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J Biol Chem, Vol. 273, Issue 45, 29745-29753, November 6, 1998
From the Departments of The tight junction protein ZO-1 belongs to a
family of multidomain proteins known as the membrane-associated
guanylate kinase homologs (MAGUKs). ZO-1 has been demonstrated to
interact with the transmembrane protein occludin, a second tight
junction-specific MAGUK, ZO-2, and F-actin, although the nature and
functional significance of these interactions is poorly understood. To
further elucidate the role of ZO-1 within the epithelial tight
junction, we have introduced epitope-tagged fragments of ZO-1 into
cultured MDCK cells and identified domains critical for the interaction
with ZO-2, occludin, and F-actin. A combination of in vitro
and in vivo binding assays indicate that both ZO-2 and
occludin interact with specific domains within the N-terminal
(MAGUK-like) half of ZO-1, whereas the unique proline-rich C-terminal
half of ZO-1 cosediments with F-actin. Consistent with these
observations, we found that a construct encoding the N-terminal half of
ZO-1 is specifically associated with tight junctions, whereas the
unique C-terminal half of ZO-1 is distributed over the entire lateral surface of the plasma membrane and other actin-rich structures. In
addition, we have identified a 244-amino acid domain within the
N-terminal half of ZO-1, which is required for the stable incorporation
of ZO-1 into the junctional complex of polarized MDCK cells. These
observations suggest that one functional role of ZO-1 is to organize
components of the tight junction and link them to the cortical actin cytoskeleton.
The tight junction forms the apical barrier to the paracellular
movement of water, solutes and immune cells in polarized epithelia (1).
Electron micrographs reveal that the paracellular seal is composed of a
series of highly ordered membrane contact sites (2), which in
freeze-fracture micrographs can be visualized as a series of
interconnected fibrils within the plasma membrane (3). Actin filaments
terminate at these membrane contact sites (4, 5), suggesting that the
cytoskeleton is involved in the structural and functional organization
of the tight junction. Although many of the molecular components of the
tight junction have now been identified, little is known about how they
interact to regulate the assembly and permeability of the
paracellular barrier.
At least one transmembrane component of the tight junction, occludin
(6), has been demonstrated to contribute to the paracellular seal in
cultured cells (7-10). More recently, investigators have identified
two novel transmembrane proteins, claudin-1 and claudin-2, which are
also presumably components of the paracellular seal (11). Both occludin
and claudins localize to the discrete membrane contact sites, or
kisses, within the freeze-fracture fibrils (6). These kisses are in
turn intimately associated with the cytosolic plaque proteins ZO-1
(12), ZO-2 (13), and ZO-3 (14). These proteins can be
co-immunoprecipitated as a complex from cultured epithelial cells (14,
15), and have been demonstrated to bind directly to the C-terminal 146 amino acids (aa)1 of occludin
(16). Other components of the cytosolic plaque include cingulin (17),
7H6 (18), AF-6 (19), and symplekin (20).
The plaque proteins ZO-1, ZO-2, and ZO-3 probably play a unique role in
the organization/regulation of tight junctions. These proteins are
members of a family of membrane-associated signaling proteins known as
the membrane-associated guanylate kinase homologs (MAGUKs), which
include the Drosophila tumor suppressor dlg (21), the
Caenorhabditis elegans signaling protein LIN2 (22), synaptic proteins hDlg and PSD95 (23-25), and the erythrocyte membrane protein p55 (26). Members of this family are distinguished by a core cassette
of protein-binding domains, which include one or more PSD95/dlg/ZO-1
(PDZ) domains, an SH3 domain, and a region of homology to guanylate
kinase (GuK) (27). All of these proteins are associated with cell-cell
contact sites such as synapses, intercalated disks, and epithelial
tight junctions. Mutations in the genes for Dlg and LIN2 cause severe
alteration in cell growth and differentiation (21, 22), suggesting that
MAGUKs are involved in signal transduction pathways controlling growth
and differentiation (27-29).
The role of MAGUKs in signal transduction may be due in large part to
the demonstrated ability of these proteins to organize protein
complexes at the plasma membrane. There are several examples in which
expression of a given MAGUK with its transmembrane binding partner
results in clustering of both proteins within the plasma membrane (30,
31). This property appears to be due to the presence of multiple
protein binding motifs within these proteins, as well as their ability
to form heterodimers with other MAGUKs. This organizational capacity
may also be enhanced by interactions with the cytoskeleton since hdlg,
p55, and the human LIN2 homolog all bind to the actin-binding protein
4.1 via a conserved motif located between the SH3 and GUK domains (23,
32, 33). Many members of this family have been demonstrated to interact
directly with ion channels, transmembrane receptors, and known
cytosolic signal transduction proteins (34-36). These observations
suggest that MAGUKs can act as a scaffold for signal transduction
complexes, organizing cytosolic signaling molecules at the plasma
membrane with transmembrane receptors and ion channels.
It is likely that the tight junction MAGUKs like ZO-1 have
organizational/functional roles analogous to other MAGUKs. ZO-1 binds
to several other tight junction components (13, 16, 37) and has also
been demonstrated to interact with several known signaling proteins,
such as the Ras substrate AF-6 (19), heterotrimeric G-proteins (38), an
unidentified serine kinase (39), and connexin 43 (40). ZO-1 has also
been shown to bind directly to F-actin in vitro (41).
