and p120 (Fig.
4B), were always expressed at the contiguous membranes between cells, that is, at the lateral pole; E-cadherin was previously shown to be located there (16), while sometimes also being
present at the apicolateral boundary. The TJ protein occludin (Fig.
4C'') was concentrated as honeycomb networks at the cell
apex, colocalizing with ZO-1 (Fig. 4C) at the apicolateral
frontier; occludin was also present in the cytoplasm. The
actin-associated protein ezrin was associated to the apical pole and to
the apicolateral boundary (Fig. 4D). These localizations are
very similar to those described for MDCK cells, typical of the simple
epithelial phenotype (42).
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WIF-B9 cells with hepatic phenotype.
From day 8 after they were plated, 60-99% (according
to plating density) of WIF-B9 cells expressed a hepatic phenotype with the formation of BC structures between two and sometimes three cells,
with the apical pole at the BC membrane and the lateral pole at the
contiguous membranes between cells. The localization of the adhesion
system catenins and of the TJ protein occludin is summarized in
Table 2 and shown in Fig. 5. The - and
-catenins (Fig. 5A) and
-catenin and p120 were always
at the lateral pole and excluded from the BC membrane, like E-cadherin;
- and
-catenin were also present at the apicolateral boundary,
although to a lesser extent than E-cadherin (70% of cells). Occludin
(Fig. 5D) was concentrated at the apicolateral frontier,
making a belt in the BC, where it colocalized (Fig. 5E')
with ZO-1 (Fig. 5E); occludin was also present in the
cytoplasm.
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Analysis of the Amount and Distribution of E-Cadherin and Catenins During WIF-B9 Polarity Establishment
We were interested to know the cytoplasmic distribution of the adhesion system proteins according to the polarized phenotype that is expressed by WIF-B9 cells. Hence, we studied their relative distribution at times corresponding to the best expression of the different phenotypes: 1) at day 1 after plating, when most cells were isolated or in doublets; 2) at day 4, when 75% of the cells expressed a simple epithelial phenotype; and 3) at days 8-10, when more than 75% of the cells expressed a hepatic phenotype.After cell extraction in 1% Triton X-100 buffer, the soluble and
insoluble fractions were studied by Western blotting with antibodies
against E-cadherin, the various catenins, and actin (Fig.
6A). To compare these
fractions, we analyzed the intensities of the bands (5 times more
protein was loaded for the insoluble fractions). On the basis of the
results of several Western blotting experiments, we found that the
total amount of E-cadherin (Triton X-100 soluble + insoluble) was
very similar at days 1, 4, and 8 of
culture; it was the same for the amounts of the three catenins, p120,
and actin. The major part of these proteins was present in the Triton
X-100-soluble fraction whatever the time in culture: 94 ± 3% for
E-cadherin, 91 ± 5% for -catenin, 89 ± 3% for
-catenin, 78 ± 3% for
-catenin, 90 ± 3% for p120,
and 77 ± 6.5% for actin. Moreover, no significant difference in
the insoluble vs. soluble ratio was found whatever the time in culture;
for example, the insoluble
-catenin accounted for 11% of total
-catenin at day 1, 13% at day 4, and 11% at
day 8.
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In summary, the total amount and the distribution in soluble-insoluble fractions of E-cadherin, all the catenins, and p120 showed little change whatever the phenotype expressed by WIF-B9 cells, with most of the proteins being Triton X-100 soluble.
Presence of Soluble E-Cadherin-Catenin Complexes During WIF-B9 Polarity Establishment
For MDCK cells, a model has been proposed in which dynamic E-cadherin-catenin complexes play an important role during initiation and maintenance of structural and functional organization (48). Spatial distribution of these complexes in the cell is correlated with the differentiation state: E-cadherin-We have looked for the presence of such soluble complexes of E-cadherin
with the various catenins in WIF-B9 cells. The Triton X-100-soluble
fractions of three independent cell extracts at days 1,
4, and 8-10 of culture were
immunoprecipitated with anti-E-cadherin in six separate experiments.
