ª
Oncogene (2002) 21, 7642 – 7655
2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00
www.nature.com/onc
Involvement of nectin in the localization of junctional adhesion molecule at
tight junctions
Atsunori Fukuhara1, Kenji Irie1, Hiroyuki Nakanishi1, Kyoji Takekuni1, Tomomi Kawakatsu1,
Wataru Ikeda1, Akio Yamada1, Tatsuo Katata1, Tomoyuki Honda1, Tatsuhiro Sato1,
Kazuya Shimizu1, Harunobu Ozaki2, Hisanori Horiuchi2, Toru Kita2 and Yoshimi Takai*,1
1
Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita
565-0871, Japan; 2Department of Geriatric Medicine, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan
Junctional adhesion molecule (JAM) is a Ca2+-independent immunoglobulin-like cell – cell adhesion molecule
which localizes at tight junctions (TJs). Claudin is a key
cell – cell adhesion molecule that forms TJ strands at
TJs. JAM is associated with claudin through their
cytoplasmic tail-binding protein, ZO-1. JAM is furthermore associated with Par-3, a cell polarity protein which
forms a ternary complex with Par-6 and atypical protein
kinase C. Nectin is another Ca2+-independent immunoglobulin-like cell – cell adhesion molecule which
localizes at adherens junctions (AJs). Nectin is associated with E-cadherin through their respective
cytoplasmic tail-binding proteins, afadin and catenins,
and involved in the formation of AJs cooperatively with
E-cadherin. We show here that nectin is furthermore
involved in the localization of JAM at TJs. During the
formation of the junctional complex consisting of AJs
and TJs in Madin-Darby canine kidney (MDCK) cells,
JAM was recruited to the nectin-based cell – cell
adhesion sites. This recruitment of JAM was inhibited
by nectin inhibitors, which inhibited the trans-interaction
of nectin. Microbeads coated with the extracellular
fragment of nectin, that interacted with cellular nectin,
also recruited JAM to the bead – MDCK cell contact
sites. Furthermore, when cadherin-deficient L fibroblasts
stably expressing both exogenous JAM and nectin
(nectin-JAM-L cells) were co-cultured with L fibroblasts
expressing only nectin (nectin-L cells), JAM was
concentrated at the cell – cell adhesion sites between
nectin-JAM-L and nectin-L cells without the transinteraction of JAM. Analyses of the localization and
immunoprecipitation of JAM revealed that it was
associated with nectin through afadin and ZO-1. These
results suggest that nectin has a role in the localization
of JAM at TJs in the process of the formation of the
junctional complex in epithelial cells.
Oncogene (2002) 21, 7642 – 7655. doi:10.1038/sj.onc.
1205875
Keywords: nectin; JAM; cadherin; adherens junctions;
tight junctions
*Correspondence: Y Takai;
E-mail:[email protected]
Received 21 June 2002; revised 8 July 2002; accepted 15 July 2002
Introduction
Junctional adhesion molecule (JAM) is a Ca2+independent immunoglobulin-like cell – cell adhesion
molecule which localizes at tight junctions (TJs) in
epithelial cells (Martin-Padura et al., 1998; Bazzoni et
al., 2000; Liu et al., 2000; Palmeri et al., 2000; Itoh et
al., 2001). At TJs, claudin is a key Ca2+-independent
cell – cell adhesion molecule that forms TJ strands
(Furuse et al., 1998a,b; Tsukita and Furuse, 1999;
Tsukita et al., 1999). Occludin is another transmembrane protein at TJs, but its function has not been
established (Tsukita and Furuse, 1999; Tsukita et al.,
1999). Claudin and occludin interact with actinfilament (F-actin)-binding scaffold molecules, ZO-1,
-2, and -3 (Itoh et al., 1993, 1997, 1999a,b; Willott et
al., 1993; Furuse et al., 1994; Haskins et al., 1998;
Wittchen et al., 2000). JAM also interacts with ZO-1
(Bazzoni et al., 2000; Ebnet et al., 2000; Itoh et al.,
2001). ZO-1 and -2 form a dimer with ZO-3 (Haskins
et al., 1998; Itoh et al., 1999b; Wittchen et al., 1999).
Furthermore, JAM directly interacts with one of the
cell polarity proteins, Par-3, which forms a ternary
complex with Par-6 and atypical protein kinase C
(aPKC), suggesting that JAM is involved in the
formation of cell polarity through these proteins
(Ebnet et al., 2001; Itoh et al., 2001). JAM modulates
monocyte transmigration (Martin-Padura et al., 1998),
and serves as a ligand of the b2 integrin LFA-1
involved in transendothelial migration of leukocytes
(Ostermann et al., 2002). JAM furthermore serves as a
receptor for reovirus (Barton et al., 2001). Two other
JAM homologs, VE-JAM/JAM-2 and JAM-3, have
been identified (Cunningham et al., 2000; Palmeri et
al., 2000; Arrate et al., 2001; Aurrand-Lions et al.,
2001). VE-JAM/JAM-2 and JAM-3 are predominantly
expressed in endothelial cells of different vascular beds
and are absent in epithelial cells (Palmeri et al., 2000;
Aurrand-Lions et al., 2001).
The formation and maintenance of TJs are
dependent on the cell – cell adhesion activity of Ecadherin in epithelial cells (Gumbiner and Simons,
1986; Gumbiner et al., 1988; Pasdar and Nelson,
1988a,b; Watabe-Uchida et al., 1998). E-Cadherin is a
key Ca2+-dependent cell – cell adhesion molecule at
adherens junctions (AJs) which localize just at the
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basal side of TJs (Takeichi, 1988, 1991; Geiger and
Ginsberg, 1991; Gumbiner, 1996, 2000). E-Cadherin is
associated with the actin cytoskeleton through peripheral membrane proteins, including a-, b-, and gcatenins, a-actinin, and vinculin (Ozawa et al., 1989;
Nagafuchi et al., 1991; Tsukita et al., 1992). Several
experiments in which E-cadherin function is inhibited
have revealed that E-cadherin is important for the
assembly of the junctional complex in epithelial cells:
incubation of epithelial cells in a low Ca2+ medium or
in a medium containing E-cadherin function-blocking
antibodies (Abs) prevents assembly of AJs and TJs
(Gumbiner and Simons, 1986; Gumbiner et al., 1988;
Pasdar and Nelson, 1988a,b; Siliciano and Goodenough, 1988); and the junctional complex does not
assemble in the cells genetically deficient of a-catenin
(Watabe et al., 1994). However, the mechanism of the
organization of the junctional complex remains
unknown.
Nectin is another immunoglobulin-like Ca2+-independent cell – cell adhesion molecule at AJs (Aoki et
al., 1994; Lopez et al., 1995; Takahashi et al., 1999;
Miyahara et al., 2000; Satoh-Horikawa et al., 2000).