However, the mechanism of these interactions and their relevance to
tight junction assembly is poorly understood. For example, it is not
known whether the binding sites for other tight junction proteins
reside within the MAGUK-like N-terminal half or within the C-terminal
domain, which is unique to tight junction MAGUKs. In addition, although ZO-1 interacts with the cytoskeleton like other MAGUKs, it lacks the
band 4.1 binding site found in other members of this family. To better
understand the role of MAGUK proteins like ZO-1 in the tight junction,
we have used epitope-tagged deletion constructs to map the binding
sites for ZO-2, occludin, and F-actin on ZO-1. Furthermore, we have
examined how these domains are involved in the assembly of ZO-1 into
the tight junction complex. Our results suggest that ZO-1 may serve as
a link between the proteins of the tight junction and the actin cytoskeleton.
Expression Constructs--
A diagram of the expression
constructs used in this study is shown in Fig. 1. To insert an epitope
tag on the C terminus of the full-length human ZO-1 (ZO1myc), a
203-base pair fragment of the C terminus was amplified from a
ZO-1 cDNA using the sense primer 5'-GAAGATGGTCATACTGTGG-3', which
is complimentary to a sequence 5' of a unique MscI site in
ZO-1, and the antisense primer 5'-GGACTAGTTTACAAGTCCTCTTCAGATATCAGCTTTTGCTCGGCAAAGTGGTCAATAAGGACAG-3'. This antisense primer contained a 20-base pair region of homology to ZO-1 (bold) followed by a sequence encoding an alanine linker, an
11-amino acid epitope from the c-Myc protein, a stop codon, and a
SpeI restriction enzyme site. This product was amplified using Taq polymerase (Promega, Madison, WI), digested with
MscI and SpeI (New England Biolabs, Beverly, MA),
and subcloned into a pSK+ Bluescript plasmid (Stratagene, La Jolla, CA)
containing ZO-1, which had been digested with MscI and
SpeI to remove the 203-base pair sequence encoding the
native C terminus. All C-terminal deletion constructs were produced
using a variation of this technique. N-terminal deletion constructs
were produced by digesting ZO1myc with restriction enzymes that removed
specific DNA sequences (for example amino acids 2-156 for del 2-156),
and replacing those sequences with an annealed pair of complimentary
oligonucleotides that reestablish the appropriate reading frame. Exact
details of plasmid construction are available from the authors by
request. The pSK+ Bluescript constructs described here were
subsequently digested with KpnI and SpeI and
subcloned into the KpnI and XbaI sites of the
eukaryotic expression vector pCB6 (Karl Matter, University of Geneva,
Geneva, Switzerland).
Cell Culture and Transfection--
MDCK cells were obtained from
Michael Caplan (Yale University, New Haven, CT) and maintained as
described previously (14). Expression constructs were introduced into
cells using a variation on standard calcium-phosphate mediated
transfection (42). For the isolation of stably transfected lines, cells
were selected in 1.0 mg/ml G418 (Life Technologies, Inc.). Colonies
were isolated using cloning loops and subsequently analyzed for
transgene expression by immunofluorescence and immunoblotting with the
anti-myc monoclonal antibody 9E10. In all assays, transgene expression
was induced by supplementing the normal growth medium with 5.0 mM sodium butyrate.
ZO-2 Co-immunoprecipitation Assays--
To examine the
interactions between ZO-1 and ZO-2, two 60-mm dishes of transfected
MDCK cells were placed on ice, washed twice with PBS, and incubated for
30 min in 1.0 ml of extraction/binding buffer (20 mM Tris,
pH 8.0, 150 mM NaCl, 2 mM EDTA, 1.0% Triton X-100, 0.05% SDS, 1.0 mg/ml bovine serum albumin, 1 mM
dithiothreitol, 20 mM phenylmethylsulfonyl fluoride, 20 mM benzamidine, 1.0 µg/ml aprotinin, 1.0 µg/ml
leupeptin, 1.0 µg/ml antipain). Cell lysates were transferred to a
microcentrifuge tube and clarified for 30 min at 18,000 × g. The supernatants were mixed with 40 µl of a 1:1 slurry
of Protein A-Sepharose CL-4B (Pharmacia Biotech, Uppsala, Sweden),
incubated for 2.0 h at 4 °C, and centrifuged for 1 min at
500 × g. Supernatants were then transferred to a new
tube, mixed with 5 µl of the anti-myc monoclonal antibody 9E10, and incubated for 2-15.0 h at 4 °C. Following this incubation, 40 µl
of a 1:1 slurry of Protein A-Sepharose were added and incubated for
2.0 h at 4 °C. The Sepharose beads were washed three times in
extraction/binding buffer, once in extraction/binding buffer without
Triton X-100 or SDS, and resuspended in 50 µl of 2× gel sample
buffer (43). These immunoprecipitates were resolved by SDS-PAGE,
transferred to nitrocellulose filters, and analyzed by Western blotting
using standard techniques (43). Filters were incubated for 2.0 h
in a 1:2500 dilution of the anti-ZO-2 polyclonal antiserum R9989, and
developed using the enhanced chemiluminescence technique (Amersham
Pharmacia Biotech). The same filters were then stripped (Amersham
Pharmacia Biotech) and incubated with a 1:1000 dilution of the anti-myc
ascites 9E10 or a 1:300 dilution of a culture supernatant from the
anti-ZO-1 hybridoma R40.76.
