* Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan; and Department of Urology,
Faculty of Medicine, Kyoto University, Kyoto 606, Japan
ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large
(dlg)-like domain and a carboxyl-terminal half domain,
is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half
(N-ZO-1), and carboxyl-terminal half (C-ZO-1) into
mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1
were concentrated at cadherin-based cell-cell adhesion
sites. In good agreement with these observations,
N-ZO-1 was specifically coimmunoprecipitated from
EL transfectants expressing N-ZO-1 (NZ-EL cells)
with the E-cadherin/,
catenin complex. In contrast,
C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus
infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant
N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with
catenin, but not to that with
catenin or the cytoplasmic domain of E-cadherin. The
dissociation constant between N-ZO-1 and
catenin
was ~0.5 nM. On the other hand, recombinant C-ZO-1
was specifically cosedimented with actin filaments in
vitro with a dissociation constant of ~10 nM. Finally,
we compared the cadherin-based cell adhesion activity
of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among
these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We
conclude that in nonepithelial cells, ZO-1 works as a
cross-linker between cadherin/catenin complex and the
actin-based cytoskeleton through direct interaction
with
catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a
functional component in the cadherin-based cell adhesion system.
ZO-1 is a peripheral membrane protein with a molecular mass of 220 kD that was first identified as a
component of tight junctions (TJ) of epithelial and
endothelial cells (Stevenson et al., 1986 We developed an isolation procedure for cell-to-cell adherens junctions (AJ) from rat liver (Tsukita and Tsukita,
1989 TJ is an element of epithelial and endothelial junctional
complexes and functions as a primary barrier to the diffusion of solutes through the paracellular pathway (Schneeberger and Lynch, 1992 AJ is a specialized region of the plasma membrane,
where cadherin molecules (uvomorulin, L-CAM, A-CAM,
etc.) function as adhesion molecules and actin filaments
are densely associated with the plasma membrane through
a well-developed plasmalemmal undercoat (Farquhar and
Palade, 1963 Our knowledge is still limited regarding the molecular
basis of the association of ZO-1 with the cadherin adhesion system in nonepithelial cells and why this association
is not observed in epithelial cells (Itoh et al., 1993 Antibodies and Cells
Mouse anti-ZO-1 mAbs, T8-754 and T7-713, and rat anti- Sf9 cells were obtained from InVitrogen (San Diego, CA) and cultured
in TC-100 medium (GIBCO BRL, Gaithersburg, MD) supplemented with
tryptose phosphate broth and 10% FCS at 27°C. Mouse L cells and E-cadherin-transfected L cells (EL cells; clone EL Constructs and Transfections
To express a protein carrying seven c-myc epitopes (EQKLISEEDL) in
tandem at its carboxyl end, a tandem array of seven copies of an oligonucleotide encoding the c-myc epitope was ligated into the expression plasmid pME18S producing pME18S-7myc. The tandem c-myc tag and
pME18S were provided by Drs. K. Green (Northwestern University, Chicago, IL) and K. Maruyama (Tokyo Medical and Dental University, Tokyo, Japan), respectively. The cDNA fragment containing the entire open
reading frame of mouse ZO-1 (
EL cells (1 × 107 cells) were cotransfected with 2 µg of each expression
vector and 0.1 µg of pSV2bsr (Funakoshi Pharmaceutical Co., Tokyo, Japan) using Lipofectamine reagent (GIBCO BRL) for 5 h in Opti-MEM
(GIBCO BRL), followed by addition of 3 ml DME supplemented with
10% FCS. After incubation for 43 h, cells were replated onto four 9-cm
dishes in the presence of 2 µg/ml of blasticidin-S to select stable transfectants. Colonies of blasticidin-resistant cells were isolated and screened by
immunoblotting with anti-c-myc tag mAb. The positive clones were recloned. Clones expressing full-length ZO-1, N-ZO-1, or C-ZO-1 were designated as FZ-EL, NZ-EL, or CZ-EL cells, respectively. Three independent clones were isolated in each transfection (e.g., FZ-EL-1, FZ-EL-2,
and FZ-EL-3) and examined. Since these three independent clones gave the same results, we will mainly discuss FZ-EL-1, NZ-EL-1, and CZ-EL-1
cells in this paper. Furthermore, we performed analyses using the Immunofluorescence Microscopy
Cells plated on glass coverslips were rinsed in PBS and fixed with 1%
formaldehyde in PBS for 15 min at room temperature. The fixed cells
were treated with 0.3% Triton X-100 in PBS for 15 min and washed three
times with PBS. After soaking in PBS containing 1% BSA, the samples
were treated with primary antibodies for 1 h in a moist chamber. They
were then washed three times with PBS, followed by incubation for 30 min
with FITC-conjugated sheep anti-mouse IgG and/or rhodamine-conjugated donkey anti-rabbit IgG (Chemicon, Temecula, CA). In some experiments, rhodamine phalloidin (Molecular Probes, Inc., Eugene OR) was
added to detect F-actin. The samples were washed with PBS three times,
embedded in 95% glycerol-PBS containing 0.1% para-phenylendiamine
and 1% n-propylgalate, and examined with a photomicroscope (model
Axiophot; Carl Zeiss, Jena, Germany).
Immunoprecipitation
Confluent monolayers of cultured EL or NZ-EL cells on 9-cm dishes were
washed three times with ice-cold HMF buffer (HCMF [Ca2+/Mg2+-free
Hepes-buffered saline, pH 7.4] containing 2 mM CaCl2) and then lysed in
2 ml of extraction buffer (0.5% NP-40, 2 mM PMSF, 2 µg/ml leupeptin in
HMF). The cell lysate clarified by centrifugation at 100,000 g for 30 min
was incubated with 100 µl of protein G-Sepharose bead slurry (Zymed
Laboratories, Inc., San Francisco, CA) coupled with rat anti-E-cadherin
mAb, ECCD-2, or mouse anti-myc epitope tag mAb, 9E10, for 3 h at 4°C.