These E-cadherin-IP were probed in several Western blotting experiments
with antibodies against the various catenins and p120. As controls, two
lanes were loaded with 30 and 15 µg of proteins of a soluble fraction
at day 8 before immunoprecipitation. We found that the
E-cadherin-IP reacted with antibodies directed against -,
-, and
-catenins and p120 (Fig. 6B). This indicates the presence
of E-cadherin-
-catenin, E-cadherin-
-catenin, E-cadherin-
-catenin, and E-cadherin-p120 soluble complexes in WIF-B9
cells at each time of culture. E-cadherin-
-catenin complexes were
present in a smaller amount than the other E-cadherin-catenins complexes as shown by the lower intensity of reaction compared with
that of the control before immunoprecipitation. As estimated by the
intensities of the bands, a similar reactivity with anti-E-cadherin was
observed in the IP at days 1, 4, and
8, which is consistent with the similar amount of E-cadherin
found in the soluble fractions before immunoprecipitation. The amounts
of each soluble complex were rather similar at days 4 and
8. In comparison, the amounts at day 1 were
lower, corresponding to 50% of the amount at day 8 for
E-cadherin-
-catenin, 55% for E-cadherin-
-catenin, 65% for
E-cadherin-
-catenin, and 45% for E-cadherin-p120. Thus
E-cadherin-catenin complexes are present in WIF-B9 cells and are more
abundant when cells express a fully polarized phenotype.
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DISCUSSION |
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Development of polarity in epithelial cells requires specialized
localization of proteins to distinct PM domains. Increasing evidence
has been gathered concerning the important role of adhesion system and
cytoskeletal components in the various processes leading to this
organization (41). Using immunofluorescence and
biochemical analysis, we have studied the expression and localization
of proteins of the adhesion system (E-cadherin, -,
-, and
-catenins, and p120), proteins of the cytoskeleton (actin-associated
ezrin and cytokeratins), and a protein specific to the TJ (occludin)
during polarity establishment in WIF-B9 cells.
Polarization: Early Events
We wanted to know the spatial distribution of proteins as soon as WIF-B9 cells were fixed on the support and as soon as two cells associated. Therefore, we studied WIF-B9 cells at 24 h of culture after they were plated, when a majority of cells were isolated and in doublets.Isolated WIF-B9 cells.
In isolated WIF-B9 cells, -tubulin and cytokeratins were organized
in cytoplasmic networks and p58 Golgi was located near the nucleus. The
other studied proteins were spread in the cytoplasm, showing no
organization. As soon as WIF-B9 were fixed on a support, they displayed
some architectural order, due in part to the structural properties of
-tubulin and cytokeratin. Bacallao et al. (5) made
similar observations in isolated MDCK cells 12 h after they were
plated: the Golgi complex and microtubules assumed an organization even
when E-cadherin and ZO-1 were still spread in the cytoplasm. Ezrin was
sometimes expressed in a cap at the WIF-B9 cell apex, consistent with
its involvement in the organization of cell membrane structures
(66) and cell shape (37).