Glutathione S-Transferase (GST)-Occludin Binding Assays--
To
assess the binding of different epitope-tagged proteins to occludin,
confluent monolayers of transfected MDCK cells were washed twice with
PBS+ (containing 1.0 mM CaCl2 and
1.0 mM MgCl2) and lysed in extraction/binding buffer as described for ZO-2 co-immunoprecipitations. The supernatants were then transferred to a new tube, mixed with 40 µl of a 1:1 slurry
of GST beads, and incubated for 2.0 h at 4 °C. The GST beads
were sedimented by a quick pulse in a microcentrifuge, and the
supernatant was transferred to a new tube. A GST fusion protein encoding aa 358-504 of chicken occludin (GST-occ) (44) was prepared as
described by Ausubel et al. (43), and 40 µl of a 1:1
slurry of GST-occ was added to the supernatant and incubated overnight (12-18 h) at 4 °C. The GST-occ beads were then washed, resolved by
SDS-PAGE, and analyzed by Western blotting with the 9E10 sera.
Actin Cosedimentation Assays--
Cells were plated into 100-mm
dishes at confluent density, grown for 2 days, and induced for 20 h with 5.0 mM sodium butyrate. For each construct, two
dishes of cells were washed twice with PBS+ and scraped on
ice into 1.0 ml of a hypotonic carbonate buffer consisting of 10 mM sodium carbonate (pH 11.0), 1.0 mM K-EGTA, 5.0 mM MgCl2, 0.2 mM
dithiothreitol, and protease inhibitors. Cells were homogenized using a
Dounce homogenizer (~50 strokes), and the resulting lysate was
clarified at 100,000 × g for 60 min at 4°/C. The pH
of the resulting supernatant was adjusted to 7.0 by the addition of 9.0 µl of 1.0 M HCl and 20 µl of 1.0 M Tris (pH
7.0) before being used in cosedimentation assays.
To polymerize F-actin, G-actin stocks (Cytoskeleton Inc., Denver, CO)
were diluted to 2.5 mg/ml in binding buffer (10 mM
imidazole, pH 7.2, 75 mM KCl, 5.0 mM
MgCl2, 0.5 mM dithiothreitol), incubated for 30 min on ice, and stabilized by adjusting to 25 µg/ml phalloidin (Molecular Probes, Eugene, OR). F-actin, at a final concentration of 22 µM, was mixed with 60 µl of the cell lysate in binding
buffer in a final volume of 200 µl and incubated for 20 min at room
temperature. Samples were subsequently spun at 100,000 × g for 20 min. Binding was tested in the presence or absence
of 2.0 mM ATP (Boehringer Mannheim) and/or 5.0 µM myosin subfragment-1 (S1) (Sigma) as indicated in the
legend of Fig. 7. Gel samples were prepared as described previously
(45), and examined by Western blotting with the 9E10 antisera.
Immunofluorescence--
To analyze the distribution of the
epitope-tagged proteins in immature and mature cell-cell contacts,
clonal cell lines expressing these constructs were plated at 80%
confluence (106 cells/ml) onto acid-washed 12-mm circular
coverslips and induced for 12 h with 5.0 mM sodium
butyrate starting 24-72 h after plating, as indicated in figure
legend. Alternatively, 106 cells were plated onto 12-mm
Transwell filter inserts (Corning Costar Corp., Cambridge, MA),
incubated for 10 days, and induced for 24 h with 5.0 mM butyrate. Coverslips or filter inserts were then washed
briefly with PBS+ and fixed in freshly prepared 1.0%
paraformaldehyde for 20 min. Subsequently, coverslips were
permeabilized for 15 min with a solution of 0.2% (w/v) Triton X-100,
2.0% donkey serum in PBS, incubated with 2.0% donkey serum (Life
Technologies, Inc.) in PBS for 60 min, and finally incubated for
2.0 h with an undiluted cell supernatant from the 9E10 hybridoma
supplemented with Texas Red-conjugated phalloidin (Molecular Probes,
1:40 dilution), a rat monoclonal antibody against ZO-1 (R40.76 cell
supernatant, 1:10 dilution), or a rabbit polyclonal against human
occludin (Zymed Laboratories Inc. Laboratories, South
San Francisco, CA). Following incubation with the appropriate secondary
antibody (Jackson Immunoresearch, West Grove, PA), coverslips were
mounted on glass slides (Corning) in Vectashield antifade solution
(Vector Laboratories, Burlingame, CA). Slides were viewed on a Nikon
Microfot FX microscope using a 60× PlanApo lens and photographed using
Kodak TMAX 400 film. Alternatively, slides were viewed using a Bio-Rad
MRC1024 confocal microscope on a Zeiss Axiovert using a 63× PlanApo
lens. Film images were digitized using a Sprintscan slide scanner
(Polaroid, Cambridge, MA). Figures were assembled using Adobe Photoshop
(Adobe Systems Inc., Mountain View, CA).
ZO-1 can be divided into an N-terminal half, which constitutes the
region of homology to other members of the MAGUK family, and a unique
proline-rich C-terminal half. Each of these halves contains several
previously identified protein domains whose specific roles are unknown
in ZO-1 (46). To address the functional role of these domains in
protein binding and tight junction assembly, we created a panel of myc
epitope-tagged expression constructs which encode overlapping fragments
of human ZO-1 (Fig. 1; see "Experimental Procedures") and introduced these constructs into cultured MDCK epithelial cells.
The Second PDZ Domain of ZO-1 Mediates the Interaction with
ZO-2--
One of the first molecular interactions to be defined at the
tight junction was that between ZO-1 and ZO-2. These proteins can be
coimmunoprecipitated from epithelial cell extracts (Fig. 2) under relatively stringent conditions,
suggesting a tight and possibly direct interaction (13, 15). However,
if ZO-1 does bind directly to ZO-2, then the striking homology between
these two proteins (53% identity between human ZO-1 and ZO-2) suggests that these proteins might also form homodimers in vivo. To
address these questions and further elucidate the interaction between ZO-1 and ZO-2, the epitope-tagged fusion proteins were
immunoprecipitated from lysates of transfected MDCK cells. The
immunoprecipitates were then resolved by SDS-PAGE and analyzed by
Western blotting with antibodies recognizing the endogenous canine ZO-1
and ZO-2 to identify which constructs coprecipitated either of these
proteins (Fig. 2). We found that all of the constructs used expressed a polypeptide of the appropriate apparent molecular weight (Fig. 2,
myc). In addition, the full-length epitope-tagged construct, ZO1myc, was targeted to the tight junction (discussed below), indicating that the myc epitope does not interfere with these properties of ZO-1.
