Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan
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Abstract |
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The interaction of cadherin-catenin complex
with the actin-based cytoskeleton through -catenin is
indispensable for cadherin-based cell adhesion activity.
We reported previously that E-cadherin-
-catenin fusion molecules showed cell adhesion and cytoskeleton
binding activities when expressed in nonepithelial
L cells. Here, we constructed deletion mutants of
E-cadherin-
-catenin fusion molecules lacking various
domains of
-catenin and introduced them into L cells. Detailed analysis identified three distinct functional domains of
-catenin: a vinculin/
-actinin-binding domain, a ZO-1-binding domain, and an adhesion-modulation domain. Furthermore, cell dissociation assay
revealed that the fusion molecules containing the
ZO-1-binding domain in addition to the adhesion-modulation domain conferred the strong state of cell adhesion activity on transfectants, although those lacking
the ZO-1-binding domain conferred only the weak state. The disorganization of actin-based cytoskeleton
by cytochalasin D treatment shifted the cadherin-based
cell adhesion from the strong to the weak state. In the
epithelial cells, where
-catenin was not precisely colocalized with ZO-1, the ZO-1-binding domain did not
completely support the strong state of cell adhesion
activity. Our studies showed that the interaction of
-catenin with the actin-based cytoskeleton through
the ZO-1-binding domain is required for the strong
state of E-cadherin-based cell adhesion activity.
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Introduction |
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THE cadherins are a family of transmembrane proteins responsible for Ca2+-dependent cell-cell adhesion (Takeichi, 1991). Intracellularly, they interact
with a group of proteins collectively termed catenins (Ozawa et al., 1989
; Nagafuchi et al., 1993
). Association
with catenins is necessary for cadherins to express their
full function as cell adhesion molecules, and this complex
is now regarded as a functional unit for cell adhesion. A
unique property of this cadherin-catenin complex is its intimate interaction with the actin-based cytoskeleton. At
cell-cell contact sites, this complex is colocalized with actin filaments and resists nonionic detergent extraction
(Hirano et al., 1987
). This complex is a major constituent of intercellular adherens junctions (AJ),1 where actin filaments are densely associated with the plasma membrane
through its well-developed plaque structure (Tsukita et al.,
1993
). Although the cytoskeletal interaction of cadherin- catenin complex is thought to be essential for adhesion,
the detailed molecular mechanism of this interaction remains elusive.
-Catenin associates with the COOH-terminal end of
the cadherin cytoplasmic domain (catenin-binding site) via
-catenin. Sequence analysis showed that
-catenin has
similarity to vinculin, another constituent of AJ (Herrenknecht et al., 1991
; Nagafuchi et al., 1991
), which interacts
with various actin-based cytoskeletal components including actin itself (Jockusch et al., 1995
). This suggested that
-catenin may interact with the actin-based cytoskeleton.
-Actinin, ZO-1, vinculin, and actin itself were reported to
interact directly with
-catenin (Rimm et al., 1995
; Itoh et al.,
1997
; Nieset et al., 1997
; Watabe-Uchida et al., 1998
; Weiss
et al., 1998
). ZO-1 was identified originally as a tight
junction (TJ)-associated peripheral membrane protein
(Stevenson et al., 1986
; Anderson et al., 1988
), but it is also
concentrated at cadherin-based cell-cell contact sites together with vinculin in nonepithelial cells lacking TJ such
as fibroblasts and cardiac muscle cells (Itoh et al., 1993
).
The functions of -catenin have been analyzed mainly
using two cell lines, the human lung carcinoma cell line
PC9, and L cells, a mouse fibroblast cell line. PC9 cells express E-cadherin and
-catenin but not
-catenin. They do
not form tight cell aggregates after rotation culture and resultant aggregates are easily dissociated into single cells
(Shimoyama et al., 1992
). The exogenous expression of
-catenin in these cells restored not only the full cell adhesion activity of cadherins but also the cadherin-cytoskeleton interaction (Hirano et al., 1992
; Watabe et al., 1994
).
Mouse L fibroblasts do not express cadherin molecules
and lack the cadherin-based cell adhesion activity. When
E-cadherin was introduced into L cells, stable transfectants showed the full cell adhesion activity through the interaction of introduced E-cadherin with endogenous
-
and
-catenins, but the mutant E-cadherin lacking the
catenin-binding site did not confer such cell adhesion activity on L cells (Nagafuchi et al., 1987
; Nagafuchi and
Takeichi, 1988
; Ozawa et al., 1989
). When the full-length
or COOH-terminal half of
-catenin was covalently connected to this nonfunctional mutant E-cadherin, these fusion molecules showed the full cell adhesion activity in L
cells without interacting with endogenous catenins. Moreover, the introduced fusion molecules were associated
with the cytoskeleton (Nagafuchi et al., 1994
). These findings indicated that
-catenin, especially its COOH-terminal half, is crucial for the full cadherin-based cell adhesion
activity as well as cadherin-cytoskeleton interaction.
The L cell transfection system is advantageous for examination of the function of the cadherin-catenin complex
in cell adhesion and cytoskeleton interaction. Since L cells
show little cell-cell adhesion activity, these functions of introduced cadherin or its fusion molecules can be selectively analyzed. Using this system, we examined the domains of -catenin responsible for the cadherin-based cell
adhesion activity and the interaction with cytoskeletal
components such as ZO-1, vinculin, and
-actinin. We also
evaluated the functions of these
-catenin functional domains in
-catenin-deficient epithelial cell lines.
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Materials and Methods |
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Cells and Antibodies
Mouse L cells were grown in DME supplemented with 10% FCS. Transfectants expressing E-cadherin (EL1; Nose et al., 1988
), nE
(1-906),
nE
N(1-508), and nE
C(509-906) (nE
L2, nE
NL28, and nE
CL1, respectively; Nagafuchi et al., 1994
), and other E-cadherin-
-catenin fusion
molecules (see Fig. 1) were grown in the same medium containing 150 µg/
ml of G418. nE
(1-906), nE
N(1-508), and nE
C(509-906) were originally called nE
, nE
N, and nE
C, respectively (Nagafuchi et al., 1994
).