Nectin comprises a family consisting of at least four
members, nectins-1, -2, -3, and -4 (Morrison and
Racaniello, 1992; Aoki et al., 1994; Lopez et al., 1995;
Eberle et al., 1995; Cocchi et al., 1998; Warner et al.,
1998; Satoh-Horikawa et al., 2000; Reymond et al.,
2001). Nectins-1, -2, and -3 have two or three splicing
variants. Nectin-1a has originally been identified as the
poliovirus receptor-related protein and has recently
been shown to serve as the a-herpes virus entry and
cell – cell spread mediator (Lopez et al., 1995; Eberle et
al., 1995; Cocchi et al., 1998, 2000; Geraghty et al.,
1998; Warner et al., 1998; Lopez et al., 2000; Sakisaka
et al., 2001). Each member of the nectin family forms
homo-cis-dimers, followed by formation of homotrans-dimers, causing cell – cell adhesion (Lopez et al.,
1998; Takahashi et al., 1999; Miyahara et al., 2000;
Satoh-Horikawa et al., 2000; Sakisaka et al., 2001).
Nectin-3 furthermore forms hetero-trans-dimers with
either nectin-1 or -2 and this formation of each heterotrans-dimer is much stronger than that of each homotrans-dimer (Satoh-Horikawa et al., 2000). Nectin-4
also forms hetero-trans-dimers with nectin-1 (Reymond
et al., 2001). Nectin is associated with the actin
cytoskeleton through afadin, an F-actin-binding
protein (Mandai et al., 1997, 1999; Takahashi et al.,
1999). Most of the nectin family members have a Cterminal conserved motif of four amino acid residues
which interacts with the PDZ domain of afadin
(Takahashi et al., 1999; Satoh-Horikawa et al., 2000;
Reymond et al., 2001). Afadin has at least two splicing
variants, l- and s-afadins. l-Afadin, the larger splicing
variant, is an F-actin-binding protein with one PDZ
domain and three proline-rich domains and connects
nectin to the actin cytoskeleton (Mandai et al., 1997;
Takahashi et al., 1999). s-Afadin, the smaller splicing
variant, has one PDZ domain but lacks the F-actinbinding domain and the third proline-rich domain
(Mandai et al., 1997). Human s-afadin is identical to
the gene product of AF-6, a gene that has been
identified as an ALL-1 fusion partner involved in acute
myeloid leukemias (Prasad et al., 1993). In this study,
l-afadin is simply referred to as afadin. The nectinafadin system is expressed not only in cells having TJs
but also cells lacking TJs, such as fibroblasts, neurons,
and spermatids (Lopez et al., 1995; Eberle et al., 1995;
Mandai et al., 1997; Takahashi et al., 1999; SatohHorikawa et al., 2000; Nishioka et al., 2000; Mizoguchi
et al., 2002; Ozaki-Kuroda et al., 2002).
Nectin has a potency to recruit the E-cadherin-bcatenin complex to the nectin-based cell – cell adhesion
sites through afadin and a-catenin in fibroblasts
(Tachibana et al., 2000). Nectin has furthermore a
potency to recruit ZO-1 to the nectin-based cell – cell
adhesion sites through afadin in a cadherin-independent manner in fibroblasts (Yokoyama et al., 2001). In
epithelial cells of afadin (7/7) mice and (7/7)
embryoid bodies, the proper organization of AJs and
TJs is impaired (Ikeda et al., 1999). Nectin-1 has
recently been determined by positional cloning to be
responsible for cleft lip/palate-ectodermal dysplasia,
which is characterized by cleft lip/palate, syndactyly,
mental retardation, and ectodermal dysplasia (Suzuki
et al., 2000). These results suggest that the nectinafadin system plays an important role in the organization of both AJs and TJs, but the direct evidence for
this role of the nectin-afadin system has not yet been
obtained.
In this study, we have examined the possible
interaction between nectin and JAM by the use of
Madin-Darby canine kidney (MDCK) cells and
cadherin-deficient L cells stably expressing nectin and
JAM, and have found that nectin is involved in the
localization of JAM at TJs in epithelial cells.
Results
Remaining of nectin-1a-based cell – cell adhesion in the
absence of the cadherin-based cell – cell adhesion
We examined the localization of JAM in comparison
with that of nectin-1a or E-cadherin during the
disruption of cell – cell AJs in MDCK cells stably
expressing FLAG-nectin-1a (nectin-1a-MDCK cells).
When the Ca2+ concentration in the culture medium is
switched from 2 mM to 2 mM, the cells gradually
detached from each other as described in wild-type
MDCK cells (Kartenbeck et al., 1991). Wild-type
MDCK cells expressed nectin-1 and -2 as estimated
by Western blotting, but none of them were stained by
any currently available Abs (data not shown). Thus, we
utilized nectin-1a-MDCK cells in this study. The
staining patterns for afadin, E-cadherin, and JAM in
wild-type MDCK cells cultured at 2 mM Ca2+ were
similar to those in nectin-1a-MDCK cells (data not
shown). The immunofluorescence signals for nectin-1a,
E-cadherin, and JAM were highly concentrated at
cell – cell adhesion sites of nectin-1a-MDCK cells
cultured at 2 mM Ca2+ as described previously
(Takahashi et al., 1999; Asakura et al., 1999) (Figure
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1; 0 min). When these cells were cultured at 2 mM Ca2+
for 15, 30, 60, and 120 min, the immunofluorescence
signals for E-cadherin and JAM gradually disappeared
and partly appeared on intracellular vesicles (Figure 1;
15, 30, 60, 120 min). The signals were first observed
just beneath the plasma membrane and gradually
translocated to the perinuclear regions. The signal for
E-cadherin first disappeared and the signal for JAM
then disappeared (Figure 1; 15, 30 min). In contrast,
the signal for nectin-1a mostly remained on the plasma
membrane and formed a ring-like structure. The
extracellular domain of nectin-1a was exposed to the
outside of the cells which had been cultured at 2 mM
Ca2+ for 120 min, as estimated by immunostaining of
non-permeabilizing cells with the anti-FLAG monoclonal antibody (mAb) which recognized the
extracellular domain of FLAG-nectin-1a (data not
shown).