For control, rat or mouse IgG was used. After five washes with the extraction buffer, immunoprecipitates were eluted with 500 µl of 1 M acetic acid
twice. These acid eluates were combined, lyophilized, and resolved in SDS
sample buffer.
Production of the Amino- and Carboxyl-terminal
Halves of ZO-1 by Recombinant Baculovirus Infection
The EcoRI/SalI-digested fragments from pBFZO-1, pBNZO-1, and pBCZO-1 were integrated into the baculovirus genome (Hirao et al., 1996 In Vitro Binding Assay Using GST Fusion Proteins
The cDNA encoding the full length of the cytoplasmic domain of mouse
E-cadherin (2,268-2,720 bp) with BamHI and EcoRI sites at its ends was
produced by PCR using pBATEM2 (Nose et al., 1988 GST fusion proteins were expressed in E. coli and purified using glutathione-Sepharose 4B beads (Pharmacia LKB Biotechnology) as previously described (Furuse et al., 1994 To estimate the dissociation constant between N-ZO-1 and
Cosedimentation Experiments with Actin Filaments
Actin was purified from rabbit skeletal muscle according to the method
of Pardee and Spudich (1982) To estimate the dissociation constant between C-ZO-1 and actin filaments, 100 µl of the actin filament solution was incubated with 100 µl of
Sf9 cell lysates containing 0.2-2 µg of C-ZO-1 for 30 min at room temperature. After centrifugation, the pellet was resolved by SDS-PAGE (see
Fig. 6). The amount of cosedimented C-ZO-1 was estimated densitrometrically by comparing the intensity of its Coomassie brilliant blue-stained
band with those of various amounts of BSA using Adobe PhotoshopTM
3.0J histogram. A Scatchard plot of the data was then generated. Experiments were repeated three times for each estimation of Kd.
Cell Aggregation and Intercellular Motility Assays
Cell aggregation assay was performed basically according to the method
developed by Takeichi (1977). Cells were treated with 0.1% trypsin in the
presence of 2 mM CaCl2 at 37°C for 20 min and then washed with HCMF
to obtain single-cell suspensions. Cells suspended in DME medium were
placed in 1% agar-coated four-well plates and rotated at 37°C on a gyratory shaker at 80 rpm for 30 min, 2 h, or 6 h.
For the intercellular motility assay, cells were labeled with DiI according to the method developed by Honig and Hume (1986) Gel Electrophoresis and Immunoblotting
One-dimensional SDS-PAGE (10-12.5% gel) was based on the method of
Laemmli (1970) Behavior of Amino-terminal and Carboxyl-terminal
Halves of ZO-1 in EL Cells
As shown previously (Itoh et al., 1993
Association of the Amino-terminal Half of ZO-1 with
the Cadherin/Catenin Complex In Vivo
The transfection results suggested that endogenous ZO-1
is concentrated at cell-cell contact sites through the direct
or indirect association of its amino-terminal dlg-like domain with the cadherin/catenin complex. The interaction
of ZO-1 with the cadherin/catenin complex in vivo was
then examined by immunoprecipitation. The E-cadherin immunoprecipitates from the 0.5% NP-40 lysate of EL
cells contained
Direct Binding of the Amino-terminal Half of ZO-1 with
Immunoprecipitation experiments suggested that the cytoplasmic domain of E-cadherin, Next, using this in vitro binding assay, we estimated the
dissociation constant between N-ZO-1 and Direct Binding of the Carboxyl-terminal Half of ZO-1
with Actin Filaments
Immunofluorescence microscopy revealed that C-ZO-1
was concentrated along stress fibers. We then examined the
binding affinity of C-ZO-1 with actin filaments in vitro.
When actin filaments were incubated with the cell lysate of
Sf9 cells expressing C-ZO-1, only the recombinant C-ZO-1
in the lysate was selectively cosedimented with actin filaments (Fig. 6 a). In control experiments, BSA was used instead of actin, and no significant amount of C-ZO-1 was sedimented. Then, using this cosedimentation system, we
determined the dissociation constant between C-ZO-1 and
actin filaments. For this purpose, the interaction of C-ZO-1
with actin filaments was examined by incubating various
concentrations of C-ZO-1 with actin filaments or BSA (for
control). The dissociation constant was estimated by subtracting the amount of pelleted C-ZO-1 in the presence of
BSA. The binding was saturable, and Scatchard analysis
revealed a single class of affinity binding sites with a Kd of
~10 nM (Fig. 6 b).