We named transfectants by combining the name of the fusion protein with
L; for example, L cells expressing nE
(1-906) were designated as nE
(1-906)L cells. Mouse PC9 cells were grown in a 1:1 mixture of DME and
Ham's F12 supplemented with 10% FCS (DH10). PC9 cells expressing
(1-184/509-643)-HA (see below) were grown in the same medium containing 150 µg/ml of G418. Human colon carcinoma DLD-1/R2/7, abbreviated to R2/7, and its transfectants expressing
E(1-890),
E(1-325/510-890),
E(1-509), and
E(1-325) (Watabe-Uchida et al., 1998
) were also
cultured in DH10 medium.
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Mouse anti-ZO-1 mAb (T8-754) was obtained and characterized as described (Itoh et al., 1991). Anti-E-cadherin mAb (ECCD-2), which was
concentrated by ammonium sulfate precipitation, was a generous gift from
Dr. M. Takeichi (Kyoto University, Kyoto, Japan; Shirayoshi et al., 1986
).
Mouse anti-vinculin mAb (hVIN-1) and mouse anti-
-actinin mAb
(BM-75.2) were purchased from Sigma Chemical Co. Mouse anti-HA-tag
mAb (12CA5) was purchased from Boehringer Mannheim Biochemicals.
Cy2-labeled anti-rat IgG and Cy3-labeled anti-mouse IgG antibodies
were purchased from Amersham. DTAF (dichlorotriazinyl amino fluorescence)-labeled anti-rat IgG was purchased from Chemicon International, Inc.
Constructs
We constructed pBATE(327-906), pBATE
(631-906), pBATE
(1-643),
pBATE
(509-643), pBATE
(1-325/509-906), and pBATE
(1-402/509-906), expression vectors for nE
(327-906), nE
(631-906), nE
(1-643), nE
(509-643), nE
(1-325/509-906), and nE
(1-402/509-906), respectively (see Fig. 1). For construction of these vectors, we used three plasmids: (a)
pBATEM2, mouse E-cadherin expression vector (Nose et al., 1988
), the
ClaI-XbaI fragment of which corresponds to the catenin-binding site and
was replaced with the
-catenin cDNA fragments in constructs; (b)
pSK102B, which contains the
-catenin cDNA with a PstI-BglII adaptor
inserted into the PstI site just before the initiation methionine codon; and
(c) pBATE
, the ClaI-XbaI fragment of pBATEM2 was replaced with
the BglII-XbaI fragment of pSK102B including the whole ORF of the
-catenin cDNA (Nagafuchi et al., 1994
). In the open reading frame of the
-catenin cDNA sequence, we used four restriction sites, PmaCI, ScaI,
ClaI, and SmaI, corresponding to amino acid residues 326, 403, 508, and 670, respectively. The XbaI site at the 3' terminal of
-catenin cDNA in
pSK102B was also used. For the production of pBATE
(327-906), the
ClaI-XbaI fragment of pBATEM2 was replaced with the PmaCI-XbaI fragment of pSK102B. For construction of pBATE
(631-906), a ClaI site
(630ClaI) was introduced at the position corresponding to amino acid residue 630 of
-catenin cDNA in pSK102B by PCR, then the ClaI-XbaI
fragment of pBATEM2 was replaced with the 630ClaI-XbaI fragment.
For production of pBATE
(1-643), an XbaI site (644XbaI) was introduced at a position corresponding to amino acid residue 644 of
-catenin
cDNA in pSK102B by PCR, then the ClaI-XbaI fragment of pBATE
was replaced with the ClaI-644XbaI fragment. For production of
pBATE
(1-325/509-906) and pBATE
(1-402/509-906), the PmaCI-ClaI and the ScaI-ClaI fragments, respectively, were excised from pBATE
. All junctions newly produced in the
-catenin coding sequence were arranged in frame using adaptors or linkers as necessary.
For construction of pEF(1-184/509-643)-HA, an EcoRI site (185 EcoRI) was introduced at the position corresponding to amino acid residue 185 of
-catenin cDNA in pSK102B by PCR. The BglII-185EcoRI fragment, ClaI-644XbaI fragment, and a HA
3' fragment, which contains an HA epitope tag sequence, a stop codon, and 3' noncoding region of
-catenin, were tandemly ligated and inserted into the pEFMC1-neo expression vector (Visvader et al., 1992
).
We also constructed pGEX-N(1-508), pGEX-
C(509-906), and
pGEX-
(671-906), expression vectors for GST-
-catenin fusion molecules, using pSK102B and pGEX vectors (Pharmacia LKB Biotechnology). For production of pGEX-
N(1-508), pGEX-
C(509-906), and
pGEX-
(631-906), the BglII-ClaI, ClaI-XbaI, and SmaI-EcoRI fragments
of pSK102B were inserted into the BamHI-SmaI sites of pGEX-2T, the
SmaI site of pGEX-3X, and the SmaI-XhoI sites of pGEX-4T-3 (Pharmacia LKB Biotechnology), respectively.
Transfection
L cells (5 × 105 per 3-cm plate) were cotransfected with 1 µg of each expression vector and 0.05 µg of pSTneoB (Katoh et al., 1987) by the lipofectamine method (Life Technologies, Inc.). After 48 h of incubation, the
cells were replated on pairs of 9-cm dishes and cultured in the presence of
400 µg/ml G418 to select stable transfectants. Colonies of G418-resistant
cells were isolated, recloned, and subsequently maintained in complete
medium with 150 µg/ml of G418. We isolated several stable clones for
each transfection experiment. Since nE
(327-906)L-11, nE
(631-906)L-7, nE
(1-643)L-9, nE
(509-643)L-32, nE
(1-325/509-906)L-2, and nE
(1-402/509-906)L-23 clones expressed relatively large amounts of fusion molecules, we mainly used these in this study.