WIF-B9 doublets. WIF-B9 doublets presented a more organized state. The striking observation was that E-cadherin, all the catenins, and p120 were always highly concentrated at the contiguous membranes between the two cells; in addition, some proteins were still present in the cytoplasm. Therefore, the chief actors of epithelial cell-cell adhesion (51, 65) colocalize at a precise membrane domain as soon as two WIF-B9 cells share contiguous membranes. Actin, ezrin, and ZO-1 were also located at these contiguous membranes, but not in all WIF-B9 doublets. This could indicate that they arrive at the membrane after E-cadherin-catenin components. The presence of actin at the submembrane plaque of cell-cell adhesion is in accordance with its role in functional adhesion by complexing with cadherin/catenins (24). Recent studies have shown the coordinate roles of E-cadherin and actin cables in initial remodeling of cell-cell contacts (2). Ezrin is also involved in the regulation of cell morphology, acting as a linker between the plasma membrane and the cytoskeleton (66). Ezrin was also shown to colocalize with actin at the apical surface of many epithelial cells and at membrane ruffles in cultured cells (10). In WIF-B9 cell doublets, ezrin colocalized with actin at the contiguous membranes. It was also expressed in a cap at the cell apex and in focal contacts at the cell periphery, where no actin was present. ZO-1 is a peripheral membrane protein enriched at the TJ of epithelial and endothelial cells (63). Linkage to a variety of proteins was shown, including colocalization with actin filaments in many cell types (28) and complex formation with catenins during TJ early state assembly in MDCK cells (55). These ZO-1-catenin complexes are localized at the adherens junctional complex before moving to the newly developed TJ (69). In WIF-B9 doublets, ZO-1 colocalized with all the above described partners responsible for the first stages in polarity establishment. Occludin, the first described protein constitutive of the TJ (20), has direct association with ZO-1, with the association with cytoskeleton through ZO-1 being required for occludin to be localized to TJ (19, 21). In WIF-B9 doublets, occludin was not associated to a particular domain of the membrane but was spread in the cytoplasm, indicating that complete TJ formation had not occurred yet.
In summary, we have shown that cell-cell or cell-substratum adhesion was sufficient to generate an initial level of asymmetry in WIF-B9 cells, which is the first critical event in the generation of epithelial polarity (68). We have evidence of a hierarchy in the localization of some of the key components during the early steps of polarity establishment: first, the adhesion proteins E-cadherin, all the catenins, and p120 localized at the site of cell-cell contact, followed by actin, then by ZO-1, and, finally, by ezrin, with occludin remaining cytoplasmic. However, these contiguous membranes could not be considered as lateral poles because the TJ were not completed; even in small cell islets at day 1, occludin was, most often, not precisely localized. Among WIF-B9 doublets formed 24 h after cells were plated, a minority had BC. In these doublets with BC, all studied proteins had a precise localization and the PM poles were formed, with occludin and ZO-1 colocalizing in belts at the TJ level. Moreover, cytoplasmic networks ofPolarization: Later Events
When the majority of WIF-B9 cells expressed a simple epithelial phenotype, we showed that all the catenins and the p120 were always localized at the lateral pole, as is usually described in cells with simple epithelial polarity (22). These proteins of the adhesion system are not always restricted to the so-called adherens junction sites (25); in WIF-B9 cells, we observed that E-cadherin could localize at the apicolateral boundary, that is, at the TJ level. Ezrin was present at the apical pole and at the apicolateral boundary, colocalizing with actin as described in a variety of epithelial cells (10). Finally, we found occludin at the TJ level, colocalizing with ZO-1 as described for human intestinal and bovine renal epithelial cells (21).After 10 days in culture, the majority of WIF-B9 cells expressed a
hepatic phenotype, with formation of BC structures between cells. Our
present study indicates that WIF-B9 cells express -,
-, and
-catenins and p120 at the lateral pole (where E-cadherin is
localized, too) and, also, sometimes at the apicolateral boundary, namely, at the TJ level where occludin and ZO-1 always colocalized. These observations confirm the close positional relationship between zonula occludens and zonula adherens previously observed in electron microscopic studies (16), an observation already made in
hepatocytes (31). Cytokeratins showed reinforced
localization at these BC levels. Previous studies on WIF-B cells have
already evidenced a highly concentrated network of actin filaments and
-tubulin around the BC (30). In rat hepatocytes,
colocalizing microtubules, actin bundles, and cytokeratin filament
sheaths at the BC level play an essential role in the formation and
maintenance of these BC (33, 50); consequently, the
cytoskeleton is important for hepatic functions that are correlated
with the polarity state of hepatocytes (18, 46). Thus the
major cytoskeleton proteins present at the BC level of WIF-B9 cells
could contribute to their structural but also functional polarity as
demonstrated by the efficient vectorial transport of a bile acid analog
(12) and by the functional specialization of stable and
dynamic microtubules in protein traffic (54).