Analysis of the epitope tag immunoprecipitates with an antibody
(R40.76) that recognizes only the endogenous canine ZO-1 isoform (Fig.
2, ZO-1), and not the transfected human polypeptide,
indicates that the endogenous protein does not coimmunoprecipitate with the full-length epitope-tagged ZO-1 construct under these conditions, nor does it interact with any of the deletion constructs. This result
strongly suggests that ZO-1 does not form homodimers in vivo, but instead forms a simple " Identification of an Occludin-binding Site within Amino Acids
633-876 of ZO-1--
Furuse et al. (16) have identified a
ZO-1 binding site within the C-terminal aa 358-504 of chicken
occludin, and have established that this domain binds directly to the
full-length ZO-1 polypeptide. To identify the reciprocal occludin
binding site on ZO-1, we performed binding assays between the
epitope-tagged ZO-1 constructs and a GST fusion protein encoding this
146-aa domain. Total lysates from MDCK cells transfected with the
epitope-tagged cDNA constructs (Fig.
3A, total) were
incubated with the immobilized GST-occludin (GST-occ) fusion protein,
and the myc-tagged proteins bound to the GST fusion protein were
identified by Western blotting with the anti-myc antisera (Fig.
3A, bound). The relative amount of protein
binding to GST-occ was assessed by comparing the amount of myc-tagged
protein in the bound fraction to that in the total lysate or unbound
fraction. In all cases, the expression of deletion constructs was
identical to or greater than that of the full-length construct
ZO1myc.
An initial comparison of tagged constructs encoding the N- and
C-terminal halves of ZO-1 (z1-876 and del 67-1033, respectively) indicates that only the N-terminal half of ZO-1 associates with the
GST-occ fusion protein (Fig. 3A). Progressive C-terminal
deletions beyond aa 876 severely diminish the ability to associate with GST-occ (Fig. 3B). For example, no binding is detected with
the constructs z1-412, z1-549, and z1-572. These observations
suggest that sequences within the 305-aa domain between aa 572 and 876 mediate a strong interaction with occludin (Fig. 1). The binding site
can be further refined from the observation that the construct del
294-633 also binds to the GST-occ fusion protein. Interactions with
PDZ1 (del 2-156), PDZ2 (del 159-252, del 1-245), PDZ3 (del 294-633), and the SH3 domain (del 550-603) do not appear to be required for binding to occludin (Fig. 3). These results suggest that
the primary occludin binding site resides within the 244-aa domain
between aa 633 and 876 (Fig. 1).
The N-terminal MAGUK-like Half of ZO-1 Associates with Tight
Junctions When Expressed in MDCK Cells--
To determine the
contribution of the N-terminal half, which interacts with ZO-2 and
occludin, and the unique C-terminal half of ZO-1 to tight junction
localization of ZO-1 in MDCK cells, we examined the subcellular
localization of the epitope-tagged constructs encoding these domains by
indirect immunofluorescence. Cells were transiently transfected with
the full-length epitope-tagged ZO-1 (ZO1myc), a construct encoding the
N-terminal half of ZO-1 (z1-876), or a construct encoding the
C-terminal half of ZO-1 (del 67-1033) and were double-labeled with
antibodies against occludin and the myc epitope. Face-on view of
confocal sections from the apical junctional complex indicate that both
the N- and C-terminal constructs were associated with the plasma
membrane at the apical margin of transfected cells at points that
colocalized with the endogenous occludin (Fig.
4). However, vertical sections from the
same fields indicate that only the full-length and N-terminal half of
ZO-1 specifically colocalized with occludin at the tight junction. In
contrast, the C-terminal construct was more uniformly distributed along
the lateral surface relative to either the full-length ZO-1, the
N-terminal construct, and occludin. The C-terminal construct appeared
less focused at the membrane, and was often observed to accumulate in
other actin-rich structures such as apical microvilli and within
discrete puncta on the basal surface (data not shown).
N-terminal Amino Acids 633-896 Are Required for Stable Association
with the Tight Junction--
To better understand how different
domains within the N-terminal half of ZO-1 direct the assembly of ZO-1
into the tight junction, we looked at the distribution of
epitope-tagged constructs at different stages during the assembly of
tight junctions. MDCK cells stably transfected with each construct were
dissociated by treatment in PBS-EDTA, trypsinized, and replated on
glass coverslips at a 80% confluent density. The expression of the
epitope-tagged construct was then induced either 24 or 72 h after
plating by the addition of sodium butyrate into the culture media. At
24 h, cells are still subconfluent, relatively flat, and have not yet developed a barrier to ions. In contrast, by 72 h cells appear columnar, densely packed, and have developed a transepithelial electrical resistance characteristic of this line of MDCK cells (110-130 ohms × cm2). Thus, we define junctions or
cell-cell contacts at the 24- and 72-h time points as functionally
immature and mature, respectively. We have observed that the myc-tagged
constructs used here are fortuitously not expressed in stable lines in
the absence of sodium butyrate (data not shown). Thus, the expression
of a construct can be induced at different points during the assembly
of the tight junction.