Trypsinized PC9 cells (105) were suspended in 500 µl of Hepes-buffered (pH 7.4) Ca2+- and Mg2+-free saline and mixed with 10 µg of expression vector and 1 µg of pSTneoB. Electroporation was performed at 960 µF, 250 V. The cells were selected in G418 (0.2 mg/ml)-containing medium.
Immunohistochemistry
All procedures were performed at room temperature. Cells cultured on coverslips were fixed with 3.5% (for hVIN-1 and BM-75.2) or 1.0% (for T8-754) formaldehyde solution in HMF (Hepes-buffered magnesium-free saline) for 15 min. After three washes with PBS, cells were soaked in blocking solution (1% BSA in PBS) for 30 min and subsequently incubated with ECCD-2 diluted with PBS containing 1% BSA for 30-60 min at room temperature. The cells were then washed three times with PBS and soaked in 0.2% Triton X-100 in PBS for 15 min. After rinsing with PBS, the cells were treated with 1% BSA in PBS for 30 min and subsequently incubated with hVIN-1, T8-754, or BM-75.2 for 30-60 min. After extensive washing with PBS, the specimens were incubated with fluorescence-labeled second antibodies (Cy2- or DTAF-labeled goat anti-rat IgG for ECCD-2 and Cy3-labeled donkey anti-mouse IgG [H&L] for hVIN-1, T8-754, and BM-75.2) diluted with PBS containing 1% BSA for 30 min at room temperature. After washing thoroughly with PBS, the preparation was mounted with 90% glycerol-PBS containing 0.1% para-phenylendiamine and 1% n-propylgalate. Samples were observed with a Zeiss Axiophot photomicroscope (Carl Zeiss). Images were recorded with a cooled CCD camera (SenSys 0400, 768 × 512 pixels; Photometrics) controlled by a Power Macintosh 7600/132 and the software package IPLab Spectrum V3.1 (Signal Analytic Corp.).
SDS-PAGE and Immunoblotting
SDS-PAGE (10 or 7.5%) and immunoblotting were performed as described previously (Nagafuchi et al., 1994). Samples were solubilized in
SDS sample buffer, separated by SDS-PAGE, and gels were stained with
Coomassie brilliant blue R-250. For immunoblotting, proteins were electrophoretically transferred onto nitrocellulose sheets. Nitrocellulose membranes were then incubated with ECCD-2, T8-754, or 12CA5. Antibody
detection was performed using an Amersham biotin-streptavidin kit with
biotinylated anti-rat or anti-mouse Ig and NBT-BCIP.
In Vitro Binding Assay Using GST Fusion Proteins
In vitro binding assays were performed as previously described (Itoh et al.,
1997). In brief, GST-
-catenin fusion proteins were expressed in Escherichia coli and purified using glutathione-Sepharose 4B beads (Pharmacia
LKB Biotechnology) as previously described (Itoh et al., 1997
). Then, 2 ml
of the cell lysate of Sf9 cells expressing N-ZO-1 was added, followed by incubation for 3 h at 4°C. The beads were again washed with PBS containing
0.1% Triton X-100, 2 mM PMSF, and 4 µg/ml of leupeptin, and then
bound proteins were eluted with 1 ml of 50 mM Tris-HCl buffer, pH 8.0, containing 10 mM glutathione. The amounts of GST fusion proteins in
each eluate were determined by SDS-PAGE.
Detergent Extraction of Cells
Confluent cultured cells (~4 × 106 cells per 6-cm dish) were extracted
with 2.5% NP-40 in HMF, then centrifuged at 100,000 rpm for 30 min as
described previously (Nagafuchi and Takeichi, 1988). To the supernatant,
2× SDS sample buffer was added to make the total volume 0.4 ml and
used as the detergent-soluble fraction. On the other hand, the pellet fraction was dissolved in 0.4 ml of 1× SDS sample buffer and used as the detergent-insoluble fraction.
Trypsin Treatment, Aggregation, and Dissociation of Cells
Cells were trypsinized by two different methods for the differential removal of E-cadherin or its fusion molecules, as described by Takeichi
(1977). In brief, cells were treated with 0.01% trypsin in the presence of
1 mM CaCl2 (TC treatment) or 1 mM EGTA (TE treatment) at 37°C for
30 min. Generally, cadherins are left intact after TC treatment, but are digested by TE treatment.
For the cell aggregation assay, cells were dispersed after TC treatment
as described by Takeichi (1977). L and R2/7 transfectants were pretreated
with 1 µM cytochalasin D in culture medium for 2 h. Although this treatment is necessary for the dissociation of L cells expressing nE
C(509-906)
and R2/7 cells expressing
E(1-890), it did not affect cell aggregation of
other transfectants. Aliquots of 5 × 105 dissociated cells were plated in
each well of a Falcon 12-well plate with 0.5 ml HMF and allowed to aggregate, as described previously (Nagafuchi et al., 1987
). The extent of cell
aggregation was represented by the index Nt/N0 where Nt is the total particle number after incubation time t and N0 is the total particle number at
the initiation of incubation.
For the cell dissociation assay, confluent cultures were treated with TC
and TE and dissociated by pipetting 10 times (Nagafuchi et al., 1994). The
extent of cell dissociation was represented by NTC/NTE, where NTC and
NTE are the total particle number after TC and TE treatment, respectively. In some experiments, confluent cultures were pretreated with 1 µM
cytochalasin D in culture medium for 2 h, then cell dissociation analyses
were performed in the presence of 1 µM cytochalasin D.