In summary, our results concerning cell-cell adhesion proteins, cytokeratin, and occludin reinforce previous studies in which other protein markers for cell polarization were used, which showed that WIF-B9 cells express a simple epithelial phenotype followed by a hepatic phenotype.
Cell-Cell Adhesion Protein Distribution and E-Cadherin-Catenin Complexes During WIF-B9 Polarity Establishment
In hepatocytes, Kim et al. (34) showed that the key components in cell-cell adhesion are also key elements in restoration of differentiated functions; rat hepatocytes induced to differentiate in Matrigel showed increased amounts of E-cadherin andHere we analyzed the distribution of the adhesion system proteins in
WIF-B9 cells according to the expressed phenotype. We found that the
total amount of E-cadherin was very similar whatever the time in
culture, which means that, whatever the expressed phenotype, this
protein is mainly present in the Triton X-100-soluble fraction. We
obtained the same distribution with the three catenins and p120. Our
results are in agreement with those of Kim et al. (34),
who showed that E-cadherin and -catenin are mostly present in the
Triton X-100-soluble fraction in rat hepatocytes. Similar distribution
was observed in other cell types: HT29 cells (60) and A6
cells (43). On the contrary, in MDCK cells 5 days after the induction of cell-cell contact, Shore and Nelson (61)
observed a conversion of E-cadherin from a soluble to an insoluble
0.5% Triton X-100 pool. This discrepancy is not due to different cell lysis conditions because we found the same relative distribution of
E-cadherin using 0.5% or 1% Triton X-100 extractions (data not
shown); however, cell culture conditions were not the same, with MDCK
cells being plated on hydrated collagen gels. In WIF-B9, another type
of cadherin could be responsible in part for adhesion; WIF-B9 cells
express a cadherin different from E-cadherin [as evidenced by
reactivity with antibodies against pan-cadherin (9)]. They could also express LI-cadherin, a cadherin specific to liver and
intestinal epithelia, the adhesive function of which is independent of
any interaction with cytoplasmic components (36). Finally, the difference in distribution between MDCK and WIF-B9 cells could be
due to the difference in cell lines.
Dynamic cadherin-catenin complexes play an important role in structural
and functional organization (48). Looking for their presence in WIF-B9 cells, we found soluble complexes of E-cadherin that
contained -,
-, and
-catenins or p120. Interestingly, these
complexes were more abundant when WIF-B9 expressed a fully polarized
phenotype. As described in MDCK cells, these complexes could be
potential targets on signaling pathways in WIF-B9 cells.
In conclusion, our study here shows that the spatiotemporal expression pattern of adhesion system proteins, cytoskeletal proteins, and junction-associated proteins correlates with morphogenetic events in WIF-B9 cells as described for other epithelial cell systems. Some of these proteins are known to take part in signal transduction pathways. It would be interesting to study the level of tyrosine phosphorylation of the cell-cell adhesion proteins and of ezrin in WIF-B9 cells. Ezrin is a substrate for the tyrosine kinase HGF (hepatic growth factor) receptor and is able to convey signals to the actin cytoskeleton machinery (15). Because the molecular mechanisms involved in hepatocyte differentiation are far from being elucidated, we believe that WIF-B9 cells will contribute to the understanding of these mechanisms during the establishment and maintenance of hepatic cell polarity and BC neoformation.
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ACKNOWLEDGEMENTS |
---|
We thank A. Forchioni for help with confocal analysis, V. Favaudon for experiments of cell counting with automatic scanning, C. Cruttwell for critical review of the manuscript, and H. Tourbez for invaluable help.
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FOOTNOTES |
---|
This work was supported in part by Association pour la Recherche sur le Cancer Grant 6551, the Institut Curie (PIC Signalisation Cellulaire Grant 914), the Centre National de la Recherche Scientifique, and the Institut National de la Santé et de la Recherche Médicale (contract PRISME 98-09).
Address for reprint requests and other correspondence: C. Decaens, INSERM U442, Signalisation Cellulaire et Calcium, Université Paris-Sud (Bat 443), Centre Universitaire, 91405 Orsay Cedex, France (E-mail: catherine.decaens{at}ibaic.u-psud.fr).