There is a dramatic difference in the distribution of different
myc-tagged constructs in immature (24 h) and mature (72 h) cell-cell
contacts. In both immature and mature cell-cell contacts, the
distribution of the full-length ZO-1 and z1-876 are indistinguishable. Both are tightly associated with the plasma membrane at sites of
cell-cell contact (Fig. 5) and colocalize
with the endogenous occludin (Fig. 4). The constructs z1-412 and
z1-572, which are progressively truncated from the C terminus, are
also targeted to cell-cell contacts in subconfluent cells (Fig. 5),
although the pattern of staining was often more discontinuous and less intense than with the full-length construct. However, neither of these
proteins were incorporated to any significant extent into mature tight
junctions. These results suggest that aa 573-876 are required for
stable incorporation of the N-terminal half of ZO-1 into the tight
junction. Significantly, this region contains aa sequences 634-876,
which are required for interaction with the transmembrane protein
occludin. The construct del 159-252 is associated with both immature
and mature cell-cell contacts, suggesting that interactions with ZO-2
mediated by the PDZ2 domain are not required for targeting to cell-cell
contacts.
The construct z1-193, which includes the PDZ1 domain, is not
incorporated into either immature or mature cell-cell contacts, but
instead has a distinct nuclear distribution. A faint but distinct nuclear localization of the full-length and N-terminal construct z1-876 was also detected in immature cell-cell contacts (Fig. 5). The
physiological relevance of nuclear localization is unclear, although
nuclear localization of the endogenous ZO-1 has been previously
reported under similar conditions of low cell density (47).
The Unique C-terminal Half of ZO-1 Associates with F-actin
Structures at the Plasma Membrane--
The distribution of the
C-terminal construct is markedly different from that of z1-876 or the
full-length construct. As previously noted, in mature cell-cell
contacts the C-terminal construct is uniformly distributed along the
lateral plasma membrane (Fig. 4), and does not concentrate within the
tight junction. Del 67-1033 is also concentrated in lamellar
structures that penetrate beneath adjacent cells (Fig. 5), and within
punctate or linear strands that colocalize with the focal adhesion
protein FAK (data not shown). The distinction between N- and C-terminal
constructs is even more dramatic at earlier times during the assembly
of tight junctions. After only 12 h in culture, the full-length
and N-terminal construct z1-876 are only found in a continuous band at
points of cell-cell contact (Fig. 6). The
distribution of these two constructs at cell-cell contacts was much
more punctate and diffuse than that found in more mature monolayers
(Fig. 5) and was indistinguishable from that of adherens junction
protein The C-terminal Half of ZO-1 Associates with F-actin in
Vitro--
The lateral plasma membrane, basal lamellae, and cell-cell
contacts sites are rich in F-actin. The co-distribution of the C-terminal construct with stress fibers and several different compartments within the actin cytoskeleton suggests that ZO-1 may
cross-link components of the tight junction like occludin and ZO-2 with
the cytoskeleton via its C-terminal domain. This would be consistent
with the recent observations of Itoh et al. (41), which
demonstrated that a baculovirus-produced fragment of the C-terminal
half bound directly to F-actin. We confirmed and extended these
results. Cell lysates from transfected cells were incubated with
F-actin, and the ability of the myc-tagged constructs to cosediment
with actin was examined by immunoblotting (Fig.
7A). In the absence of
F-actin, little if any of the myc-tagged protein is found in the
pellet. However, addition of F-actin results in a significant shift of
both the full-length ZO-1 and the C-terminal construct into the pellet
fraction. In contrast, there is no detectable sedimentation of the
N-terminal construct. These results confirm that ZO-1 interacts with
F-actin through its C-terminal domain. Interestingly, addition of
skeletal muscle myosin S1 at equimolar concentrations with actin
results in a loss of ZO-1 from the pellet fraction. The addition of
ATP, which effectively removes myosin from F-actin, results in the
reattachment of ZO-1 to F-actin. ATP alone has no effect on the binding
of ZO-1 to F-actin, and the addition of myosin S1, ATP, and cell lysate
have no effect on the polymerization of F-actin (Fig. 7B).
These results suggest that ZO-1 binds specifically to F-actin in an
ATP-independent manner, and suggest that the binding site on F-actin
might overlap with that of myosins.
In the present study, we have identified domains in the tight
junction protein ZO-1 that mediate interactions with ZO-2, occludin, and F-actin. The N-terminal half of ZO-1, which is structurally similar
to other members of the MAGUK family of membrane-associated signaling
proteins, binds to the tight junction proteins occludin and ZO-2, while
the interaction with the actin cytoskeleton is mediated by the unique
proline-rich C-terminal half. Furthermore, we have identified a 244-aa
domain within the N-terminal half of ZO-1, which is required for the
stable association of ZO-1 with the tight junction and which includes
the occludin-binding site. Thus, the N-terminal half of ZO-1 has a
functional role analogous to other MAGUK proteins organizing
transmembrane and cytosolic components at specialized cell-cell contact
sites. Unlike other MAGUKs, ZO-1 has a unique C-terminal domain that
mediates direct interaction with actin. We suggest that ZO-1 forms a
functional link between the paracellular seal and the actin cytoskeleton.