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Results |
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Involvement of -Catenin in the Recruitment of
Vinculin and ZO-1 to Cadherin-based Cell-Cell
Adhesion Sites
In L cell transfectants expressing E-cadherin, two cytoskeletal proteins, vinculin and ZO-1, are precisely colocalized with E-cadherin at cell-cell contact sites (Itoh et al.,
1993). In parental L cells, vinculin is concentrated exclusively at cell-substrate AJ, and ZO-1 does not show specialized localization but some condensation at tips of cellular processes (data not shown).
To determine whether -catenin is involved in the recruitment of vinculin and ZO-1 to E-cadherin-based cell
adhesion sites, we used L cell transfectants expressing
nE
(1-906), which is a fusion molecule consisting of nonfunctional E-cadherin lacking its catenin-binding domain
and full-length
-catenin (Fig. 1 B; Nagafuchi et al., 1994
).
As previously reported, this molecule showed similar cell
adhesion and cytoskeleton interaction activities to the normal E-cadherin-catenin complex. Immunocytochemical
analysis clearly revealed that both vinculin and ZO-1 were
precisely colocalized with nE
(1-906) at cell-cell contact
sites in L cell transfectants (Fig. 2). As reported previously, nonfunctional E-cadherin does not interact with the
cytoskeleton and nE
(1-906) is not associated with endogenous
-catenin (Nagafuchi and Takeichi, 1988
; Ozawa et al.,
1989
; Nagafuchi et al., 1994
). These observations indicated that
-catenin was crucial for the recruitment of vinculin
and ZO-1 to cell-cell contact sites in transfected L cells.
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Involvement of Residues 327-402 of -Catenin in the
Recruitment of Vinculin
To determine the domain of -catenin necessary for the
recruitment of vinculin or ZO-1, we constructed several
expression vectors encoding various E-cadherin-
-catenin
fusion molecules, in which distinct domains of
-catenin
were deleted (Fig. 1 B). These expression vectors were introduced into mouse L cells, and stable transfectant clones
were isolated for each construct. Each mutant molecule
expressed in transfectants had the expected apparent molecular mass (Fig. 1 C).
The subcellular localization of vinculin was compared
with those of expressed fusion molecules in various transfectants (Fig. 3). nE(327-906) in which the NH2-terminal
326 residues had been truncated was precisely colocalized
with vinculin (Fig. 3, a and a'). However, nE
C(509-906)
with a longer NH2-terminal deletion showed no colocalization with vinculin (Fig. 3, b and b'). In cells expressing
nE
N(1-508) with truncation of the COOH-terminal 398 residues, expressed fusion molecule was also colocalized
with vinculin at sites where it was heavily condensed (data
not shown). These observations suggested that residues
327-508 are important for the recruitment of vinculin.
Consistently, vinculin was not colocalized with nE
(1-325/
509-906) lacking residues 326-508 (Fig. 3, c and c'). Residues 326-508 of
-catenin include the direct
-actinin-binding site (325-394 residues) reported previously (Nieset
et al., 1997
). When this
-actinin-binding site was added to
nE
(1-325/509-906), the resultant fusion molecule nE
(1-402/509-906) retained the ability to colocalize with vinculin
(Fig. 3, d and d'). Double-immunostaining for E-cadherin
and
-actinin revealed that the constructs which recruited
vinculin, such as nE
(327-906) and nE
(1-402/509-906),
could recruit
-actinin (Fig. 4, a, a', c, and c'). In contrast,
nE
(1-325/509-906) was not colocalized with either vinculin or
-actinin (Fig. 4, b and b'). These results demonstrated that residues 327-402 of
-catenin are crucial for the recruitment of vinculin as well as
-actinin to cadherin-based cell adhesion sites of L cell transfectants (see
Fig. 11 A).
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Involvement of the COOH-terminal Domain of
-Catenin (Residues 631-906) in the Recruitment
of ZO-1
To determine the domain(s) of -catenin involved in the
recruitment of ZO-1, we compared the subcellular localization of ZO-1 with that of expressed fusion molecules in
transfectants. In contrast with vinculin, ZO-1 was not precisely colocalized with nE
N(1-508) but with nE
C(509-906) (Fig. 5, a, a', b, and b'). nE
(631-906), which had the
longest NH2-terminal deletion and showed no cell adhesion activity (see below), was also colocalized with ZO-1 at
cell-cell boundaries (Fig. 5, c and c'), although their condensation at cell-cell boundaries was not as exclusive compared with those in other transfectants. These observations strongly suggested that the COOH-terminal domain
of
-catenin (631-906 residues) is crucial for the recruitment of ZO-1 (see Fig. 11 A).
|
We have reported previously that the NH2-terminal half
of ZO-1 (N-ZO-1) directly interacts with -catenin (Itoh
et al., 1997
). Therefore, it was expected that N-ZO-1
would directly interact with the COOH-terminal domain
of
-catenin. To test this possibility, we produced four
GST fusion proteins, GST-
(1-906), GST-
N(1-508), GST-
C(509-906), and GST-
(671-906), which contained the
full-length
-catenin, its NH2-terminal half, its COOH-terminal half, or the COOH-terminal 236 residues, respectively (Fig. 6 A). Then, we analyzed in vitro binding abilities of these fusion molecules with recombinant N-ZO-1
produced in Sf9 cells by baculovirus infection. As shown
in Fig. 6 B, N-ZO-1 bound to not only GST-
(1-906) but also GST-
C(509-906) and GST-
(671-906), although the
binding affinities to the latter two were lower than that
to the former. N-ZO-1 did not bind to GST-
N(1-508).
These results strongly suggested that the COOH-terminal
276 amino acids (residues 631-906) of
-catenin recruited
ZO-1 to cadherin-based cell-cell contact sites through its
direct binding to ZO-1 in transfected L cells.