The disappearance of the immunofluorescence
signals for E-cadherin and JAM from the plasma
membrane might be due to their internalization, their
degradation, or to their diffusion on the plasma
membrane. To examine which is the case, the
homogenate was prepared from nectin-1a-MDCK cells
cultured at 2 mM Ca2+ for indicated periods of time
and subjected to SDS-polyacrylamide gel electrophor-
esis (PAGE), followed by Western blotting with an Ab
against each protein. The total amount of nectin-1a, Ecadherin, or JAM was not changed (Figure 2). The
post-nuclear supernatant was then prepared from the
cells precultured at 2 mM Ca2+ for 120 min and
Figure 2 Stability of nectin-1a, E-cadherin, and JAM during the
culture at low Ca2+. Nectin-1a-MDCK cells were cultured at
2 mM Ca2+ for indicated periods of time. The cells were homogenized and the lysates of the cells (10 mg of protein each) were
subjected to SDS – PAGE (8% polyacrylamide gel), followed by
Western blotting with the anti-FLAG mAb, the anti-E-cadherin
mAb, and the anti-JAM pAb. The results are representative of
three independent experiments
Figure 1 Disappearance of the immunofluoresence signals for E-cadherin and JAM from the plasma membrane by reduction of
medium Ca2+ from a normal to a low concentration. Nectin-1a-MDCK cells were cultured at 2 mM Ca2+ for indicated periods
of time. The cells were triple stained with the anti-FLAG mAb, the anti-JAM polyclonal Ab (pAb), and the anti-E-cadherin
mAb. Arrows, nectin-1a-based cell – cell adhesion sites remained at 2 mM Ca2+; and Bars, 10 mm. The results are representative
of three independent experiments
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subjected to sucrose density gradient ultracentrifugation, followed by Western blotting of each fraction
with an Ab against each protein. Nectin-1a, Ecadherin, and JAM were partly detected in the
intracellular vesicle fraction where early or late
endosomes were enriched, but mostly detected in the
plasma membrane fraction even after the cells were
cultured at 2 mM Ca2+ for 120 min (data not shown).
These results indicate that the disappearance of the
immunofluorescence signals for E-cadherin and JAM
from the plasma membrane was mainly due to the
lateral diffusion of these proteins on the plasma
membrane and partly due to their internalization.
We examined in detail whether the nectin-1a-based
cell – cell adhesion remained after the cadherin-based
cell – cell adhesion was disrupted by culturing nectin1a-MDCK cells at 2 mM Ca2+ for 120 min (Figure 3).
The immunofluorescence signal for nectin-1a was
diffusely observed along the free surface of the plasma
membrane at the apical side, but it was concentrated at
the cell – cell adhesion sites at the basal side of the cells
(Figure 3; arrow). The signals for afadin and ZO-1
colocalized with that for nectin-1a not only at the free
surface of the plasma membrane but also at the cell –
cell adhesion sites in the cells which had been cultured
at 2 mM Ca2+ for 120 min (Figure 3). These results
indicate that nectin-1a forms cell – cell adhesion even in
the absence of the cadherin-based cell – cell adhesion,
and afadin and ZO-1 still remain at the nectin-1a-based
cell – cell adhesion sites.
Recruitment of JAM to the nectin-1a-based cell – cell
adhesion sites by increase of Ca2+ from a low to a high
concentration
Nectin-1a-MDCK cells precultured at 2 mM Ca2+ for
120 min were recultured at 2 mM Ca2+ for 15, 30, and
60 min, and the time courses of the accumulation of Ecadherin and JAM at the nectin-1a-based cell – cell
adhesion sites were examined in detail. Before the
incubation at 2 mM Ca2+, the immunofluorescence
signal for E-cadherin or JAM was not observed at the
nectin-1a-based cell – cell adhesion sites where the
signals for nectin-1a, afadin, and ZO-1 were observed
(Figure 4A – C; 0 min). After the incubation at 2 mM
Ca2+, the signals for E-cadherin and JAM were
concentrated at the nectin-1a-based cell – cell adhesion
sites (Figure 4A; 30 min, arrow and arrowhead). After
60 min of the incubation at 2 mM Ca2+, the signals for
nectin-1a, E-cadherin, JAM, afadin, and ZO-1 were
concentrated at the cell – cell adhesion sites (Figure 4A –
C; 60 min). The signals for both JAM and ZO-1 were
more concentrated at the apical side than at the basal
side, whereas the signals for nectin-1a and E-cadherin
were more concentrated at the basal side than at the
apical side, indicating that the signals for both JAM and
ZO-1 were localized at the apical side of the nectin-1abased cell – cell adhesion sites (Figure 4A – C; 60 min).
To obtain the evidence that the nectin-afadin system
is necessary for the recruitment of JAM, we utilized the
inhibitors of nectin-1a, the chimeric proteins of
Figure 3 Remaining of the nectin-1a-based cell – cell adhesion sites and colocalization of afadin and ZO-1 with nectin-1a during the
culture at low Ca2+. Nectin-1a-MDCK cells were cultured at 2 mM Ca2+ for 120 min, and the cells were double stained with the
anti-nectin-1a pAb (a1, b1) and the anti-afadin mAb (a2, b2) (left panel) or with the anti-nectin-1a pAb (c1, d1) and the anti-ZO-1
mAb (c2, d2) (right panel). Two optical sections are shown for each staining. Confocal images of b1, b2, b3, d1, d2, and d3 are the
apical side of the images of a1, a2, a3, c1, c2, and c3, respectively. Arrows, nectin-1a-based cell – cell adhesion sites remained at 2 mM
Ca2+; and Bars, 10 mm. The results are representative of three independent experiments
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Figure 4 Recruitment of E-cadherin and JAM to the nectin-1a-based cell – cell adhesion sites by increase of Ca2+ from a low to a
high concentration. Nectin-1a-MDCK cells were cultured at 2 mM Ca2+ for 120 min and then incubated with 2 mM Ca2+ for inOncogene
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extracellular fragments of glycoprotein D and nectin-3
fused with human IgG Fc, gD and Nef-3. Glycoprotein
D is an envelope protein of herpes simplex virus type 1,
one of a-herpes viruses (Campadelli-Fiume et al., 2000;
Spear et al., 2000). gD binds to nectin-1a and inhibits
not only the formation of homo-trans-dimers of nectin1a but also the formation of hetero-trans-dimers
between nectin-1a and -3a (Sakisaka et al., 2001). We
have recently found that Nef-3 has also similar activity
(Honda et al., submitted). As the mixture of gD and
Nef-3 has more inhibitory activity than gD or Nef-3
alone in nectin-1a-MDCK cells (data not shown), we
utilized the mixture of gD and Nef-3 as nectin
inhibitors in this study. Nectin-1a-MDCK cells were
first cultured at 2 mM Ca2+ for 120 min and then
cultured at 2 mM Ca2+ in the presence or absence of
the mixture of gD and Nef-3. In the absence of gD or
Nef-3, the immunofluorescence signals for nectin-1a, Ecadherin, and JAM were detected at 93, 78, and 79%
of the cell – cell adhesion sites, respectively (n=105)
(Figure 5; control and see Figure 4A; 60 min).