Dominant-negative Effects of the Amino-terminal Half
of ZO-1 on the Cadherin-based Cell Adhesion System
If in EL cells the endogenous ZO-1 was functionally involved in the cadherin-based cell adhesion, some dominant-negative effect of N-ZO-1 in cell adhesion was expected
in NZ-EL cells. Conventional cell aggregation assay (Takeichi, 1977
To detect the qualitative changes in cadherin-based cell
adhesion, the intercellular motility assay was used (Nagafuchi et al., 1994
Cadherins are responsible for Ca2+-dependent cell-cell
adhesion (Gumbiner et al., 1988 In nonepithelial cells lacking TJ, ZO-1 is exclusively
concentrated at cell-cell adhesion sites, where the functioning cadherins are intimately associated with the actin-based cytoskeletons (Itoh et al., 1991 In fully polarized epithelial cells bearing TJ, ZO-1 is not
localized at AJ but at TJ. At the initial phase of polarization of these epithelial cells, however, small spotlike AJ
are formed, where ZO-1 is coconcentrated with E-cadherin (Yonemura et al., 1995 ZO-1 belongs to the MAGUK family, members of which
are characterized by PDZ (DHR) domains. Although the
physiological relevance of this domain remains unclear,
PSD95, one of the MAGUK family members, was reported to facilitate the lateral aggregation of its membrane
binding partners, such as the NMDA receptor and the K+
channel (Kim et al., 1995 Recently, ZO-1 was reported to be localized in the nucleus, suggesting its possible involvement in intracellular
signaling (Gottardi et al., 1996 Considering that ZO-1 works as a cross-linker and that
N-ZO-1 is concentrated at cadherin-based cell adhesion
sites, some dominant-negative effect of N-ZO-1 in the cadherin-based cell adhesion is expected. Various deletion
mutants of ZO-1 have been introduced into various types
of cells, but no obvious dominant-negative effects on cadherin-based cell adhesion were observed (Fanning, A.S.,
B.T. Jameson, and J.M. Anderson. 1996. Mol. Biol. Cell. 7:
607a; Itoh, M., and Sh. Tsukita, unpublished data). This
may be partly explained by the occurrence of some functionally redundant ZO-1-like proteins. ZO-2 is one candidate for such proteins, and L cells (and EL cells) do not
express ZO-2. Furthermore, the lack of occludin in EL
cells is also advantageous for analysis and interpretation of
the possible dominant-negative effects of truncated ZO-1
molecules. In terms of the conventional cell aggregation activity, however, in EL cells N-ZO-1 did not show any detectable dominant-negative effects. To evaluate dynamic
aspects of cadherin-based cell adhesion, we previously developed an assay system for intercellular motility, i.e., the
cell movement in a confluent sheet, and reported that the
expression of E-cadherin in L cells facilitates intercellular motility (Nagafuchi et al., 1994; Anderson et al.,
1988
). As a ZO-1-binding protein, another peripheral membrane protein called ZO-2 with a molecular mass of
160 kD has been identified (Gumbiner et al., 1991
). Sequence analysis of the cDNAs encoding mammalian ZO-1
and ZO-2 revealed that both show similarity to the product of lethal (1) discs large-1 (dlg),1 one of the tumor suppressor molecules in Drosophila (Itoh et al., 1993
; Tsukita
et al., 1993
; Willott et al., 1993
; Jesaitis and Goodenough,
1994
), and that there are at least two isotypes of ZO-1 generated by alternative splicing (
+ and
) (Willot et al.,
1992). Recently, in addition to these proteins, many dlg-like proteins have been identified, indicating the existence of a novel gene family named membrane-associated guanylate kinase homologues (MAGUKs) (Woods and Bryant,
1993
; Kim, 1995
; Anderson, 1995
).
). Using this fraction, we identified a 220-kD peripheral membrane protein that was highly concentrated at AJ
of cardiac muscle cells (intercalated discs) and cultured fibroblasts (Itoh et al., 1991
), suggesting that this 220-kD
protein is involved in some function of AJ. Cloning of its
cDNA, however, revealed that this protein is identical to
ZO-1, indicating that ZO-1 is concentrated not only at TJ
in epithelial and endothelial cells but also at AJ in cardiac
muscle and fibroblastic cells (Itoh et al., 1993
). Similar observations were also reported by other laboratories (Jesaitis and Goodenough, 1994
).
; Gumbiner, 1987
, 1993
) as well as
a fence between the apical and basolateral plasma membrane domains to create and maintain their polarity (Rodriguez-Boulan and Nelson, 1989
). ZO-1 and ZO-2 are
thought to constitute the undercoat structure of TJ together with other peripheral membrane proteins such as
cingulin, 7H6 antigen, and symplekin (Citi et al., 1988
;
Zhong et al., 1993
; Keon et al., 1996
). An integral membrane protein localized at TJ was recently identified and
named occludin (Furuse et al., 1993
; Ando-Akatsuka et al.,
1996
). Occludin has four transmembrane domains in its
amino-terminal half and a long carboxyl-terminal cytoplasmic domain. ZO-1 is directly associated with the carboxyl-terminal 150 amino acids (aa) of occludin in TJ (Furuse et al., 1994
).
; Volk and Geiger, 1984
; Boller et al., 1985
;
Takeichi, 1988
, 1991
). This type of junction is important for the formation and maintenance of tissues. AJ appears
beltlike in simple epithelial cells and endothelial cells, but
appears spotlike in the intercalated discs of cardiac muscle
cells. Small spot-like AJ also occur in cultured fibroblasts
(Heaysman and Pegrum, 1973
; Yonemura et al., 1995
). At
least two cytoplasmic proteins,
and
catenins, are
tightly associated with the cytoplasmic domain of cadherins (Ozawa et al., 1989
, 1990
; Nagafuchi and Takeichi, 1989
). Those catenins show a similarity to vinculin and the
Drosophila armadillo gene product, respectively (Herrenknecht et al., 1991
; Nagafuchi et al., 1991
; McCrea et al.,
1991
), and constitute the undercoat of AJ together with
other cytoplasmic proteins such as vinculin and
-actinin.
As described above, only in nonepithelial cells, ZO-1 is
also concentrated in the undercoat of AJ.
). To examine this issue, mouse L cell transfectants expressing exogenous E-cadherin (EL cells; Nagafuchi et al., 1987
) provide an advantageous experimental system. They express
neither endogenous cadherins nor occludin and show strong
cell adhesion activity only through the exogenous E-cadherin. They express endogenous
and
catenin, which
form a stable complex with E-cadherin (Nagafuchi et al.,
1991
). Furthermore, they express endogenous ZO-1 but
not ZO-2. In this study, we analyzed the behavior of introduced exogenous full-length, amino-terminal half and carboxyl-terminal half ZO-1 molecules in EL cells and also
examined the binding abilities of these molecules to E-cadherin,
catenin,
catenin, and actin filaments in vitro.
These analyses led us to conclude that ZO-1 is involved in
the cadherin-based cell adhesion system through its direct
binding to
catenin and actin filaments.
Materials and Methods
catenin mAb,
18, have been described (Itoh et al., 1991
, 1993
; Nagafuchi et al., 1991
).
T8-754 recognizes the amino-terminal half of ZO-1, while T7-713 is specific for its carboxyl-terminal half. Rabbit anti-E-cadherin pAb and rat
anti-E-cadherin mAb, ECCD-2, were provided by Dr. M. Takeichi (Kyoto University, Kyoto, Japan). Mouse anti-
catenin mAb and mouse
anti-c-myc tag mAb, 9E10, were purchased from Transduction Labs (Lexington, KY), and Sigma Chemical Co. (St. Louis, MO), respectively.