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. Section 1734 solely to indicate this fact.
Received 5 September 2000; accepted in final form 23 October 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aberle, H,
Schwartz H,
and
Kemler R.
Cadherin-catenin complex: protein interactions and their implications for cadherin function.
J Cell Biochem
61:
514-523,
1996[ISI][Medline].
2.
Adams, CL,
Chen YT,
Smith SJ,
and
Nelson WJ.
Mechanisms of epithelial cell-cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin-green fluorescent protein.
J Cell Biol
142:
1105-1119,
1998
3.
Adams, CL,
Nelson WJ,
and
Smith SJ.
Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion.
J Cell Biol
135:
1899-1911,
1996[Abstract].
4.
Arpin, M,
Algrain M,
and
Louvard D.
Membrane-actin microfilament connections: an increasing diversity of players related to band 4.1.
Curr Opin Cell Biol
6:
136-141,
1994[ISI][Medline].
5.
Bacallao, R,
Antony C,
Dotti C,
Karsenti E,
Stelzer EH,
and
Simons K.
The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium.
J Cell Biol
109:
2817-2832,
1989[Abstract].
6.
Barth, AI,
Nathke IS,
and
Nelson WJ.
Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways.
Curr Opin Cell Biol
9:
683-690,
1997[ISI][Medline].
7.
Behrens, J,
von Kries JP,
Kuhl M,
Bruhn L,
Wedlich D,
Grosschedl R,
and
Birchmeier W.
Functional interaction of -catenin with the transcription factor LEF-1.
Nature
382:
638-642,
1996[ISI][Medline].
8.
Bender, V,
Bravo P,
Decaens C,
and
Cassio D.
The structural and functional polarity of the hepatic human/rat hybrid WIF-B is a stable and dominant trait.
Hepatology
30:
1002-1010,
1999[ISI][Medline].
9.
Bender, V,
Buschlen S,
and
Cassio D.
Expression and localization of hepatocyte domain-specific plasma membrane proteins in hepatoma × fibroblast hybrids and in hepatoma dedifferentiated variants.
J Cell Sci
111:
3437-3450,
1998
10.
Berryman, M,
Franck Z,
and
Bretscher A.
Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells.
J Cell Sci
105:
1025-1043,
1993
11.
Boyer, JL.
Isolated rat hepatocyte couplets.
In: Hepatic Transport and Bile Secretion: Physiology and Pathophysiology, edited by Tavoloni N,
and Berk PD.. New York: Raven, 1993, p. 597-606.
12.
Bravo, P,
Bender V,
and
Cassio D.
Efficient in vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells.
Hepatology
27:
576-583,
1998[ISI][Medline].
13.
Cadigan, KM,
and
Nusse R.
Wnt signaling: a common theme in animal development.
Genes Dev
11:
3286-3305,
1997
14.
Cassio, D,
Hamon-Benais C,
Guerin M,
and
Lecoq O.
Hybrid cell lines constitute a potential reservoir of polarized cells: isolation and study of highly differentiated hepatoma-derived hybrid cells able to form functional bile canaliculi in vitro.
J Cell Biol
115:
1397-1408,
1991[Abstract].
15.
Crepaldi, T,
Gautreau A,
Comoglio PM,
Louvard D,
and
Arpin M.
Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells.
J Cell Biol
138:
423-434,
1997
16.
Decaens, C,
Rodriguez P,
Bouchaud C,
and
Cassio D.
Establishment of hepatic cell polarity in the rat hepatoma-human fibroblast hybrid WIF-B9. A biphasic phenomenon going from a simple epithelial polarized phenotype to an hepatic polarized one.
J Cell Sci
109:
1623-1635,
1996
17.
Drubin, DG,
and
Nelson WJ.
Origins of cell polarity.
Cell
84:
335-344,
1996[ISI][Medline].
18.
Dunn, JC,
Yarmush ML,
Koebe HG,
and
Tompkins RG.
Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration.
FASEB J
3:
174-177,
1989
19.
Fanning, AS,
Jameson BJ,
Jesaitis LA,
and
Anderson JM.
The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton.
J Biol Chem
273:
29745-29753,
1998
20.
Furuse, M,
Hirase T,
Itoh M,
Nagafuchi A,
Yonemura S,
and
Tsukita S.
Occludin: a novel integral membrane protein localizing at tight junctions.
J Cell Biol
123:
1777-1788,
1993[Abstract].
21.
Furuse, M,
Itoh M,
Hirase T,
Nagafuchi A,
Yonemura S,
and
Tsukita S.
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].
22.
Geiger, B,
and
Ayalon O.
Cadherins.
Annu Rev Cell Biol
8:
307-332,
1992[ISI].
23.
Graf, J,
and
Boyer JL.
The use of isolated rat hepatocyte couplets in hepatobiliary physiology.
J Hepatol
10:
387-394,
1990[ISI][Medline].
24.
Gumbiner, BM.
Cell adhesion: the molecular basis of tissue architecture and morphogenesis.
Cell
84:
345-357,
1996[ISI][Medline].
25.
Gumbiner, B,
Stevenson B,
and
Grimaldi A.
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].
26.
Hinck, L,
Nathke IS,
Papkoff J,
and
Nelson WJ.
-Catenin: a common target for the regulation of cell adhesion by Wnt-1 and Src signaling pathways.
Trends Biochem Sci
19:
538-542,
1994[ISI][Medline].
27.
Hinck, L,
Nathke IS,
Papkoff J,
and
Nelson WJ.
Dynamics of cadherin/catenin complex formation: novel protein interactions and pathways of complex assembly.
J Cell Biol
125:
1327-1340,
1994[Abstract].
28.
Howarth, AG,
and
Stevenson BR.
Molecular environment of ZO-1 in epithelial and non-epithelial cells.
Cell Motil Cytoskeleton
31:
323-332,
1995[ISI][Medline].
29.
Hulsken, J,
Birchmeier W,
and
Behrens J.
E-cadherin and APC compete for the interaction with -catenin and the cytoskeleton.
J Cell Biol
127:
2061-2069,
1994[Abstract].
30.
Ihrke, G,
Neufeld EB,
Meads T,
Shanks MR,
Cassio D,
Laurent M,
Schroer TA,
Pagano RE,
and
Hubbard AL.
WIF-B cells: an in vitro model for studies of hepatocyte polarity.
J Cell Biol
123:
1761-1775,
1993[Abstract].
31.
Itoh, M,
Nagafuchi A,
Yonemura S,
Kitani-Yasuda T,
and
Tsukita S.
The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy.
J Cell Biol
121:
491-502,
1993[Abstract].
32.
Itoh, M,
Yonemura S,
Nagafuchi A,
and
Tsukita S.
A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites.
J Cell Biol
115:
1449-1462,
1991[Abstract].
33.
Kawahara, H,
Cadrin M,
Perry G,
Autilio-Gambetti L,
Swierenga SH,
Metuzals J,
Marceau N,
and
French SW.
Role of cytokeratin intermediate filaments in transhepatic transport and canalicular secretion.
Hepatology
11:
435-448,
1990[ISI][Medline].
34.
Kim, TH,
Bowen WC,
Stolz DB,
Runge D,
Mars WM,
and
Michalopoulos GK.
Differential expression and distribution of focal adhesion and cell adhesion molecules in rat hepatocyte differentiation.
Exp Cell Res
244:
93-104,
1998[ISI][Medline].
35.
Korinek, V,
Barker N,
Morin PJ,
van Wichen D,
de Weger R,
Kinzler KW,
Vogelstein B,
and
Clevers H.
Constitutive transcriptional activation by a -catenin-Tcf complex in APC
/
colon carcinoma.
Science
275:
1784-1787,
1997
36.
Kreft, B,
Berndorff D,
Bottinger A,
Finnemann S,
Wedlich D,
Hortsch M,
Tauber R,
and
Gessner R.