However, in the presence of the mixture of gD and
Nef-3, the immunofluorescence signal for nectin-1a was
reduced to 20% of the cell – cell adhesion sites (n=164)
(Figure 5; inhibitors, Nectin-1). The signal for Ecadherin at the cell – cell adhesion sites was reduced to
36% of the cell – cell adhesion sites (n=164) in the
presence of the mixture of gD and Nef-3 (Figure 5;
inhibitors, E-Cad). The signal for JAM at the cell – cell
adhesion sites was also reduced to 31% of the cell – cell
adhesion sites (n=164) in the presence of the mixture
of gD and Nef-3 (Figure 5; inhibitors, JAM). When the
cells were incubated at 2 mM Ca2+ for a longer time,
the immunofluorescence signals for nectin-1a, Ecadherin, and JAM were re-concentrated at the cell –
cell adhesion sites even in the presence of the mixture
of gD and Nef-3 (data not shown). These results
indicate that the mixture of gD and Nef-3 inhibits the
formation of the trans-dimers of nectin-1a and thereby
inhibits the recruitment of E-cadherin and JAM to the
nectin-1a-based cell – cell adhesion sites.
Phorbol ester-induced recruitment of JAM to the
nectin-1a-based cell – cell adhesion sites
It has been shown that when nectin-1a-MDCK cells
precultured at 2 mM Ca2+ for 120 min is incubated with
a phorbol ester, 12-o-tetradecanoyl-phorbol-13-acetate
(TPA), a TJ-like structure is formed, and that nectin-1a,
afadin, ZO-1, but not E-cadherin, accumulate there
(Balda et al., 1993; Asakura et al., 1999). We next
examined whether JAM accumulates at the TPAinduced TJ-like structure in nectin-1a-MDCK cells.
The immunofluorescence signal for JAM as well as that
for nectin-1a, but not that for E-cadherin, accumulated
at the TPA-induced TJ-like structure (Figure 6; 30 min,
60 min). The sites of recruited JAM were apparently
indistinguishable from the nectin-1a-based cell – cell
adhesion sites by confocal microscopy, because the cells
Figure 5 Inhibition by the nectin inhibitors of the recruitment of
E-cadherin and JAM to the nectin-1a-based cell – cell adhesion
sites. Nectin-1a-MDCK cells were cultured at 2 mM Ca2+ for
120 min and then incubated with 2 mM Ca2+ for 60 min in the
absence (Control) or presence (Inhibitors) of 60 mg/ml gD and
60 mg/ml Nef-3. The cells were triple stained with the anti-FLAG
mAb, the anti-JAM pAb, and the anti-E-cadherin mAb. Bars,
10 mm. The results are representative of three independent experiments
dicated periods of time. (A) The cells were triple stained with the anti-FLAG mAb (a1, b1, c1, d1, e1, f1, g1, h1), the anti-JAM pAb
(a2, b2, c2, d2, e2, f2, g2, h2), and the anti-E-cadherin mAb (a3, b3, c3, d3, e3, f3, g3, h3); (B) The cells were double stained with the
anti-nectin-1a pAb (a1, b1, c1, d1, e1, f1, g1, h1) and the anti-ZO-1 mAb (a2, b2, c2, d2, e2, f2, g2, h2); and (C) The cells were
double stained with the anti-nectin-1a pAb (a1, b1, c1, d1, e1, f1, g1, h1) and the anti-afadin mAb (a2, b2, c2, d2, e2, f2, g2,
h2). Two optical sections are shown for each staining. Confocal images of b1, b2, b3, d1, d2, d3, f1, f2, f3, h1, h2, and h3 are
the apical side of the images of a1, a2, a3, c1, c2, c3, e1, e2, e3, g1, g2, and g3, respectively. Arrows, the signal for E-cadherin
concentrated at the nectin-1a-based cell – cell adhesion sites; Arrowheads, the signal for JAM concentrated at the nectin-1a-based
cell – cell adhesion sites; and Bars, 10 mm. The results are representative of three independent experiments
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Figure 6 TPA-induced recruitment of JAM to the nectin-1a-based cell – cell adhesion sites. Nectin-1a-MDCK cells were cultured at
2 mM Ca2+ for 120 min and then incubated with 100 nM TPA for indicated periods of time. The cells were triple stained with the
anti-FLAG mAb (a1, b1, c1, d1, e1, f1), the anti-JAM pAb (a2, b2, c2, d2, e2, f2), and the anti-E-cadherin mAb (a3, b3, c3, d3, e3,
f3). Two optical sections are shown for each staining. Confocal images of b1, b2, b3, d1, d2, d3, f1, f2, and f3 are the apical side of
the images of a1, a2, a3, c1, c2, c3, e1, e2, and e3, respectively. Arrows, the signal for JAM, but not that for E-cadherin, concentrated at the nectin-1a-based cell – cell adhesion sites; and Bars, 10 mm. The results are representative of three independent experiments
became flattened by the action of TPA and the cell – cell
adhesion sites were not thick.
Then we examined whether this TPA-induced
recruitment of JAM to the nectin-1a-based cell – cell
adhesion sites was inhibited by the mixture of gD and
Nef-3. In the absence of gD or Nef-3, the immunofluorescence signals for nectin-1a and JAM were
detected at 96 and 89% of the cell – cell adhesion sites,
respectively (n=46) (Figure 7; control and see Figure
6; 60 min). However, in the presence of the mixture of
gD and Nef-3, the immunofluorescence signal for
nectin-1a was reduced to 19% of the cell-cell adhesion
sites (n=32) (Figure 7; inhibitors, Nectin-1). The
immunofluorescence signal for JAM was also reduced
to 31% of the cell-cell adhesion sites (n=32) (Figure 7;
inhibitors, JAM). These results indicate that the
mixture of gD and Nef-3 inhibits the formation of
the trans-dimers of nectin-1a and thereby inhibits the
recruitment of JAM to the TPA-induced TJ-like
structure.
Recruitment of JAM to the Nef-3-coated bead – cell
contact sites
To obtain more definitive evidence that nectin-1a
recruits JAM to the nectin-1a-based cell – cell adhesion
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sites, we utilized microbeads coated with Nef-3. We
have recently shown that the microbeads coated with
Nef-3 recruit first the nectin-afadin complex and then
the E-cadherin-catenin complex to the bead – cell
contact sites using L fibroblasts stably expressing both
E-cadherin and nectin-1a (Honda et al., submitted).
Nectin-1a-MDCK cells were cultured at 2 mM Ca2+ for
120 min in the presence of the Nef-3-coated beads, and
further incubated for various periods of time at 2 mM
Ca2+ (Figure 8). The cells were then fixed, followed by
immunostaining for cellular nectin-1a, afadin, ZO-1,
and JAM. The immunofluorescence signals for cellular
nectin-1a and ZO-1 were concentrated at the bead – cell
contact sites at 60 min (Figure 8). The signals for
afadin and JAM were also concentrated at the bead –
cell contact sites (Figure 8). The time courses of the
concentration of the signals of nectin-1a, ZO-1, and
afadin at the bead – cell contact sites were similar, but
the signal for JAM at the bead – cell contact sites was
not observed at 60 min, but started to be concentrated
at 180 min and gradually increased (data not shown).