1) were provided by Dr. M. Takeichi and cultured in DME supplemented with 10% FCS (Nose et al.,
1988
).
+ isotype) was produced by PCR using
clone 220-1 as a template (Itoh et al., 1993
). The first half of this fragment
was amplified using the sense primer from position 404 containing an
EcoRI site (5
-CAGAATTCATGTCCGCCAGGGCCGCG-3
) and the
antisense primer from position 2686 (5
-AAGCTTATGAGAGCGTTC-3
), which contained an internal HindIII site. The second half was amplified using the sense primer from position 2681 (5
-AAGCTTCTGAAGAACAAT-3
) and the antisense primer from position 5638 containing a
SalI site (5-CCGTCGACAAAGTGGTCAATCAGGAC-3
). The first
half was digested with EcoRI and HindIII, the second half was digested
with HindIII and SalI, and then both fragments were ligated. This single
recombinant fragment was subcloned into the EcoRI/SalI sites of pBluescript SK(
) (designated as pBFZO-1), and then the EcoRI/SalI-digested
fragment was inserted into pME18S-7myc (Fig. 1). The cDNA fragments
encoding the ZO-1 amino-terminal half dlg-like (N-ZO-1;1-862 aa) and
carboxyl-terminal half (C-ZO-1;863-1745 aa) domains were also produced by PCR using clone 220-1 as a template (Itoh et al., 1993
). As primers, 5
-CAGAATTCATGTCCGCCAGGGCCGCG-3
(sense for amino-terminal half), 5
-CCGTCGACAAGAGTTTCATCTAGTTC-3
(antisense
for amino-terminal half), 5
-CCGAATTCAATGATGAGGTGGGGACT-3
(sense for carboxyl-terminal half), and 5
-CCGTCGACAAAGTGGTCAATCAGGAC-3
(antisense for carboxyl-terminal half) were
used. Each amplified fragment was subcloned into EcoRI/SalI sites of
pBluescript SK(
) (designated as pBNZO1 and pBCZO1, respectively)
and then into pME18S-7myc (Fig. 1).
Fig. 1.
Structure of c-myc-
tagged full-length ZO-1 (F-ZO-1), dlg-like amino-terminal half
ZO-1 (N-ZO-1), and carboxyl-terminal half ZO-1 (C-ZO-1). A
tandem array of seven c-myc
epitopes (myc; EQKLISEEDL) was tagged to the carboxyl terminus of each molecule. F-ZO-1
contains three PDZ domains
(PDZ1-3), one SH3 domain
(SH3), one guanylate kinase domain (GUK), and a proline-rich
domain (proline-rich). The +
isotype with an insert (
) was
used in this study.
[View Larger Version of this Image (13K GIF file)]
isotype of ZO-1 and obtained the same results (data not shown).
). The
recombinant virus containing each cDNA was isolated and condensed using a MAXBAC kit (InVitrogen). Insect Sf9 cells were infected with recombinant viruses and cultured in 100 ml spinner flasks at 27°C for 72 h.
Cells were collected and washed with PBS. After suspension in PBS containing 0.1% Triton X-100, 2 mM PMSF, and 4 µg/ml of leupeptin, the
cells were homogenized in a tight-fitting Dounce homogenizer, sonicated,
and clarified by centrifugation at 100,000 g for 1 h at 4°C. Immunoblotting revealed that the level of expression of the full-length ZO-1 in Sf9 cells
was too low to be used for in vitro binding assay with glutathione-S-transferase (GST) fusion proteins or actin filaments. Therefore, we used only
recombinant N-ZO-1 and C-ZO-1, which were abundantly expressed in
Sf9 cells, for the in vitro binding analyses.
) as the template
and subcloned into pBluescript SK(
). The insert was then subcloned into
the pGEX2T expression vector (Pharmacia LKB Biotechnology, Uppsala,
Sweden), which was designed to express a GST fusion protein in Escherichia coli. The cDNAs containing the entire coding regions of mouse
or
catenin with BamHI and EcoRI sites at the ends were produced by PCR
using PSK102B (Nagafuchi and Tsukita, 1994
) or pM
cat (Nagafuchi et al.,
1994
), respectively, as the template and subcloned into pBluescript
SK(
). The inserts were then subcloned into the pGEX2T expression
vector. The GST fusion protein with the cytoplasmic domain of occludin
(358-504 aa; GST-Oc358) was previously described (Furuse et al., 1994
).
). After the 200 µl of glutathione-Sepharose bead slurry containing GST fusion proteins was washed with
100 vol of PBS containing 0.1% Triton X-100, 2 mM PMSF, and 4 µg/ml
of leupeptin by brief centrifugation, 2 ml of the cell lysate of Sf9 cells expressing N-ZO-1 or C-ZO-1 was added, followed by incubation for 3 h at
4°C. The beads were washed with 40 vol of the same solution, 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. An appropriate amount of
each eluate was again applied to SDS-PAGE to contain the same amount of GST fusion proteins, and the amount of N-ZO-1 or C-ZO-1 in each eluate was evaluated by CBB staining and/or immunoblotting.
catenin,
200 µl of the glutathione-Sepharose bead slurry containing 40 µg of GST-
catenin was incubated with 2 ml of the Sf9 cell lysate containing 0.012-0.6 µg
of N-ZO-1. The beads were washed, and bound proteins were eluted with
1 ml of 50 mM Tris-HCl buffer, pH 8.0, containing 10 mM glutathione.
The amounts of N-ZO-1 in the cell lysate or in each eluate were estimated
as follows: N-ZO-1 was precipitated out from the Sf9 cell lysate using
GST-occludin (GST-Oc358) as shown in Fig. 5 a. This was tentatively
called `purified' N-ZO-1, and the amount of this purified N-ZO-1 was determined densitrometrically by comparing the intensity of its band after
Coomassie brilliant blue staining with those of various amounts of BSA
using Adobe PhotoshopTM 3.0J histogram (San Jose, CA). We then compared the intensity of immunoblotted N-ZO-1 bands in cell lysates or in
eluates with those of immunoblotted bands of various amounts of purified
N-ZO-1 resolved and immunoblotted in the same gel. The intensity of the immunoblotted band was also measured by densitrometry using Adobe PhotoshopTM 3.0J histogram, and a Scatchard plot of the data was generated. Experiments were repeated three times for each estimation of Kd.