LI-cadherin-mediated cell-cell adhesion does not require cytoplasmic interactions.
J Cell Biol
136:
1109-1121,
1997
37.
Lamb, RF,
Ozanne BW,
Roy C,
McGarry L,
Stipp C,
Mangeat P,
and
Jay DG.
Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts.
Curr Biol
7:
682-688,
1997[ISI][Medline].
38.
Leighton, J,
Estes LW,
Mansukhani S,
and
Brada Z.
A cell line derived from normal dog kidney (MDCK) exhibiting qualities of papillary adenocarcinoma and of renal tubular epithelium.
Cancer
26:
1022-1028,
1970[ISI][Medline].
39.
Marrs, JA,
and
Nelson WJ.
Cadherin cell adhesion molecules in differentiation and embryogenesis.
Int Rev Cytol
165:
159-205,
1996[ISI][Medline].
40.
Maurice, M,
Rajho Meerson N,
Durand-Schneider AM,
and
Delautier D.
Polarity of epithelial cells of the liver. Cellular and molecular mechanisms, and pathologic changes.
Gastroenterol Clin Biol
22:
530-540,
1998[ISI][Medline].
41.
Mays, RW,
Beck KA,
and
Nelson WJ.
Organization and function of the cytoskeleton in polarized epithelial cells: a component of the protein sorting machinery.
Curr Opin Cell Biol
6:
16-24,
1994[ISI][Medline].
42.
Mays, RW,
Nelson WJ,
and
Marrs JA.
Generation of epithelial cell polarity: roles for protein trafficking, membrane-cytoskeleton, and E-cadherin-mediated cell adhesion.
Cold Spring Harb Symp Quant Biol
60:
763-773,
1995[ISI][Medline].
43.
McCrea, PD,
and
Gumbiner BM.
Purification of a 92-kDa cytoplasmic protein tightly associated with the cell-cell adhesion molecule E-cadherin (uvomorulin). Characterization and extractability of the protein complex from the cell cytostructure.
J Biol Chem
266:
4514-4520,
1991
44.
McNeill, H,
Ozawa M,
Kemler R,
and
Nelson WJ.
Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity.
Cell
62:
309-316,
1990[ISI][Medline].
45.
McNeill, H,
Ryan TA,
Smith SJ,
and
Nelson WJ.
Spatial and temporal dissection of immediate and early events following cadherin-mediated epithelial cell adhesion.
J Cell Biol
120:
1217-1226,
1993[Abstract].
46.
Moghe, PV,
Berthiaume F,
Ezzell RM,
Toner M,
Tompkins RG,
and
Yarmush ML.
Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function.
Biomaterials
17:
373-385,
1996[ISI][Medline].
47.
Morin, PJ,
Sparks AB,
Korinek V,
Barker N,
Clevers H,
Vogelstein B,
and
Kinzler KW.
Activation of -catenin-Tcf signaling in colon cancer by mutations in
-catenin or APC.
Science
275:
1787-1790,
1997
48.
Nathke, IS,
Hinck L,
Swedlow JR,
Papkoff J,
and
Nelson WJ.
Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells.
J Cell Biol
125:
1341-1352,
1994[Abstract].
49.
Nelson, WJ,
Shore EM,
Wang AZ,
and
Hammerton RW.
Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin-Darby canine kidney epithelial cells.
J Cell Biol
110:
349-357,
1990[Abstract].
50.
Novikoff, PM,
Cammer M,
Tao L,
Oda H,
Stockert RJ,
Wolkoff AW,
and
Satir P.
Three-dimensional organization of rat hepatocyte cytoskeleton: relation to the asialoglycoprotein endocytosis pathway.
J Cell Sci
109:
21-32,
1996
51.
Ozawa, M,
Baribault H,
and
Kemler R.
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].
52.
Ozawa, M,
and
Kemler R.
Molecular organization of the uvomorulin-catenin complex.
J Cell Biol
116:
989-996,
1992[Abstract].
53.