The signal for nectin-1a, ZO-1, afadin, or JAM at the
bead – cell contact sites was not observed when the
control microbeads coated with human IgG were used
(Figure 8 and data not shown). These results suggest
that Nef-3 on the beads first forms trans-dimers with
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Figure 7 Inhibition by the nectin inhibitors of the TPA-induced
recruitment of JAM to the nectin-1a-based cell – cell adhesion
sites. Nectin-1a-MDCK cells were cultured at 2 mM Ca2+ for
120 min and then incubated with 100 nM TPA for 60 min in
the absence (Control) or presence (Inhibitors) of 60 mg/ml gD
and 60 mg/ml Nef-3. The cells were double stained with the
anti-FLAG mAb and the anti-JAM mAb. Bars, 10 mm. The results are representative of three independent experiments
cellular nectin-1a, which might form a complex with
afadin and ZO-1, and that JAM then accumulates at
the bead – cell contact sites. Taken together, these
results indicate that JAM is recruited to the nectin1a-mediated cell – cell or cell – bead adhesion sites, and
that the trans-interaction of nectin-1a is necessary and
sufficient for this recruitment of JAM.
Afadin- and ZO-1-dependent recruitment of JAM to the
nectin-based cell – cell adhesion sites without the transinteraction of JAM
Next we examined whether JAM is recruited to the
nectin-based cell – cell adhesion sites through their
cytoplasmic tail-binding proteins, afadin and ZO-1.
For this purpose, we utilized cadherin-deficient L cells
stably expressing both full-length nectin-2a and fulllength JAM (nectin-2a-JAM-L cells), both C-terminal
four amino acids (aa)-deleted nectin-2a and full-length
JAM (nectin-2a- DC-JAM-L cells), or both full-length
nectin-2a and C-terminal two aa-deleted JAM (nectin2a-JAM-DC-L cells). Nectin-2a-DC-L cells forms
nectin-2a-DC-based cell – cell adhesion sites, but afadin
does not accumulate at the adhesion sites, while afadin
accumulates at the nectin-2a-based cell – cell adhesion
sites in nectin-2a-L cells (Miyahara et al., 2000;
Tachibana et al., 2000). Similarly, endogenous ZO-1
is recruited to the JAM-based cell – cell adhesion sites
in JAM-L cells, whereas ZO-1 is not recruited to the
JAM-DC-based cell – cell adhesion sites in JAM-DC-L
cells (Itoh et al., 2001).
When nectin-2a-JAM-L cells were co-cultured with L
cells stably expressing both full-length nectin-3a
(nectin-3a-L cells), the immunofluorescence signal for
nectin-2a was concentrated at the adhesion sites
between two nectin-2a-JAM-L cells and between
nectin-2a-JAM-L and nectin-3a-L cells (Figure 9A).
The signal for JAM was concentrated at the adhesion
sites between two nectin-2a-JAM-L cells. In addition,
the signal for JAM was concentrated at 42% of the
adhesion sites between nectin-2a-JAM-L and nectin-3aL cells (n=106) (Figure 9A). These results indicate that
JAM is recruited to the nectin-2a/-3a-based cell – cell
adhesion sites without the trans-interaction of JAM in
L cells. When nectin-2a-DC-JAM-L cells were cocultured with nectin-3a-L cells, the immunofluorescence
signal for nectin-2a-DC was also concentrated at the
adhesion sites between two nectin-2a-DC-JAM-L cells
and between the nectin-2a-DC-JAM-L and nectin-3a-L
cells (Figure 9B). However, the signal for JAM at the
adhesion sites between nectin-2a-DC-JAM-L and
nectin-3a-L cells was significantly reduced (6% of the
adhesion sites between nectin-2a-DC-JAM-L and
nectin-3a-L cells, n=62) when compared to that of
nectin-2a-JAM-L cells. These results indicate that the
binding of nectin-2a to afadin is required for the
recruitment of JAM to the nectins-2a/-3a-based cell –
cell adhesion sites. When nectin-2a-JAM-DC-L cells
were co-cultured with nectin-3a-L cells, the immunofluorescence signal for JAM-DC was not concentrated
at the adhesion sites between nectin-2a-JAM-DC- and
nectin-3a-L cells (less than 2% of the adhesion sites
between nectin-2a- DC-JAM-L and nectin-3a-L cells,
n=53) (Figure 9C). These results indicate that the
binding of JAM to ZO-1 is required for the
recruitment of JAM to the nectins-2a/-3a-based cell –
cell adhesion sites. Taken together, JAM is recruited to
the nectins-2a/-3a-based cell – cell adhesion sites without the trans-interaction of JAM, and this recruitment
of JAM requires afadin and ZO-1.
We next examined whether JAM is associated with
nectin-2a through afadin and ZO-1. For this purpose,
we performed immunoprecipitation analysis of nectin2a-JAM-L, nectin-2a-DC-JAM-L, and nectin-2a-JAMDC-L cells. When the cell extracts of nectin-2a-JAM-L
and nectin-2a-DC-JAM-L cells were subjected to
immunoprecipitation by the use of the nectin-2 mAb,
similar amounts of nectins-2a and -2a-DC were
precipitated (Figure 10A, lanes 3, 4, Nectin-2). Afadin
and ZO-1 were co-immunoprecipitated with nectin-2a,
whereas they were not co-immunoprecipitated with
nectin-2a-DC (Figure 10A, lanes 3, 4, Afadin and ZO1), as described previously (Yokoyama et al., 2001). It
should be noted that afadin was more efficiently coimmunoprecipitated than ZO-1 with nectin-2a (Figure
10A, lanes 3, 4, Afadin and ZO-1). However, JAM was
not co-immunoprecipitated with nectin-2a (Figure 10A,
lanes 3, 4, JAM). When the cell extracts of nectin-2aOncogene
A role of nectin in the localization of JAM at TJs
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Figure 8 Recruitment of JAM and ZO-1 to the Nef-3-coated bead – cell contact sites. The Nef-3-coated or control microbeads were
added to nectin-1a-MDCK cells, followed by incubation at 2 mM Ca2+ for 120 min. After the incubation, the cells were washed with
DMEM and further incubated for various periods of time (360 min for nectin-1a/JAM staining, 60 min for nectin-1a/ZO-1 and
nectin-1a/afadin staining) at 2 mM Ca2+. The cells were then fixed, followed by immunostaining for cellular nectin-1a, afadin,
JAM, and ZO-1 using the anti-FLAG mAb or the anti-nectin-1a pAb, the anti-afadin mAb, the anti-ZO-1 mAb, and the antiJAM pAb, respectively. The samples were analysed using a differential interference contrast and confocal laser scanning microscope.