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Involvement of Residues 509-643 of -Catenin in Cell
Adhesion Activity of Fusion Molecules
We have reported previously that nEC(509-906) shows
similar cell adhesion activity to the normal E-cadherin-
catenin complex (Nagafuchi et al., 1994
). To determine the
domain which is required for this function, we compared
the reaggregative properties of L cell transfectants expressing nE
(509-643) and nE
(631-906); the former was
an nE
C(509-906) derivative lacking the ZO-1-binding domain, and the latter contained only the ZO-1-binding
domain (see Fig. 1 B). Cells expressing nE
(509-643) aggregated as rapidly as those expressing nE
C(509-906). In
contrast, the reaggregation of cells expressing nE
(631-906) was indistinguishable from that of parent L cells (Fig.
7 A). These observations suggested that residues 509-643 of
-catenin are required for cell adhesion activity of fusion molecules. Indeed, when residues 509-643 were added to nE
N(1-508), cells expressing the resultant fusion molecule nE
(1-643) aggregated rapidly, although nE
N(1-508) did not (Fig. 7 A). The levels of expression of
nE
(631-906) or nE
N(1-508) in the transfectants examined were relatively low among the fusion molecules examined (Fig. 1 C, lanes 4 and 5). However, the reduced level of fusion molecule expression did not seem to be the
cause of loss of their cell adhesion activity since L cells expressing similar or lesser amounts of nE
(1-643) still aggregated rapidly (data not shown). These results demonstrated that residues 509-643 of
-catenin are required for
cell adhesion activity of E-cadherin-
-catenin fusion molecules in transfected L cells (see Fig. 11 A). We tentatively called this an adhesion-modulation domain (see Fig. 11 B).
|
The cell adhesion activities of E-cadherin and its -catenin fusion molecules were reported to be associated with
their interactions with the cytoskeleton (Nagafuchi et al.,
1994
; Sako et al., 1998
). For example, about half of the
nE
C(509-906) was resistant to extraction with NP-40
(Fig. 8) Interestingly, most of the nE
(509-643) carrying
only the adhesion-modulation domain was extracted with NP-40, suggesting that this fusion molecule did not interact with the cytoskeleton (Fig. 8). In contrast, nE
N(1-508) (data not shown; see Nagafuchi et al., 1994
) and
nE
(631-906) (Fig. 8) did not show cell adhesion activity
but interacted with the cytoskeleton as judged from their
resistance to NP-40 extraction. In the cell aggregation assay, the disorganization of actin-based cytoskeleton by cytochalasin D treatment essentially did not affect the cell aggregating activity of any of the transfectants (data not
shown). These results suggested that cadherin-based
cell adhesion activity and cadherin-cytoskeleton interaction are regulated independently by distinct domains
of
-catenin.
|
Requirement of ZO-1-Binding Domain for the Strong State of Cell Adhesion
We demonstrated previously that cells assumed two states
of cadherin-based cell adhesion, strong and weak (Takeda
et al., 1995). The cell-cell adhesion in the strong state
could hardly be dissociated by pipetting, although that in
the weak state was easily dissociated. Using the cell dissociation assay, we examined the states of cell adhesion of
various L cell transfectants. As reported previously, L cell
transfectants expressing nE
C(509-906), which contained
both the adhesion-modulation and ZO-1-binding domains
of
-catenin, were hardly dissociated in the cell dissociation assay, indicating that these cells showed the strong
state of cell adhesion activity (Fig. 7 B; Nagafuchi et al.,
1994
). Intact E-cadherin or other fusion molecules containing both of these domains also showed the strong state
of cell adhesion activity (data not shown; see Fig. 11 A). In
contrast, cells expressing nE
(509-643), which contained
only the adhesion-modulation domain of
-catenin, were
easily dissociated into single cells under the conditions of
the cell dissociation assay (Fig. 7 B), indicating that these
cells showed the weak state of cell adhesion activity. Interestingly, cells expressing nE
(1-643) lacking only the ZO-1-binding domain also showed the weak state of cell adhesion activity (Fig. 7 B). It should be noted that nE
(1-643)
was hardly extracted with NP-40 (Fig. 8), suggesting its
interaction with the cytoskeleton probably through the
vinculin/
-actinin-binding domain. In the presence of cytochalasin D, nE
C(509-906)L cells were also easily dissociated under the same condition (Fig. 7 C), although the amount of expressed nE
C(509-906) was not altered in
the presence or absence of cytochalasin D (data not
shown). These results indicated that the ZO-1-binding domain, but not the vinculin/
-actinin-binding domain, in addition to the adhesion-modulation domain was required for the strong state of cell adhesion activity. It was also
suggested that the intact actin-based cytoskeleton is required for this type of adhesion activity.
Functions of -Catenin in Epithelial Cells
We examined the role of -catenin in epithelial cell adhesion using
-catenin-deficient epithelial cell lines and their
transfectants expressing
-catenin deletion mutants. Since
cadherin-catenin complex is not colocalized with ZO-1 in
epithelial cells, the roles of
-catenin in epithelial cells are
expected to be different, at least in some aspects, from
those in nonepithelial cells such as L cells. DLD-1/R2/7,
abbreviated to R2/7, is an
-catenin-deficient colon carcinoma line. R2/7 transfectants expressing
-catenin deletion mutants were previously reported (Watabe-Uchida
et al., 1998
). We used four of these transfectants in this
study.
-Catenin deletion mutants expressed in these
transfectants are shown in Fig. 9 A. Using the cell aggregation assay, we compared the reaggregative properties of
R2/7 and its transfectants (Fig. 9 B). R2/7 itself showed
aggregation activity which was blocked in the presence of E-cadherin blocking antibodies (data not shown, see
Watabe-Uchida et al., 1998
). R2/7 transfectants expressing
E(1-890), which is indistinguishable from cells expressing
intact
-catenin, aggregated more rapidly than parental
R2/7 cells. Not only cells expressing
E(1-325/510-890) but
also those expressing
E(1-509), which lacks the adhesion-modulation domain, also aggregated more rapidly than parental R2/7 cells. R2/7 cells expressing
E(1-325) showed
similar aggregation activity to the parental cell line R2/7.