Pinto, M,
Appay M,
Simon-Assman P,
Chevallier G,
Dracopoli N,
Fogh J,
and
Zweibaum A.
Enterocytic differentiation of cultured human colon cancer cells by replacement of glucose by galactose in the medium.
Biol Cell
44:
193-196,
1982[ISI].
54.
Pous, C,
Chabin K,
Drechou A,
Barbot L,
Phung-Koskas T,
Settegrana C,
Bourguet-Kondracki ML,
Maurice M,
Cassio D,
Guyot M,
and
Durand G.
Functional specialization of stable and dynamic microtubules in protein traffic in WIF-B cells.
J Cell Biol
142:
153-165,
1998
55.
Rajasekaran, AK,
Hojo M,
Huima T,
and
Rodriguez-Boulan E.
Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions.
J Cell Biol
132:
451-463,
1996[Abstract].
56.
Reynolds, AB,
Daniel J,
McCrea PD,
Wheelock MJ,
Wu J,
and
Zhang Z.
Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes.
Mol Cell Biol
14:
8333-8342,
1994[Abstract].
57.
Rimm, DL,
Koslov ER,
Kebriaei P,
Cianci CD,
and
Morrow JS.
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].
58.
Ringwald, M,
Schuh R,
Vesweber D,
Eistetter H,
and
Lottspeich F.
The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca++-dependent cell adhesion.
EMBO J
6:
3647-3653,
1987[Abstract].
59.
Shanks, MR,
Cassio D,
Lecoq O,
and
Hubbard AL.
An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo.
J Cell Sci
107:
813-825,
1994
60.
Shibamoto, S,
Hayakawa M,
Takeuchi K,
Hori T,
Miyazawa K,
Kitamura N,
Johnson KR,
Wheelock MJ,
Matsuyoshi N,
Takeichi M,
and
Ito F.
Association of p120, a tyrosine kinase substrate, with E- cadherin/catenin complexes.
J Cell Biol
128:
949-957,
1995[Abstract].
61.
Shore, EM,
and
Nelson WJ.
Biosynthesis of the cell adhesion molecule uvomorulin (E-cadherin) in Madin-Darby canine kidney epithelial cells.
J Biol Chem
266:
19672-19680,
1991
62.
Smith, AJ,
Stern HS,
Penner M,
Hay K,
Mitri A,
Bapat BV,
and
Gallinger S.
Somatic APC and K-ras codon 12 mutations in aberrant crypt foci from human colons.
Cancer Res
54:
5527-5530,
1994[Abstract].
63.
Stevenson, BR,
Siliciano JD,
Mooseker MS,
and
Goodenough DA.
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].
64.
Su, LK,
Vogelstein B,
and
Kinzler KW.
Association of the APC tumor suppressor protein with catenins.
Science
262:
1734-1737,
1993[ISI][Medline].
65.
Takeichi, M.
Cadherin cell adhesion receptors as a morphogenetic regulator.
Science
251:
1451-1455,
1991[ISI][Medline].
66.
Vaheri, A,
Carpen O,
Heiska L,
Helander TS,
Jaaskelainen J,
Majander-Nordenswan P,
Sainio M,
Timonen T,
and
Turunen O.
The ezrin protein family: membrane-cytoskeleton interactions and disease associations.
Curr Opin Cell Biol
9:
659-666,
1997[ISI][Medline].
67.
Watabe-Uchida, M,
Uchida N,
Imamura Y,
Nagafuchi A,
Fujimoto K,
Uemura T,
Vermeulen S,
van Roy F,
Adamson ED,
and
Takeichi M.
Alpha-catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells.
J Cell Biol
142:
847-857,
1998
68.
Yeaman, C,
Grindstaff KK,
and
Nelson WJ.
New perspectives on mechanisms involved in generating epithelial cell polarity.
Physiol Rev
79:
73-98,
1999
69.
Yonemura, S,
Itoh M,
Nagafuchi A,
and
Tsukita S.
Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells.
J Cell Sci
108:
127-142,
1995
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