The position of the bead is marked with an asterisk. Arrow, bead – cell contact sites; and Bars, 10 mm. The results shown are representative of three independent experiments
JAM-L and nectin-2a-JAM-DC-L cells were subjected
to immunoprecipitation by the use of the JAM mAb,
similar amounts of JAM and JAM-DC were precipitated (Figure 10B, lanes 3, 4, JAM). Afadin and ZO-1
were co-immunoprecipitated with JAM, whereas they
were not co-immunoprecipitated with JAM-DC (Figure
10B, lanes 3, 4, Afadin and ZO-1). In this case, ZO-1
was more efficiently co-immunoprecipitated than
afadin with JAM (Figure 10B, lanes 3, 4, Afadin and
ZO-1). Nectin-2a was not co-immunoprecipitated with
JAM (Figure 10B, lanes 3, 4, Nectin-2). Although
nectin-2a was not co-immunoprecipitated with JAM,
the co-immunoprecipitation of afadin and ZO-1 with
both nectin and JAM suggest that JAM is associated
with nectin-2a through afadin and ZO-1.
It has been reported that JAM directly binds AF-6,
the splicing variant of afadin, through the interaction
of the C-terminal PDZ binding motif of JAM with the
PDZ domain of AF-6 (Ebnet et al., 2000). This result
suggests that JAM directly binds afadin and nectin
may be associated with JAM through afadin only.
Thus, we next examined whether JAM directly binds
afadin. A GST-fusion protein of the cytoplasmic region
of JAM (GST-JAM-CP) did not bind an MBP-fusion
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protein of the PDZ domain of afadin (MBP-afadinPDZ) immobilized on amylose resin beads (Figure 10C,
lane 3). A GST-fusion protein of the cytoplasmic
region of nectin-2a (GST-nectin-2a-CP) bound MBPafadin-PDZ and the stoichiometry of this interaction
was 1 : 1 (Figure 10C, lane 4), as described previously
(Yokoyama et al., 2001). These results indicate that
JAM does not directly bind afadin, and suggest that
JAM is indirectly associated with nectin-2a through
afadin and ZO-1.
Discussion
We have previously shown that nectin has a potency to
recruit the E-cadherin-b-catenin complex and ZO-1 to
the nectin-based cell – cell adhesion sites in fibroblasts
(Tachibana et al., 2000; Yokoyama et al., 2001). In this
paper, we show several lines of evidence that nectin has
a potency to recruit JAM to the nectin-based cell – cell
adhesion sites. First, JAM was recruited to the nectin1a-based cell – cell adhesion sites during the formation
of the junctional complex in nectin-1a-MDCK cells.
This recruitment of JAM was inhibited by the nectin
A role of nectin in the localization of JAM at TJs
A Fukuhara et al
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Figure 9 Afadin- and ZO-1-dependent recruitment of JAM to the nectin-2a- and -3a-based cell – cell adhesion sites without the
trans-interaction of JAM. (A) Nectin-2a-JAM-L cells were co-cultured with nectin-3a-L cells and triple stained with the anti-nectin-2a pAb, the anti-JAM mAb, and the anti-nectin-3a mAb.; (B) Nectin-2a-DC-JAM-L cells were co-cultured with nectin-3a-L cells
and triple stained with the anti-nectin-2a pAb, the anti-JAM mAb, and the anti-nectin-3a mAb.; and (C) Nectin-2a-JAM-DC-L cells
were co-cultured with nectin-3a-L cells and triple stained with the anti-nectin-2a pAb, the anti-JAM mAb, and the anti-nectin-3a
mAb. Arrow, cell – cell adhesion sites between the same cells; Arrowhead, cell – cell adhesion sites between two different cells;
N2/JAM, Nectin-2a-JAM-L cell; N3, nectin-3 a-L cell; N2DC/JAM, Nectin-2a-DC-JAM-L cell; N2/JAMDC, Nectin-2a-JAM-DCL cell; and Bars, 10 mm. The results shown are representative of three independent experiments
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Figure 10 Indirect association of JAM and nectin-2a through
afadin and ZO-1. (A) Association of nectin-2a with afadin and
ZO-1. The cell extracts of nectin-2a-JAM-L and nectin-2a-DCJAM-L cells (each 500 mg of protein) were separately subjected
to immunoprecipitation with the anti-FLAG mAb. The immunoprecipitate was then subjected to SDS – PAGE (10% polyacrylamide gel), followed by Western blotting with the anti-nectin-2a
pAb, the anti-ZO-1 mAb, the anti-afadin mAb, and the antiJAM pAb. 1, 2; total extract, and 3, 4; immunoprecipitates of
the anti-FLAG mAb. 1, 3; nectin-2a-JAM-L cells; and 2, 4; nectin-2a-DC-JAM-L cells. (B) Association of JAM with afadin and
ZO-1. The cell extracts of nectin-2a-JAM-L and nectin-2a-JAMDC-L cells (each 500 mg of protein) were separately subjected to
immunoprecipitation with the anti-JAM mAb. The immunoprecipitate was then subjected to SDS – PAGE (10% polyacrylamide
gel), followed by Western blotting with the anti-nectin-2a pAb,
the anti-ZO-1 mAb, the anti-afadin mAb, and the anti-JAM
pAb. 1, 2; total extract, and 3, 4; immunoprecipitates of the
anti-JAM mAb. 1, 3; nectin-2a-JAM-L cells; and 2, 4; nectin2a-JAM-DC-L cells. (C) Inability of afadin to directly interact
with JAM. MBP-afadin-PDZ (20 mg of protein) was immobilized
on amylose resin beads. GST-Nectin-2a-CP and GST-JAM-CP
(each 100 mg of protein) were applied to the affinity beads. After
the beads were extensively washed, elution was performed with
10 mM maltose. The eluate was subjected to SDS – PAGE (15%
polyacrylamide gel), followed by protein staining with Coomassie
brilliant blue. 1, GST-JAM-CP (input); 2, GST-Nectin-2a-CP (input); 3, MBP-afadin-PDZ+GST-JAM-CP (eluate); and 4, MBPafadin-PDZ+GST-Nectin-2a-CP (eluate). The results shown are
representative of three independent experiments
Oncogene
inhibitors, gD and Nef-3, which inhibited the transinteraction of nectin-1a. Secondly, microbeads coated
with the extracellular fragment of nectin-3, Nef-3, that
interacts with cellular nectin-1a, also recruited ZO-1
and JAM to the bead – cell contact sites. Thirdly, when
nectin-2a-JAM-L cells were co-cultured with nectin-3aL cells, JAM was concentrated at the adhesion sites
between nectin-2a-JAM-L and nectin-3a-L cells without the trans-interaction of JAM. Analyses of the
localization and immunoprecipitation of JAM revealed
that it was associated with nectin through afadin and
ZO-1. Taken together, our results indicate that the
trans-interaction of nectin is necessary and sufficient
for the recruitment of JAM, and that the nectin-afadin
system plays a role in the recruitment of JAM in the
process of the formation of the junctional complex in
epithelial cells.