These results demonstrated that the residues 325-509 including the vinculin/
-actinin-binding domain are also involved
in cadherin-dependent cell aggregation activity in R2/7. In
the cell dissociation assay, R2/7 was readily dissociated but
R2/7 transfectants expressing
E(1-890) were hardly dissociated (Fig. 9 C). Although cells expressing
E(1-325/
510-890) showed some degree of resistance to pipetting,
the strong state of cell adhesion activity was not fully restored (Fig. 9 C). This partial restoration of the strong
state of cell adhesion activity was also observed in cells expressing not only
E(1-509) but also
E(1-325) (Fig. 9 C).
These results suggested that multiple domains of
-catenin
were required for the strong state of cell adhesion activity
in R2/7.
|
To confirm the importance of adhesion-modulation domain in epithelial cells, we used PC9 cells, a human lung
carcinoma cell line lacking -catenin expression. It was reported that PC9 showed aggregation activity to some extent and that this activity was dependent on E-cadherin-
-catenin complex without
-catenin (Shimoyama et al.,
1992
). We constructed an expression vector encoding
(1-184/509-643), in which only an adhesion-modulation
domain was covalently connected to the NH2-terminal
-catenin-binding domain of
-catenin (Fig. 10 A). This
vector was introduced into PC9 cells, and several transfectant clones were isolated.
(1-184/509-643) with the expected size was expressed in the transfectants (Fig. 10 B)
and colocalized with E-cadherin-
-catenin complex (data
not shown). Cell aggregation assay revealed that cells expressing
(1-184/509-643) aggregated more rapidly and
more extensively than parental PC9 cells (Fig. 10 C).
These aggregates were readily dissociated into single cells under the dissociation assay conditions (data not shown).
These observations indicated that an adhesion-modulation
domain is involved in the weak state of cell adhesion activity even in epithelial cell lines.
|
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Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is generally accepted that the cadherin-catenin cell adhesion complex plays fundamental roles not only in the
formation of cell-cell junctions but also in the morphogenesis of tissue and organs, dependent on its strong state of
cell-cell adhesion activity and its interaction with the
actin-based cytoskeleton (Takeichi, 1991). We identified
three distinct functional domains of
-catenin required for
the interaction with vinculin, for direct binding to ZO-1,
and for the adhesion activity of E-cadherin-
-catenin fusion molecules (Fig. 11 B). Here, we will discuss possible
functions of each domain of
-catenin and the relationship
between the cell adhesion activity and the interaction of
cadherin-catenin complex with the cytoskeleton. We will
also discuss the difference of
-catenin function in nonepithelial and in epithelial cells.
Functional Domains of -Catenin
-Catenin was reported to be directly associated with
-catenin,
-actinin, and actin filaments, and the domains responsible for their binding have
been narrowed down on the
-catenin molecule (Rimm
et al., 1995
; Nieset et al., 1997
; Obama and Ozawa, 1997
). It was reported recently that ZO-1 bound to
-catenin directly (Itoh et al., 1997
), but the domain responsible remained elusive. In this study, using deletion constructs, we
showed that the COOH-terminal domain (residues 631-906) of
-catenin recruited ZO-1 to the cell adhesion sites
and directly bound to NH2-terminal half of ZO-1 in vitro.
We also demonstrated that this ZO-1-binding domain interacted with cytoskeletons judging from the resistance of
fusion molecules to NP-40 extraction. It was reported previously that the COOH-terminal halves of ZO-1 are directly associated with actin filaments (Itoh et al., 1997
).
Based on these properties, we can imagine that the ZO-1-binding domain interact with the actin-based cytoskeleton through ZO-1. We demonstrated that this domain is
essential for the strong state of cadherin-based cell adhesion in L cell transfectants, which was dependent on the intact actin-based cytoskeleton. The functional importance
of the ZO-1-binding domain was also reported using mouse
embryos expressing mutant
-catenin (Torres et al., 1997
).
Taken together, we concluded that the ZO-1-binding domain (COOH-terminal 276 residues) of
-catenin plays a
fundamental role in the cadherin-catenin cell adhesion
system probably through its interaction with ZO-1 and/or
the actin-based cytoskeleton.
We cannot exclude the possibility that other cytoskeletal proteins interact with the ZO-1-binding domain. This
domain is known to interact with actin in vitro (Rimm et al.,
1995). The physiological role of this interaction remains to
be elucidated. The ZO-1-binding domain was also shown
to directly interact with vinculin in vitro (Weiss et al.,
1998
). However, we found that the ZO-1-binding domain
in fusion molecules did not recruit vinculin to the cell-cell boundaries in L cell transfectants. Since the reported binding constant of the ZO-1-binding domain to vinculin was
lower than that to ZO-1 (Itoh et al., 1997
; Weiss et al.,
1998
), the binding of vinculin to the ZO-1-binding domain
may be prevented by ZO-1 in L cell transfectants. ZO-2
and ZO-3, homologues of ZO-1, are other candidates as
binding proteins to the ZO-1-binding domain. In fact,
it was reported recently that ZO-2 showed very similar properties with ZO-1 and directly bound to
-catenin
(Itoh et al., 1999
). However, these proteins are not involved in the cadherin-based cell adhesion in L cell transfectants, since their expression was not detected in L cell
transfectants (our unpublished observation).