Fig. 5.
Association of N-ZO-1 with catenin in vitro. (a) GST fusion proteins with the cytoplasmic domain of occludin, the cytoplasmic domain of E-cadherin,
catenin, and
catenin, which were bound to
glutathione-Sepharose beads, were incubated with the lysate of Sf9
cells expressing N-ZO-1. After washing, the proteins associated with
GST fusion proteins were eluted from the beads with a buffer containing glutathione. The Sf9 cell lysate (Sf9 lysate), the eluate from GST-
occludin beads (GST/Occ-eluate), the eluate from GST-E-cadherin
beads (GST/Cad-eluate), the eluate from GST-
catenin beads (GST/
cat-eluate), the eluate from GST-
catenin beads (GST/
cat-eluate),
and purified GST-
catenin (GST/
cat) were separated by SDS-PAGE followed by immunoblotting with anti-ZO-1 mAb, T8-754. Although the N-ZO-1 band was not resolved from the GST-
catenin
band by CBB staining, immunoblotting identified N-ZO-1 (arrowhead) in the eluate from GST-
catenin beads at the same amount as
in that from GST-occludin beads (arrow), in which the N-ZO-1 band
was clearly visualized by CBB staining (asterisk). N-ZO-1 was not detected in the eluates from GST-E-cadherin or GST-
catenin beads.
Comparison of the CBB staining patterns between the GST-
catenin
beads eluate (GST/
cat-eluate) and the purified GST-
catenin
(GST/
cat) revealed that only N-ZO-1 was precipitated out from the
crude Sf9 lysate by GST-
catenin, indicating the direct
catenin/N-ZO-1 interaction. 1, 2, 3, and 4 indicate the bands of GST-occludin, GST-E-cadherin, GST-
catenin, and GST-
catenin, respectively,
and the other lower molecular mass bands are their degradation products. (b) GST fusion protein with
catenin, which was bound by
glutathione-Sepharose beads, was incubated with the lysate of Sf9 cells expressing C-ZO-1. After washing, the proteins associated with
the GST fusion protein were eluted from the beads with a buffer containing glutathione. The Sf9 cell lysate (Sf9-lysate) and the eluate
from GST-
-catenin beads (GST/
cat-eluate) were separated by SDS-PAGE followed by immunoblotting with anti-C-ZO-1 mAb, T7-713. 5, the band of GST-
catenin. No specific binding was detected between GST-
-catenin and C-ZO-1 (arrow). (c) Quantitative analysis of the binding between N-ZO-1 and
catenin. Glutathione-Sepharose bead slurry containing 40 µg of GST-
catenin was incubated with the Sf9 cell lysate containing 0.012-0.6 µg of N-ZO-1. The amounts of N-ZO-1 in the Sf9 cell lysate and in each eluate (inset)
were estimated as described in Materials and Methods. Each point represents the mean ± SD of triplicate determinations. The binding
was saturable, and Scatchard analysis (inset) indicated that the Kd was ~0.5 nM.
[View Larger Versions of these Images (64 + 32 + 23K GIF file)]
and further purified by gel filtration on a
Sephacryl S-200 column (Pharmacia LKB Biotechnology). Gel-filtered
G-actin was stored in buffer A (2 mM Tris-HCl, pH 8.0, 0.2 mM ATP,
0.5 mM DTT, 0.2% NaN3) and diluted at room temperature to 2-3 µM
with buffer F (20 mM Tris-HCl, pH 7.4, 75 mM KCl, 10 mM NaCl, 2 mM
DTT, 2.5 mM MgCl2) to initiate polymerization. The cell lysate of Sf9 cells
expressing C-ZO-1 was dialyzed against buffer F at 4°C overnight and
centrifuged at 100,000 g for 30 min. After actin filaments were polymerized for 30 min, 100 µl of the supernatant was added to 100 µl of the actin
filament solution and incubated for 30 min at room temperature. After
centrifugation at 100,000 g for 30 min at 20°C, the supernatant and pellet
were resolved by SDS-PAGE, followed by immunoblotting with anti-ZO-1 mAb.
Fig. 6.
Association of C-ZO-1 with actin filaments in vitro. (a)
Sf9 cell lysate containing C-ZO-1 (Sf9-lysate) was incubated with actin filaments (+Actin) or BSA (+BSA). After centrifugation, the supernatant (S) and pellet (P) were resolved by SDS-PAGE, followed by immunoblotting with anti-ZO-1 mAb, T7-713. From
crude Sf9 cell lysate, only the C-ZO-1 band was precipitated out
by actin filaments (arrows), but not by BSA. Arrowheads indicate actin or BSA. (b) Quantitative analysis of the binding between C-ZO-1 and actin filaments. Actin filaments were incubated with the Sf9 cell lysate containing 0.2-2 µg of C-ZO-1. The
amounts of C-ZO-1 in the Sf9 cell lysate and in each pellet (inset)
were estimated as described in Materials and Methods. Each
point represents the mean ± SD of triplicate determinations. The
binding was saturable, and Scatchard analysis (inset) indicated that
the Kd was ~10 nM.
[View Larger Versions of these Images (55 + 26K GIF file)]
and treated with
trypsin. Aliquots of 1 × 103 labeled cells were seeded on 6-cm dishes with
a monolayer of 2 × 106 cells. After 48 h of culture, four sister cells that
seemed to be derived from one seeded cell were examined by fluorescence microscopy. When cell line A was seeded on confluent cultures of
cell line B, we designate the experiment as A/B analysis. For quantification of intercellular motility, intercellular distances of all combinations between four cells were measured and summed as Dc. As a control experiment, labeled cells were seeded on the dishes in the absence of a cell
sheet. In this case, the intercellular distances were summed as Dd. The degree of intercellular motility was represented as Dc/Dd. At least 24 independent samples were picked up to determine Dc or Dd for each transfectant cell line.