In the initial stage during the formation of AJs and
TJs, primordial spot-like cell – cell junctions are first
formed at the tips of the cellular protrusions radiating
from adjacent cells (Yonemura et al., 1995; Adams et
al., 1998; Ando-Akatsuka et al., 1999; Vasioukhin et
al., 2000). The spot-like junctions fuse to form short
belt-like junctions. The cadherin-catenin and nectinafadin systems colocalize to the spot-like junctions
where occludin is not concentrated (Ando-Akatsuka et
al., 1999; Asakura et al., 1999; Sakisaka et al., 1999).
Claudin is not concentrated at the spot-like junctions
(Fukuhara et al., manuscript in preparation), but ZO-1
and JAM appear early at the spot-like junctions (Ebnet
et al., 2001). Our results that nectin has a potency to
recruit JAM to the nectin-based cell – cell adhesion sites
suggest that nectin first accumulates at the spot-like
junction, and then JAM accumulates there. However,
when we compared the concentrations of nectin and
JAM at the spot-like junction, we could not detect
which one accumulates first in our conventional
staining procedure. More detailed analyses with timelapse imaging should be required for the analysis of the
ordered assembly of the junctional complex.
How does nectin recruit JAM to the cell – cell
adhesion sites? JAM interacts with ZO-1 through its
C-terminal (Bazzoni et al., 2000; Ebnet et al., 2000;
Itoh et al., 2001). We have previously found that
nectin recruits ZO-1 to the nectin-based cell – cell
adhesion sites through afadin in a cadherin-independent manner (Yokoyama et al., 2001). Our studies here
show that the recruitment of JAM requires afadin and
ZO-1. Afadin and ZO-1 were co-immunoprecipitated
with either nectin or JAM, and JAM did not directly
bind to afadin. Thus, nectin recruits JAM through
their cytoplasmic-tail binding proteins, afadin and ZO1. As afadin does not directly interact with ZO-1
(Yokoyama et al., 2001), both proteins may interact
through an unidentified factor. When nectin-1aMDCK cells were cultured at 2 mM Ca2+ for
120 min, nectin-1a still formed cell – cell adhesion at
the basal sites of the cells and afadin and ZO-1
colocalized with nectin-1a. However, the signal for
JAM mostly disappeared from the nectin-1a-based
cell – cell adhesion sites. Thus, the interaction between
A role of nectin in the localization of JAM at TJs
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JAM and ZO-1 could be regulated in response to Ca2+
switch. In addition, in polarized epithelial cells, ZO-1
and JAM localize at TJs, while nectin localizes at AJs,
indicating that there must be unknown mechanisms
that regulate the translocation of ZO-1 and JAM from
the nectin-based cell – cell adhesion sites to TJs. It is of
crucial importance to clarify the molecular linkages
among the nectin-afadin complex and the JAM-ZO-1
complex to understand how the junctional complex is
formed in epithelial cells.
Materials and methods
Materials and chemicals
Rabbit anti-nectin-1a and anti-nectin-2a pAb were prepared
as previously described (Takahashi et al., 1999). Rat antinectin-2 and anti-nectin-3 mAbs were prepared as previously
described (Takahashi et al., 1999; Satoh-Horikawa et al.,
2000). A mouse anti-afadin mAb was prepared as described
(Sakisaka et al., 1999). A mouse anti-ZO-1 mAb (Itoh et al.,
1993) was kindly supplied from Dr Tsukita (Kyoto
University, Kyoto, Japan). A rat anti-E-cadherin mAb
(ECCD-2) was supplied by Dr M Takeichi (Center for
Developmental Biology, RIKEN, Kobe, Japan). A rabbit
anti-human JAM pAb and a mouse anti-human JAM mAb
were raised and characterized previously (Ozaki et al., 1999;
Itoh et al., 2001). A mouse anti-FLAG mAb was from
SIGMA. Mouse anti-EEA1 and anti-E-cadherin mAbs were
from Transduction. A rabbit anti-Rab11 pAb was from
Santa Cruz. Secondary Abs for immunofluorescence microscopy were obtained from Chemicon International, Inc. gD
and Nef-3 (the chimeric proteins of extracellular fragments of
glycoprotein D and nectin-3 fused with human IgG Fc) were
prepared as described (Tachibana et al., 2000; Sakisaka et al.,
2001; Honda et al., submitted).
1a-MDCK cells (16105) were seeded on 18-mm glass cover
slips in 12-well culture dishes. Forty-eight hours later, the
cells were washed with phosphate buffered saline (PBS) and
cultured at 2 mM Ca2+ in DMEM without serum for 60 min.
The cells were then cultured at 2 mM Ca2+ (DMEM with
5 mM EGTA) in the presence or absence of 60 mg/ml gD and
60 mg/ml Nef-3 for indicated periods of time. After the
culture, cells were washed with PBS and cultured at 2 mM
Ca2+ in DMEM without serum in the presence or absence of
60 mg/ml gD and 60 mg/ml Nef-3 for indicated periods of
time. When the cells were treated with TPA, the cells were
washed with PBS and cultured at 2 mM Ca2+ in DMEM
without serum for 60 min. The cells were then cultured at
2 mM Ca2+ in DMEM with 5 mM EGTA in the presence or
absence of 60 mg/ml gD and 60 mg/ml Nef-3 for 120 min.
After the culture, 100 nM TPA was added to the medium and
the cells were further cultured in the presence or absence of
60 mg/ml gD and 60 mg/ml Nef-3 for indicated periods of
time.
Immunofluorescence microscopy
Immunofluorescence microscopy was done as described
(Mandai et al., 1997, 1999). Briefly, the cells were fixed in
the mixture of 50% acetone and 50% methanol at 7208C for
1 min. The fixed cells were then washed three times with PBS.
After blocking in PBS containing 1% BSA for 60 min, the
cells were incubated in the same buffer with various
combinations of the anti-afadin, anti-E-cadherin, anti-FLAG,
anti-JAM, anti-nectin-2, anti-nectin-3 and anti-ZO-1 mAbs,
and the anti-JAM, anti-nectin-1a and anti-nectin-2a pAbs for
60 min. The samples were washed three times with PBS for
5 min and incubated for 30 min in PBS containing 1% BSA
with the secondary pAbs. The samples were then washed
three times with PBS for 5 min and mounted in Immunofluore mounting medium (ICN Biomedicals, Inc.). The
samples were analysed by a Radiance 2000 confocal laser
scanning microscope (Bio-Rad Laboratories).