The N-terminal MAGUK-like Half of ZO-1 Mediates the Interactions
with ZO-2 and Occludin--
The nature of the interaction of ZO-1 with
ZO-2 and occludin suggests that ZO-1 has a role in the organization of
protein complexes analogous to that of other MAGUKs. Using
coprecipitation assays, we found that ZO-2 specifically interacts with
the second PDZ domain of ZO-1. PDZ domains are a conserved motif found
in a diverse array of proteins, and have been shown to mediate
protein-protein interactions at the plasma membrane (48, 49). The
majority of the PDZ domains examined to date bind to consensus motifs
located at the C terminus of transmembrane proteins (50, 51), although several PDZ domains also interact directly with other PDZ domains (31,
36). Since ZO-2 does not have a C-terminal sequence resembling any of
the known consensus motifs, we predict that ZO-1 might bind directly to
one of the three PDZ domains in ZO-2. The ability to
coimmunoprecipitate ZO-1 and ZO-2 under harsh conditions without stoichiometric coprecipitation of another protein supports the idea of
a direct interaction between these two proteins (13, 15), and several
other MAGUKs have been demonstrated to form heterodimers by direct
interaction between PDZ domains (31). However, we have been unable to
confirm the interaction between ZO-1 and any of the three PDZ domains
in ZO-2 using yeast two-hybrid, blot overlay, or fusion protein binding
assays (data not shown). These in vitro experiments are
difficult to interpret because of an inability to confirm proper
folding and post translational processing of the polypeptides used.
Nevertheless, they raise the possibility that the interaction between
ZO-1 and ZO-2 is mediated by a third protein.
Occludin also interacts with the N-terminal half of ZO-1, but unlike
other MAGUKs the interaction with this transmembrane protein is not
mediated by a PDZ domain. Instead, our binding assays indicate that
there is a strong interaction between GST-occ and the 244-aa domain
between aa 633 and 876 of ZO-1. This region contains both the GUK
domain (the region of homology to guanylate kinase) and an acidic
domain of unknown function. Several proteins have recently been
identified that bind to the GUK region of other MAGUKs, although none
of these proteins shows any homology to occludin (52, 53). Both domains
are also conserved in ZO-2 (63% identity) and ZO-3 (43% identity),
suggesting that these proteins may also bind directly to occludin.
Indeed, ZO-3 has recently been demonstrated to bind directly to the
cytosolic tail of occludin (37), although the nature of the binding
site has not been determined. However, the interaction between ZO-1 and occludin does not require ZO-2, since the deletion of the PDZ2 domain
does not interfere with the ability of ZO-1 to bind occludin.
Differential Localization of ZO-1 Constructs Supports a Multistep
Assembly of Tight Junctions--
The localization of ZO-1 to the
plasma membrane coincides with the formation of cell-cell contacts (54,
55), although development of a measurable paracellular barrier
sometimes lags behind this initial association by days. Previous
localization studies in MTD-1A cells have demonstrated that ZO-1 is
initially assembled into actin-rich cadherin based cell-cell contacts,
and subsequently segregated into a distinct apical compartment as cells
became polarized (56). Thus, the assembly of tight junctions is
presumably a multistep process that may involve distinct
protein-protein interactions over time. At the earliest time that we
can detect ZO-1 at the plasma membrane (12-24 h; see Figs. 5 and 6),
it is diffusely distributed at points of cell-cell contact, sometimes focused within discrete puncta. By 72 h after plating, when a electrically resistive barrier has formed, the distribution appears as
a tight band at the apical junction complex (Fig. 5). Although any
construct containing aa 194-876 is found in immature cell-cell contacts (z1-412, z1-572, and z1-876; see Fig. 1), only constructs containing 573-876 are localized to the mature tight junction in
polarized epithelial cells (z1-876, Fig. 1). One interpretation of
these data is that some domain or domains within aa 194-572 is
responsible for the association with immature cell-cell contacts, while
a distinct domain within aa 573-876 is required to remain stably
incorporated in mature tight junctions. Thus, the association of ZO-1
with the plasma membrane during different stages of assembly might be
mediated by distinct protein-protein interactions.
The specific interactions that mediate association with immature
versus mature cell-cell contacts is still unresolved. One model suggests that junction formation is initiated by cadherin engagement, and that ZO-1 is recruited to new cell-cell contacts by
interacting with a cadherin-binding protein like ZO-1 Links Actin Filaments to Proteins of the Tight
Junction--
The present study demonstrates that ZO-1 not only binds
to F-actin in vitro, as previously reported (41), but also
associates specifically with actin filaments at the plasma membrane of
cultured MDCK cells in vivo. The C-terminal half of ZO-1,
when expressed in subconfluent cells, preferentially targets to actin
filaments that terminate at the plasma membrane in focal contacts,
cell-cell contacts, microvilli, and within lamellae. This is similar to the observations of Howarth et al. (60, 61) and Yonemura
et al. (56), who found that ZO-1 localizes with a subset of
actin filaments at cell-cell contacts and in lamellae of cultured
astrocytes and transformed fibroblasts. In contrast, Itoh et
al. (41) have reported that a construct encoding the C-terminal
half of ZO-1 is uniformly distributed along stress fibers in cultured
fibroblasts. This may be due in part to a difference in cell types,
expression levels, or the exact nature of the constructs used. In our
own studies in the NRK fibroblast line, we find that our C-terminal construct concentrates primarily within the sites of focal and cell-cell contact (data not shown). These results taken together suggest that ZO-1 may have some mechanism which promotes association with a specific subset of actin filaments at the membrane that is
independent of interactions with ZO-2, occludin, or
It is not clear to what extent interactions with F-actin are involved
in assembly of ZO-1 into the tight junction, or if the cytoskeleton has
a general role in the assembly of the tight junction. The N-terminal
construct, which lacks ability to bind to F-actin, is still efficiently
incorporated into both immature and mature cell-cell contacts. However,
there is considerable evidence suggesting that the perijunctional actin
is involved in the maintenance or assembly of the paracellular seal
(62, 63). Signaling pathways that affect the organization of
perijunctional actin (64-67) often modify tight junction permeability
(63). For example, activation of small GTP-binding proteins of the Rho
family triggers a marked reorganization of perijunctional actin and
increase in paracellular permeability (65, 68). In addition, treatment
of cultured epithelial cells with cytochalasins causes a decrease in
the size and complexity of tight junction fibrils (69-72). However, as
most of these interventions cause widespread alterations of the
cytoskeleton, the results are difficult to attribute to specific
affects on tight junctions.