We
found that residues 327-402 of -catenin were required for
fusion molecules to recruit not only
-actinin but also vinculin in transfected L cells. This is consistent with previous
data that this domain directly binds to both vinculin
(Watabe-Uchida et al., 1998
) and
-actinin (Nieset et al.,
1997
), and these two molecules interact with each other
(Wachsstock et al., 1987
). It is not clear whether vinculin
and
-actinin interact with this short domain with 76 residues simultaneously or competitively in vivo. E-cadherin-
-catenin fusion molecules conferred full adhesion activity
in L cell transfectants even if they lacked the vinculin/
-actinin-binding domain. This raised the question of what
is the function of the vinculin/
-actinin-binding domain. We reported previously that intact E-cadherin conferred a
flexible adhesive phenotype upon L cells, but E-cadherin-
-catenin fusion molecules conferred inflexible phenotypes (Nagafuchi et al., 1994
). If the vinculin/
-actinin-binding domain is involved in this flexible adhesion activity,
its function would not be observed using E-cadherin-
-catenin molecules expressed in L cells. On the other
hand, it was reported recently that this domain is involved
in the organization of apical junctional complex and the
activation of cadherin-based cell adhesion in epithelial
cells (Watabe-Uchida et al., 1998
). It has been reported
also that vinculin is colocalized with the cadherin-catenin
complex in epithelial cells but not in some fibroblastic cell
lines (Knudsen et al., 1995
), and that vinculin is one of the
major components of cell-cell AJ in epithelial cells (Geiger et al., 1980
). These observations suggest that the vinculin/
-actinin-binding domain is required for the function of
cadherin-catenin complex, especially for junctional complex formation, only in epithelial cells but not in fibroblastic cells.
Deletion constructs showed that residues 509-643 of -catenin are required for fusion molecules to function as cell
adhesion molecules. We tentatively called this domain
an adhesion-modulation domain. When mutant
-catenin
containing the
-catenin-binding and the adhesion-modulation domains was expressed in
-catenin-deficient PC9
cells, such cells aggregated more rapidly than parental PC9 cells. These results suggested that the adhesion-modulation domain is involved in cell adhesion in the "natural"
cadherin/catenin complex.
Although several sites of -catenin were reported to be
required for the interaction with the cytoskeleton, the adhesion-modulation domain does not correspond to these
cytoskeletal interaction sites. Moreover, the fusion molecule carrying only this domain was easily extracted with
NP-40, suggesting that this molecule did not interact with
the cytoskeleton. These findings indicated that the adhesion-modulation domain might function without the interaction with the cytoskeleton. It has been accepted that the
insoluble fraction of E-cadherin was active in cell adhesion
and the soluble one was not, since E-cadherin molecules
which could not be extracted with NP-40 were strictly localized at cell-cell contact sites (Nagafuchi and Takeichi, 1988
). However, our present results suggested that some
fraction of soluble E-cadherin-catenin complex was also
active in cell adhesion.
The molecular mechanism of the activation of E-cadherin extracellular domain remains unclear. One simple
interpretation is that the adhesion-modulation domain
supports the lateral aggregation of E-cadherin molecules,
which may mediate the weak state of cell adhesion. Alternatively, this domain may trigger off the other adhesion-modulation system. Fusion molecules used contained the membrane proximal, p120-binding domain of E-cadherin
(Yap et al., 1998) and are colocalized with endogenous
p120 protein in transfected L cells (our unpublished observation). It was reported that this membrane proximal
domain might positively or negatively regulate cadherin-based cell adhesion (Ozawa and Kemler, 1998
; Yap et al., 1998
). It is possible that the adhesion-modulation domain
of
-catenin affects the potential activity of the membrane
proximal domain of E-cadherin.
Some fusion molecules lacking the adhesion-modulation
domain were detected at cell-cell boundaries in transfectants, although nonfunctional E-cadherin itself was not
(Nagafuchi and Takeichi, 1988). These observations raised
the question of how these fusion molecules were condensed at cell-cell boundaries, although they did not
function as cell adhesion molecules. The main difference between these fusion molecules and nonfunctional E-cadherin is that the former interacted with cytoskeletal proteins such as ZO-1 or vinculin/
-actinin but the latter did
not. As discussed below, ZO-1 is likely to facilitate the lateral aggregation of its membrane binding partners. Vinculin is also expected to form clusters through its interaction
with various cytoskeletal components (Otto, 1990
). So, the
interaction with cytoskeletal components may cause the
clustering of fusion molecules in the plasma membrane, which then induces the association of cadherin complexes
on apposed cell membranes (Shapiro et al., 1995
).
Relationship between Interaction with the Cytoskeleton and Adhesion Activity of Cadherin-Catenin Complex
As previously reported, cadherin-based cell adhesion can
be classified into the strong state and the weak state, using
cell dissociation and aggregation assays (Takeda et al.,
1995). In the cell aggregation assay, cells form aggregates
in both adhesive states. In the cell dissociation assay, however, cells in the strong state were hardly dissociated into
single cells but those in the weak state were dissociated
readily. It is known that the adhesive state is regulated by
the phosphorylation level of the cytoplasmic components
(Matsuyoshi et al., 1992
; Behrens et al., 1993
). We also
demonstrated that disorganization of the actin-based cytoskeleton shifted the cadherin-based cell adhesion from
the strong to the weak state. Since the level of cadherin expression on the cell surface was not affected in either case,
the strong state and the weak state may reflect qualitative
differences in cell adhesion activity but not quantitative
differences in cell adhesion molecules. The present results
are consistent with this idea, since cells in both adhesive
states aggregated in a similar manner.