Cell culture and DNA transfection
MDCK cells were kindly supplied by Dr W Birchmeier
(Max-Delbruck-Center for Molecular Medicine, Berlin,
Germany). Nectin-1a-MDCK cells were prepared as
described (Takahashi et al., 1999). Briefly, MDCK cells were
transfected with pCAGI-puro-FLAG-nectin-1a using the
LipofectAMINE reagent (Gibco BRL). The cells were then
cultured for 24 h, replated, and selected by culturing in the
presence of 5 mg/ml puromycin (SIGMA). JAM-L and JAMDC-L cells were kindly supplied by Dr S Tsukita (Kyoto
University, Kyoto, Japan). Nectin-3a-L cells were prepared as
described (Satoh-Horikawa et al., 2000). Nectin-2a-JAM-L or
nectin-2a-JAM-DC-L cells were prepared as follows: JAM-L
or JAM-DC-L cells were transfected with pPGKIH-FLAGnectin-2a (Miyahara et al., 2000) using LipofectAMINE
reagent. The cells were cultured for 24 h, replated, and
selected by culturing in the presence of 500 mg/ml hygromicin
B (Gibco BRL). Nectin-2a-DC-JAM-L cells were prepared as
follows: JAM-L cells were transfected with pPGKIH-FLAGnectin-2a-DC (Miyahara et al., 2000) using LipofectAMINE
reagent. The cells were cultured for 24 h, replated, and
selected by culturing in the presence of 500 mg/ml hygromicin
B (Gibco BRL).
Ca2+ switch assay
Ca2+ switch experiments using nectin-1a-MDCK cells were
done as described (Kartenbeck et al., 1991). Briefly, nectin-
Preparation of Nef-3-coated beads
Latex-sulfate microspheres (36108, 5.2-mm diameter; Interfacial Dynamics Corporation, Portland, OR, USA) were
washed, resuspended in 0.2 ml of 0.1 M borate buffer, pH 8.0,
and incubated with 100 mg of a goat anti-human IgG (Fcspecific) Ab (Sigma) with gentle mixing at room temperature
for 18 h. The beads were then centrifuged at 16 000 g at 48C
for 10 min and washed three times with 1 ml PBS. The beads
were then incubated with 0.2 ml PBS containing 5 mg/ml
BSA (BSA/PBS) at room temperature for 3 h. Aliquots of
0.2 ml of the bead suspension (66107 microspheres) were
then incubated with 30 mg of Nef-3 or human IgG at room
temperature for 3 h. After the incubation, the beads were
washed three times with 1 ml of BSA/PBS and re-suspended
in 0.2 ml of BSA/PBS.
Bead – cell adhesion assay
Nectin-1a-MDCK cells (36104) were seeded on 14-mm glass
cover slips in 24-well culture dishes. Forty-eight hours later,
the cells were washed with PBS and cultured at 2 mM Ca2+
in DMEM without serum for 60 min. The cells were then
cultured at 2 mM Ca2+ (DMEM with 5 mM EGTA) with
latex-sulfate microspheres (36104) coated with human IgG
or Nef-3 for 120 min. After the culture, the cells were washed
with DMEM and cultured at 2 mM Ca2+ in DMEM with
10% FCS at 378C for indicated periods of time.
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Co-culture of various L cells
Affinity chromatography
Nectin-3a-L, nectin-2a-JAM-L, nectin-2a-DC-JAM-L, and
nectin-2a-JAM-DC-L cells were washed with PBS, incubated
with 0.2% trypsin and 1 mM EDTA at 378C for 5 min, and
dispersed by gentle pipetting to obtain single cell suspensions.
The cell number of each cell line was counted, and nectin-2aJAM-L, nectin-2a-DC-JAM-L, or nectin-2a-JAM-DC-L cells
were mixed with an equal number of nectin-3a-L cells. Three
combinations of nectin-3a-L and nectin-2a-JAM-L cells,
nectin-3a-L and nectin-2a-DC-JAM-L cells, and nectin-3a-L
and nectin-2a-JAM-DC-L cells were prepared. These mixed
cells (66104) were seeded on 18-mm glass cover slips in 12well culture dishes and cultured for 4 h.
To examine the interaction of afadin with nectin-2a or JAM,
MBP-afadin-PDZ (20 mg of protein) was immobilized on
amylose resin beads (20 ml of wet volume). GST-Nectin-2aCP or GST-JAM-CP (each 100 mg of protein) was applied to
the MBP-fusion protein-immobilized beads equilibrated with
PBS. After the beads were extensively washed with PBS,
elution was performed with PBS containing 10 mM maltose.
Each eluate was subjected to SDS – PAGE (15% polyacrylamide gel), followed by protein staining with Coomassie
brilliant blue.
Immunoprecipitation
Nectin-2a-JAM-L, nectin-2a-DC-JAM-L, or nectin-2a-JAMDC-L cells were sonicated in an ice-cold lysis buffer (25 mM
Tris/HCl at pH 7.5, 1 mM CaCl2, 1 mM MgCl2, 100 mM
NaCl, 1% Triton X-100, 2 mg/ml Aprotinin, 10 mg/ml
leupeptin, and 100 mg/ml PMSF), followed by centrifugation
at 100 0006g at 48C for 15 min. The supernatant (500 mg of
protein) was incubated with the anti-FLAG mAb or the antiJAM mAb bound to protein G-Sepharose beads (20 mg of
Ab/20 ml of beads) (Amersham Pharmacia Biotech) at 48C
overnight. After the beads were extensively washed with the
lysis buffer, the bound proteins were eluted by boiling the
beads in an SDS sample buffer (60 mM Tris/Cl at pH 6.7, 3%
SDS, 2% (v/v) 2-mercaptoethanol, and 5% glycerol), and
subjected to SDS – PAGE (10% polyacrylamide gel), followed
by Western blotting.
Other procedures
Protein concentrations were determined with BSA as a
reference protein (Bradford, 1976). SDS – PAGE was done
as described (Laemmli, 1970).
Acknowledgments
We thank Dr M Takeichi (Center for Developmental
Biology, RIKEN, Kobe, Japan) for providing us with the
anti-E-cadherin mAb, Dr Sh Tsukita (Kyoto University,
Kyoto, Japan) for providing us with the JAM-L and JAMDC-L cells and the anti-ZO-1 mAb, and Dr W Birchmeier
(Max-Delbruck-Center for Molecular Medicine, Berlin,
Germany) for providing us with MDCK cells. The
investigation at Osaka University Medical School was
supported by grants-in-aid for Scientific Research and for
Cancer Research from the Ministry of Education, Science,
Sports, Culture, and Technology, Japan (2000, 2001).
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