Several recent studies suggest that actomyosin contraction is also
directly involved in the physiological regulation of paracellular permeability. The tight junction is adjacent to a perijunctional ring
of actin that is capable of circumferential contraction both in
vitro and in vivo (5, 73). Studies in both isolated
tissues and cultured cells have demonstrated that activation of the
sodium glucose transporter leads to cytoskeletal contraction and a
concurrent increase in paracellular permeability (5, 74, 75). In
cultured cells this has been shown to occur in concert with the
phosphorylation of myosin light chain, which is known to activate
nonmuscle myosin contractility (76). In addition, Hecht et
al. (77) have shown that the introduction of a constitutively
activated myosin light chain kinase into cultured cells results in a
contraction of the cortical cytoskeleton, a decrease in transepithelial
electrical resistance, and increased paracellular flux. These
observations have led to the idea that paracellular permeability is
directly regulated by cortical tension generated by actomyosin contraction.
The results of this study are consistent with the idea that ZO-1 has an
organizational role analogous to that of other MAGUKs. For example,
ZO-1 binds to both the transmembrane protein occludin and the
peripheral membrane protein ZO-2, suggesting that it may be involved in
the organization of these proteins at the tight junction. This role is
supported by the recent observation that ZO-1 is involved in the
targeting and incorporation of occludin into tight
junctions.2 Like other
MAGUKs, ZO-1 also has the potential to act as a scaffold for the
organization of signal transduction proteins (19, 38-40). Finally,
ZO-1 binds to F-actin (41), suggesting that it may mediate the
anchoring or assembly of actin filaments at the tight junction. To date
neither ZO-2, ZO-3, nor occludin have been demonstrated to interact
directly with F-actin. The unique C-terminal regions of ZO-2 and ZO-3
show little similarity to ZO-1, and neither protein contains the
conserved protein 4.1 binding motif found in other MAGUKs. Thus, it is
possible that actin-binding is limited to ZO-1, and that ZO-1 may have
the unique potential among tight junction MAGUKs to organize both
structural and signaling components of the paracellular seal.
We are indebted to Daniel Goodenough
(National Institutes of Health Grant GM18974), Bruce Stevenson, Peter
Novick, Sandra Wolin, and Karl Matter for providing reagents, and to
Pat Brennwald, David Lewin, Min Kim, Tama Hasson, Joe Wolenski, Bill
Bement, and Maria Susanna Balda for technical advice. Special thanks to Eveline Schneeberger for her part in a collaborative effort
complementing observations made in this paper. We acknowledge the
expert technical assistance of Alexandra R. Cohen.
*
This work was supported in part by National Institutes of
Health Grants DK45134 and CA66263 (to J. M. A.) and Grant
DK34989 to the Yale Liver Center.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by National Institutes of Health Grant NRSA DK09261 and
the Irwin M. Arias Postdoctoral Research Fellowship from the American
Liver Foundation. To whom all correspondence should be addressed: Dept.
of Internal Medicine, Digestive Diseases, Yale School of Medicine, 333 Cedar St., P. O. Box 208019, New Haven, CT 06520-8019. Tel.:
203-785-4133; Fax: 203-785-7273; E-mail: alan.fanning{at}yale.edu.
The abbreviations used are:
aa, amino acid(s); GST, glutathione S-transferase; MAGUK, membrane-associated
guanylate kinase homolog; SH3, Src homology 3; MDCK, Madin-Darby canine
kidney; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel
electrophoresis; PDZ, PSD95/dlg/ZO-1.
2
L. L. Mitic, E. S. Schneeberger, A. S. Fanning, and J. M. Anderson, submitted for publication.
The Tight Junction Protein ZO-1 Establishes a Link between the
Transmembrane Protein Occludin and the Actin Cytoskeleton*
§,,
Internal Medicine and
Cell Biology, Yale School of Medicine, New Haven, Connecticut
06510 and the ¶ Department of Plant and Microbial Biology,
University of California, Berkeley, California 94720
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (40K):
[in a new window]
Fig. 1.
Diagram of ZO-1 protein and myc-tagged
expression constructs. Constructs are designated by the amino
acids included (e.g. z1-193) or deleted (e.g.
del 294-633). Black lines denote the extent of
the sequences encoded by each construct. The results of the ZO-2 and
occludin-binding studies are indicated at left. The
vertical dark shaded box
denotes the boundaries of PDZ2. The vertical
light shaded box denotes the
boundaries of the occludin-binding region. 
,
1, and 2 are alternatively spliced domains.
The 
symbol denotes an acidic domain; GuK, the region
of homology to guanylate kinase; SH3, Src homology 3 domain;
PDZ, PSD95/dlg/ZO-1 domain. All constructs are tagged at the
C terminus with an 11-amino acid epitope from the c-myc
proto-oncogene and subcloned into the pCB6 eukaryotic expression
vector.
View larger version (51K):
[in a new window]
Fig. 2.