We found that all of the fusion molecules that showed
the strong state of cell adhesion activity interacted with
the cytoskeleton, suggesting that the interaction with the
cytoskeleton is required for the strong state of cell adhesion activity. This was supported by the present observation that cytochalasin D treatment shifted cadherin-based
cell adhesion to the weak state. Interestingly, the fusion
molecule lacking only the ZO-1-binding domain showed the weak state of cell adhesion activity, although it contained a vinculin/-actinin-binding domain and may interact with the cytoskeletons, judging from the refractoriness
to NP-40 extraction. Thus, we concluded that the cytoskeleton interaction through the ZO-1-binding domain,
but not through the vinculin/
-actinin binding domain or
other domain(s), is required for the strong state of cell adhesion activity in L cell transfectants. As discussed above, ZO-1 and actin are possible binding proteins to this domain in L cell transfectants. ZO-1 is a member of the
MAGUK family. Another member of this family, PSD95,
is known to facilitate the lateral aggregation of its membrane binding partners such as NMDA receptors and K+
channels (Kim et al., 1995
; Niethammer et al., 1996
). Thus,
it is possible that ZO-1 strengthens the cell-cell adhesion
activity not only by cross-linking
-catenin to actin filaments (Itoh et al., 1997
) but also by facilitating the lateral
aggregation of E-cadherin or its fusion molecules in L cell
transfectants. The role of actin binding to this domain remains unclear. Furthermore, we cannot exclude the possibility that unknown factor(s) interacted with the ZO-1-binding domain and supported the strong state of cell
adhesion activity.
Function of -Catenin in Epithelial Cells
At the immunoelectron microscopic level, cadherin-catenin complex is known to be colocalized with ZO-1 in nonepithelial cells such as L cells but not in epithelial cells. In
epithelial cells, ZO-1 is highly condensed at the TJ and is
thought to directly interact with TJ membrane proteins including occludin (Furuse et al., 1994). These results suggested that the ZO-1-binding domain of
-catenin may
have different functions in nonepithelial cells and in epithelial cells. In fact, it was demonstrated that this domain
could not cause redistribution of ZO-1 in R2/7, an epithelial colon carcinoma cell line (Watabe-Uchida et al., 1998
).
It remains unclear why the ZO-1-binding domain does not
associate with ZO-1 and how this domain functions in epithelial cells. Further studies to address this question will
provide important information regarding the mechanisms
of junctional complex formation in epithelial cells.
It was reported recently that the vinculin/-actinin-binding domain directly binds to vinculin and this interaction
functions to organize the apical junctional complex, including TJ, in R2/7 cells (Watabe-Uchida et al., 1998
). This
domain, instead of the ZO-1-binding domain, functions to
recruit ZO-1 at cell-cell boundaries in R2/7 cells. Since we
demonstrated that this domain did not interact with ZO-1
directly, the redistribution of ZO-1 may be dependent on
the reorganization of TJ caused by
-catenin-vinculin interaction in R2/7 cells. This is consistent with the previous observation that, when the vinculin/
-actinin binding domain was replaced with the vinculin tail domain, the resultant
-catenin-vinculin fusion molecule recruited ZO-1
to cell-cell boundaries (Watabe-Uchida et al., 1998
).
The role of -catenin in epithelial cell adhesion also
seems to be different from that in nonepithelial cells, although the adhesion-modulation domain seems to be generally used in nonepithelial and epithelial cells. We found
that the ZO-1-binding domain, which cannot cause redistribution of ZO-1, also failed to fully restore the strong
state of cell adhesion activity in epithelial cells, supporting
our speculation that ZO-1 is required for the strong state
of cell adhesion activity. In epithelial cells, both the NH2-terminal half and COOH-terminal half domains are partially involved in the strong state of cell adhesion activity
and these two domains cooperatively support this activity.
Thus,
-catenin in epithelial cells supports the strong state
of cell adhesion activity in a different manner from that in
nonepithelial cells. In fact, it was reported recently that
the COOH-terminal 236 residues, the in vitro binding domain to ZO-1, were not involved in the cadherin-based cell
adhesion activity in epithelial cells, although the COOH-
terminal 273 residues of
-catenin were involved in this activity (Ozawa, 1998
). The 37 residues between 634 and 670 of
-catenin were not involved in the adhesion activity or
binding to ZO-1 in L cell transfectants (our unpublished
observation). The mechanism by which
-catenin is involved in these processes in epithelial cells remains to be elucidated.
Of course, here, we cannot completely exclude the possibility that differences of -catenin functions observed in
L cells and R2/7 cells are not due to differences of the cell
type (nonepithelium versus epithelium) but that of the expressed molecule (fusion versus nonfusion). However, our
recent observations using
-catenin-deficient F9 cells did
not favor this possibility (Maeno, Y., and A. Nagafuchi, unpublished observations).
In this study, we identified three functional domains of
-catenin, i.e., the ZO-1-binding domain, vinculin/
-actinin-binding domain and adhesion-modulation domain. We
also clarified the effects of the cytoskeleton interaction on
the different states of cadherin-based cell adhesion activity. Furthermore, we demonstrated that
-catenin may
have different functions in nonepithelial and epithelial
cells. Further studies of the regulatory mechanism of cadherin-based cell adhesion by
-catenin will lead to a better
understanding of the physiological functions of the cadherin-catenin complex which plays a pivotal role in morphogenesis in multicellular organisms.
![]() |
Footnotes |
---|
Address correspondence to Akira Nagafuchi, Ph.D., Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: 81-75-753-4375. Fax: 81-75-753-4660. E-mail: naga-san{at}mfour.med.kyoto-u.ac.jp
Received for publication 9 June 1998 and in revised form 27 January 1999.
We would like to thank all the members of our laboratory for their helpful discussions. Our thanks are also due to Drs. M. Takeichi, M. Watabe-Uchida, and S. Aono for their generous gift of anti-E-cadherin mAb, ECCD-2, R2/7 transfectants, and helpful discussions.
This work was supported in part by a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research (to A. Nagafuchi, M. Itoh, and S. Tsukita) from the Ministry of Education, Science, Sports and Culture of Japan, and by Special Coordination Funds for promoting Science and Technology (to A. Nagafuchi) from the Science and Technology Agency of Japan.
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Abbreviations used in this paper |
---|
AJ, adherens junctions; TJ, tight junction.
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