. Gels were stained with Coomassie brilliant blue R-250 or
with a silver staining kit (Wako Pure Chemical Industries, Osaka, Japan).
For immunoblotting, proteins separated by SDS-PAGE were electrophoretically transferred onto nitrocellulose sheets, which were then incubated with primary antibodies. The antibodies were detected with a blotting detection kit (Amersham Corp., Arlington Heights, IL).
Results
), endogenous ZO-1
is concentrated at the cadherin-based cell adhesion sites in
EL cells (L cells expressing exogenous E-cadherin) (Fig. 2,
a and b). When the myc-tagged full-length mouse ZO-1
was introduced into EL cells, it was also concentrated at
the cadherin-based cell adhesion sites in a serrated pattern
(Figs. 1 and 2, c and d). Then, to narrow down the region
responsible for this characteristic distribution, we divided
the ZO-1 molecule into two parts: the amino-terminal half
dlg-like domain (N-ZO-1) and the carboxyl-terminal half
domain (C-ZO-1) (see Fig. 1). After the myc tag was fused
with their carboxyl-end, they were transfected into EL cells.
As shown in Fig. 3, a, b, and b
, the introduced N-ZO-1
was concentrated at cell-cell contact sites together with
E-cadherin. In these transfectants (NZ-EL), N-ZO-1 and
E-cadherin showed a tendency to concentrate in a linear
fashion, suggesting a dominant-negative effect of N-ZO-1
in the cadherin-based junction distribution. However, since depending on the culture conditions they showed a serrated concentration pattern (Fig. 3 b, inset), it was difficult
to conclusively evaluate the dominant-negative effect in
this transfection system. In contrast, C-ZO-1 was distributed along the stress fibers (Fig. 3, c, d, and d
). Of course,
the possibility was not excluded that C-ZO-1 was also
present at the serrated points of cell contact in these transfectants (CZ-EL). N-ZO-1 and C-ZO-1 showed the same
behavior when transfected into the rat fibroblast cell line,
3Y1 cells (data not shown).
Fig. 2.
Concentration of full-length ZO-1 at cadherin-based cell adhesion sites. When parent EL cells were doubly stained with anti- ZO-1 mAb, T8-754 (a), and anti-E-cadherin pAb (b), both signals were colocalized at the cell-cell border. Introduced myc-tagged full-length ZO-1 was similarly distributed in EL transfectants (FZ-EL cells) as revealed by anti-c-myc tag mAb, 9E10 (c). This mAb gave a
signal in nuclei but not in cell-cell borders of parent EL cells (d). Bar, 10 µm.
[View Larger Version of this Image (126K GIF file)]
Fig. 3.
Subcellular distribution of N-ZO-1 and C-ZO-1 in EL transfectants (NZ-EL and CZ-EL, respectively). NZ-EL cells (a, b, and b) were singly stained with anti-c-myc tag mAb, 9E10 (a), or doubly stained with 9E10 (b) and anti-E-cadherin pAb (b
). In addition to
the diffuse cytoplasmic distribution, introduced N-ZO-1 was significantly concentrated at the cell-cell border, where E-cadherin was colocalized. N-ZO-1 and E-cadherin showed a tendency to distribute in a linear fashion, but depending on the culture conditions they
showed a serrated concentration pattern (inset, 3,000×). CZ-EL cells (c, d, and d
) were singly stained with anti-c-myc tag mAb, 9E10
(c), or doubly stained with 9E10 (d) and rhodamine phalloidin (d
). Introduced C-ZO-1 was distributed along actin stress fibers visualized with rhodamine phalloidin. Bars: (a-c) 10 µm; (d and d
) 20 µm.
[View Larger Version of this Image (153K GIF file)]
and
catenins as major components,
whereas ZO-1 was hardly detected even by immunoblotting (Fig. 4 a). Considering that most of endogenous ZO-1
is resistant against detergent extraction (Itoh et al., 1991
),
it is expected that the cadherin/catenin complex-associated ZO-1 was not extracted by 0.5% NP-40. Then, E-cadherin was immunoprecipitated from NZ-EL cells. As
shown in Fig. 4 a, in the E-cadherin immunoprecipitate containing
and
catenins, N-ZO-1 was detected by immunoblotting. Furthermore, when the introduced N-ZO-1
was immunoprecipitated with anti-myc mAb, E-cadherin,
catenin, and
catenin were coimmunoprecipitated (Fig.
4 b). By contrast, when E-cadherin was immunoprecipitated from CZ-EL cells, C-ZO-1 was not detected in the
precipitated cadherin/catenin complex (data not shown).
We then concluded that N-ZO-1 (and probably endogenous ZO-1) was concentrated at cadherin-based cell adhesion sites in EL cells through its interaction with the cadherin/catenin complex in vivo.
Fig. 4.
Association of
N-ZO-1 with the E-cadherin/
catenin complex in NZ-EL
cells. (a) Silver staining pattern of E-cadherin immunoprecipitates from EL or NZ-EL cells and accompanying
immunoblots with anti-ZO-1 mAb, T8-754, specific for the
amino-terminal half of ZO-1.
When E-cadherin (Cad) was
immunoprecipitated with
rat anti-E-cadherin mAb,
ECCD-2 (anti-cad), from the
NP-40 extract of EL or NZ-EL cells, and
catenins
were coimmunoprecipitated.
(
and
, respectively). In
the E-cadherin immunoprecipitates from NZ-EL cells,
catenin appeared to be degraded to some extent. Rat
IgG (r-IgG) was used for
control immunoprecipitation.
Asterisks indicate IgG. Immunoblotting with anti-ZO-1
mAb detected N-ZO-1 (arrowhead) in the E-cadherin immunoprecipitate from NZ-EL
cells but did not detect endogenous full-length ZO-1 in the E-cadherin immunoprecipitate from either NZ-EL or EL cells. (b) Immunoprecipitation with anti-c-myc tag mAb, 9E10 (anti-myc), from the NP-40 extract of NZ-EL cells. Silver staining detected the N-ZO-1
band (arrow) in addition to IgG bands (asterisks), and immunoblotting identified E-cadherin,
catenin, and
catenin in the immunoprecipitate. Mouse IgG (m-IgG) was used for control immunoprecipitation.
[View Larger Version of this Image (52K GIF file)]
Catenin In Vitro
catenin, or
catenin directly binds to the amino-terminal half of ZO-1. To determine the binding partner for ZO-1 in the cadherin/catenin
complex, we produced GST fusion proteins with the cytoplasmic domain of E-cadherin,
catenin, and
catenin
and performed in vitro binding assays with the recombinant N-ZO-1 produced in Sf9 cells by baculovirus infection (Fig. 5 a). In this assay, the electrophoretic mobility of
recombinant N-ZO-1 was almost the same as those of
GST-
catenin and GST-
catenin fusion proteins, making it difficult to estimate the bound N-ZO-1 by CBB
staining. Thus, to estimate the amount of N-ZO-1 by immunoblotting, N-ZO-1 was first purified from the crude
lysate of Sf9 cells using the GST/cytoplasmic domain of occludin, as previously reported (Furuse et al., 1994
). By comparison with the amount of this purified N-ZO-1 determined by CBB staining and immunoblotting, we estimated
the amounts of
catenin- or
catenin-bound N-ZO-1, if
any. As shown in Fig. 5 a, N-ZO-1 was directly and specifically bound to GST-
catenin with similar affinity to the
cytoplasmic domain of occludin, but not to the cytoplasmic
domain of E-cadherin or to
catenin. Using the same system, we checked the possible interaction of recombinant
C-ZO-1 with GST fusion proteins with the cytoplasmic domain of E-cadherin,
catenin, and
catenin, but no specific binding was detected (Fig. 5 b).
catenin. For
this purpose, the interaction of N-ZO-1 with
catenin was
examined by incubating various concentrations of N-ZO-1
with beads bearing GST-
catenin fusion protein or GST
(for control). The dissociation constant was estimated by
subtracting the binding to the GST-conjugated Sepharose
beads. The binding was saturable, and Scatchard analysis revealed a single class of affinity binding sites with a Kd of ~0.5 nM (Fig. 5 c).
) was then performed to compare the cadherin-dependent cell adhesion activity between EL and NZ-EL
cells. However, no significant difference was detected (Fig. 7).
Fig. 7.
Cell aggregation
assay of EL and NZ-EL cells.
Single cells were rotated for
30 min, 2 h, or 6 h on a gyratory shaker at 80 rpm. No significant differences were detected in cell aggregation activity between EL and NZ-EL cells. Bar, 50 µm.
[View Larger Version of this Image (126K GIF file)]
) (Fig. 8). In this assay, a single L, EL,
or NZ-EL cell labeled with a fluorescent dye (DiI) was
seeded onto a confluent culture of nonlabeled L, EL, or
NZ-EL cells, respectively, and after 48 h in culture (twice
the doubling time), the cell scattering of four labeled sister
cells was analyzed by measuring the mass distance among
these four cells. As shown previously (Nagafuchi et al.,
1994
), when the labeled EL cells were seeded on the EL cell sheet, the four sister cells were more scattered as compared to labeled L cells on the L cell sheet. In contrast,
when the labeled NZ-EL cells were seeded on the NZ-EL
cell sheet, the scattering of the labeled cells was significantly decreased. The intercellular motility of NZ-EL cells
was comparable to that of L cells on the L cell sheet. Fig. 8
b summarizes the quantitative results of the intercellular
motility assay, where the index was calculated as described
in Materials and Methods.
Fig. 8.
Intercellular motility of L, EL, and NZ-EL
cells. (a) In the intercellular
motility assay (on cell sheet),
the fluorescence microscopic
images of four DiI-stained
sister cells (white) were superimposed on the phase
contrast image of a confluent cell sheet. As compared
to EL/EL analysis (see Materials and Methods for details), the intercellular motility in the NZ-EL/NZ-EL
analysis was suppressed down to the level of that in L/
L analysis. These cell lines
showed similar scattering
properties on plastic dishes
in the absence of a cell sheet
(on dish). (b) Quantitative
analysis of the intercellular motility. Higher values on
the abscissa (intercellular
motility index [Dc/Dd]; see
Materials and Methods for
details) represent the higher
degrees of intercellular motility. At least 24 independent samples were picked up
to determine the intercellular
motility index for each analysis. Bar, 10 µm.
[View Larger Versions of these Images (99 + 13K GIF file)]
Discussion
; Takeichi, 1988
, 1991
;
Kemler, 1992
; Birchmeier, 1995
). One of the major characteristics of cadherins is that they interact intimately with
the actin-based cytoskeleton to exert their physiological
function, i.e., strong cell adhesion (Hirano et al., 1987
; Nagafuchi and Takeichi, 1988
). The identification of cytoplasmic proteins responsible for cadherin/actin interaction is thus required to better understand how the functions of
cadherin are regulated. E-cadherin has been immunoprecipitated from the detergent extracts of various cell types,
and in the immunoprecipitate at least two proteins, named
and
catenins, have been identified (Ozawa et al., 1989
;
Nagafuchi and Takeichi, 1989
). It is now accepted that the
cytoplasmic domain of cadherins directly binds to
catenin, which then binds to
catenin (Ozawa and Kemler, 1992
; Aberle et al., 1994
; Hinck et al., 1994
; Näthke et al., 1994
; Oyama et al., 1994
; Jou et al., 1995
). However, immunoprecipitation analysis along this line is expected to
face technical difficulties, since the functioning cadherins
that show strong cell adhesion activity are associated with
actin-based cytoskeletons and resist detergent extraction
(Nagafuchi and Takeichi, 1988
; Tsukita et al., 1992
).
, 1993
). Furthermore,
ZO-1 in these cells is highly resistant to detergent extraction,
suggesting that ZO-1 is a good candidate as a cross-linker
between the functioning cadherin/catenin complex and the
actin-based cytoskeleton. In this study, we divided the
ZO-1 molecule into the amino-terminal half (N-ZO-1)
and the carboxyl-terminal half (C-ZO-1). In EL cells expressing these truncated ZO-1 constructs (NZ-EL and
CZ-EL cells, respectively), N-ZO-1 was concentrated at
the cadherin-based cell-cell adhesion sites, whereas C-ZO-1
was localized along the actin stress fibers. From NZ-EL
cells, N-ZO-1, but not the endogenous full-length ZO-1, was coimmunoprecipitated with the cadherin/catenin complex. In this case, we circumvented the above technical difficulty in cadherin immunoprecipitation, probably because
N-ZO-1 lost the cross-linking activity while retaining its
binding activity to the cadherin/catenin complex. Actually,
recombinant N-ZO-1 and C-ZO-1 directly bound in vitro
to
catenin and actin filaments, respectively. The dissociation constants of N-ZO-1/
catenin and C-ZO-1/actin filaments in vitro were ~0.5 and ~10 nM, respectively. Both
of these values are small enough to postulate that binding
occurs in vivo. Taken together, we conclude that ZO-1
functions as a cross-linker between
catenin and actin filaments to insolubilize the cadherin/catenin complex in
nonepithelial cells. On the other hand,
catenin has been reported to bear at least two cytoskeleton-interacting domains (Nagafuchi et al., 1994
) and directly binds to actin
filaments and to
-actinin, an actin-binding protein (Knudsen et al., 1995
; Rimm et al., 1995
). The physiological significance of these possible multiple linkage pathways from
catenin to actin filaments remains elusive.
). It is then possible that also
in epithelial cells ZO-1 functions as a cross-linker between
catenin and the actin-based cytoskeleton at the initial
stage of the junction formation. In good agreement with
this,
and
catenins were reported to be coimmunoprecipitated with ZO-1 only at the initial stage of junction formation in Madin-Darby canine kidney (MDCK) cells, although cadherins were not identified in the immunoprecipitate (Rajasekaran et al., 1996
). During the maturation
process of beltlike AJ of fully polarized epithelial cells,
ZO-1 may be transferred from AJ to TJ through its direct
interaction with the cytoplasmic domain of occludin. Taking it into consideration that the dissociation constant between occludin and ZO-1 is ~1 nM (Itoh, M., and Sh. Tsukita, manuscript in preparation), the molecular mechanism
for this peculiar behavior of ZO-1 is one of the most interesting issues from the viewpoint of epithelial polarization.
; Kornau et al., 1995
; Niethammer
et al., 1996
). Thus, it is possible that ZO-1 is also involved
in the lateral aggregation of cadherin/catenin complexes in
nonepithelial cells as well as in epithelial cells at the initial
stage of junction formation. Furthermore, ZO-1 is possibly
involved in the lateral aggregation of occludin at TJ in
fully polarized epithelial cells.
). The sequence similarity of
ZO-1 to the dlg gene product, one of the tumor suppressors in Drosophila, also suggests this possibility. On the
other hand, evidence has accumulated that
catenin is directly involved in intracellular signaling to the nucleus
(Funayama et al., 1995
; Gumbiner, 1995
; Behrens et al.,
1996
; Huber et al., 1996
; Miller and Moon, 1996
). Furthermore, as discussed above,
catenin as well as
catenin were reported to be coimmunoprecipitated with ZO-1 in
MDCK cells at the initial phase of junction formation (Rajasekaran et al., 1996
). It is thus tempting to speculate that
catenin directly interacts with ZO-1. However, at least in
vitro, this direct interaction was not detected. Recently, we
introduced a fusion protein between nonfunctional E-cadherin and
catenin into L fibroblasts (Nagafuchi et al.,
1994
). This fusion molecule lacked the
catenin-binding site but functioned as an adhesion molecule with strong
adhesion activity at the cell-cell adhesion sites. ZO-1, but
not
catenin, was concentrated at these cell-cell adhesion
sites (Nagafuchi, A., and Sh. Tsukita, manuscript in preparation). This observation favors the notion that
catenin
is responsible for the concentration of ZO-1 at the cadherin-based cell adhesion sites. Of course, considering that
catenin appears to cross-link
catenin to N-ZO-1 in the
E-cadherin immunoprecipitate from NZ-EL cells, it is possible that
catenin interacts with ZO-1 indirectly via
catenin.
). Interestingly, N-ZO-1
suppressed this elevated intercellular motility of EL cells
down to the level of parent L cells. This indicates that the
cross-linking activity of ZO-1 between the cadherin/catenin complex and the actin-based cytoskeleton is functionally involved in cadherin-based cell adhesion, although detailed molecular mechanisms of this dominant-negative effect of N-ZO-1 in EL cells still remain elusive. Further
analyses, including the targeted disruption of ZO-1 gene,
will lead us to better understand the functional implications of ZO-1 in the cadherin-based cell adhesion system.
Received for publication 4 March 1997 and in revised form 30 April 1997.
1. Abbreviations used in this paper: aa, amino acid(s); AJ, adherens junctions; C-ZO-1 and N-ZO-1, carboxyl-terminal half ZO-1 and amino-terminal half dlg-like ZO-1; dlg, discs large; GST, glutathione-S-transferase; MAGUK, membrane-associated guanylate kinase homologues; TJ, tight junctions.We would like to thank all the members of our laboratory (Department of Cell Biology, Faculty of Medicine, Kyoto University) for their helpful discussions throughout this study. Our thanks are also due to Drs. M. Takeichi, K. Green, and K. Maruyama for their generous gift of anti- E-cadherin antibodies, the tandem c-myc tag, and the expression plasmid pME18S, respectively.
This work was supported in part by a Grant-in-Aid for Cancer Research and a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Science, and Culture of Japan (to Sh. Tsukita).
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