ZO-2 coprecipitates only with those
myc-tagged ZO-1 constructs that contain the PDZ2 domain. The
myc-tagged constructs were immunoprecipitated from transiently
transfected MDCK cells using an antibody directed against the myc
epitope, resolved by SDS-PAGE, and transferred to nitrocellulose as
described under "Experimental Procedures." A ZO-1 immunoprecipitate
from untransfected MDCK cells is included as a positive control
(ZO-1 I.P.). The filter was subsequently probed with
antibodies against ZO-2 (R9989), stripped, reprobed with an antibody
that recognizes only the endogenous canine ZO-1 isoforms (R40.76),
stripped again, and reprobed with the antibody that recognizes the myc
epitope (9E10). Molecular size standards are included on the
right (kDa).
" heterodimer with
ZO-2. When the same blots were probed with a polyclonal antisera
against ZO-2, we found that only deletion constructs which still
contained the second PDZ domain of ZO-1 (PDZ2) could coprecipitate the
endogenous ZO-2 polypeptide (Fig. 2, ZO-2). Furthermore, a
construct in which only PDZ2 was specifically deleted (del 159-252)
also fails to coprecipitate ZO-2. These results, summarized in Fig. 1,
indicate that the PDZ2 domain mediates the interaction with ZO-2.
View larger version (47K):
[in a new window]
Fig. 3.
Binding of the myc-tagged ZO-1 constructs to
a GST-occludin fusion protein. A GST fusion protein encoding
C-terminal amino acids 358-504 of the chicken occludin was attached to
glutathione beads and mixed with cell lysates from MDCK cells
transfected with the myc-tagged ZO-1 constructs. Specifically bound
proteins were detected by immunoblotting for the myc epitope. In
panel A, a sample of the total cell lysates
(total) from stable cell lines expressing the full-length
epitope-tagged ZO-1 (ZO1myc), the N-terminal half of ZO-1
(z1-876), the C-terminal half of ZO-1 (del
67-1033), or transfected with the vector alone (pCB6)
are compared with the fraction of the lysate that is bound to GST-occ
(bound). Note that the full-length and N-terminal half of
ZO-1 interact with GST-occ, but that the C-terminal half fails to bind.
Panel B shows the bound proteins from a survey of
all of the epitope-tagged ZO-1 constructs. In each case expression was
confirmed either by examination of the total cell lysate or by Western
blotting of the unbound fractions and determined to be equivalent to
that seen with the full-length protein.
View larger version (101K):
[in a new window]
Fig. 4.
Distribution of myc-tagged proteins in
polarized MDCK cell monolayers. Stable MDCK lines expressing the
full-length (ZO1myc), N-terminal (z1-876), or
C-terminal half (del 67-1033) of ZO-1 were plated on
collagen treated filter inserts and cultured for 10 days before
fixation and staining with antibodies against the myc epitope
(myc) and the endogenous occludin (occludin). A
scanning laser confocal microscope was used to collect sequential
0.36-µm-thick face-on (top) or vertical
(bottom) sections. Note that both the full-length and
N-terminal constructs colocalize with occludin at the tight junction,
whereas the C-terminal construct is distributed uniformly along the
lateral plasma membrane. The horizontal field size is 87.5 µm.
View larger version (73K):
[in a new window]
Fig. 5.
Localization of the myc-tagged constructs in
immature and mature cell-cell junctions. Expression of the tagged
constructs was induced in stable MDCK lines either 24 (immature) or
72 h (mature) after plating at approximately 80% density. Cells
were induced for approximately 12 h with sodium butyrate, and
subsequently fixed and stained with the anti-myc antibody. Transgene
expression is completely absent before sodium butyrate induction, as
assayed by immunofluorescence (data not shown). All of the constructs,
except for a construct that only contains the first PDZ domain,
z1-193, are incorporated into immature cell-cell contacts (24 h).
However, only z1-876 and del 159-252 are incorporated into the tight
junctions of mature cell-cell contacts. Constructs z1-193, z1-412,
and z1-572 remain primarily within the cytosol. The C-terminal
construct, del 67-1033, is associated with mature cell-cell contacts
along the entire lateral surface, as previously noted. Bar,
50 µm.
catenin (data not shown). In contrast, the C-terminal
construct was distributed along actin stress fibers at points where
they terminated at the basal plasma membrane, and appeared to be only
weakly associated with the plasma membrane at cell-cell contacts (Fig.
6). In rare instances of very high level expression, the full-length
construct ZO1myc could also be found in basal lamellae, focal contacts,
and stress fibers, as well as sites of cell-cell contacts. Such a
pattern was never observed with z1-876. These results suggest that the
C-terminal half of ZO-1 mediates interactions with the actin
cytoskeleton at the plasma membrane while the N-terminal half, which
binds to both ZO-2 and occludin, mediates the specific localization of
ZO-1 to tight junctions.
View larger version (116K):
[in a new window]
Fig. 6.
The C-terminal half of ZO-1 colocalizes with
actin filaments at the plasma membrane of MDCK cells. MDCK cells
stably transfected with constructs encoding the full-length
(ZO1myc), N-terminal half (z1-876), or the
C-terminal half (del 67-1033) of ZO-1 were
trypsinized and plated in the presence of sodium butyrate to induce
expression of the tagged constructs during cell adhesion and spreading.
After 12 h the cells were fixed and double labeled with antibodies
against the myc epitope (myc) and Texas Red phalloidin
(actin). Note that when cells are still relatively flat and
well spread, the C-terminal construct is primarily distributed along
actin stress fibers, particularly where these stress fibers appear to
terminate at the plasma membrane. Bar, 50 µm.
View larger version (57K):
[in a new window]
Fig. 7.
The C-terminal half of ZO-1 mediates the
interaction with F-actin. A, cell lysates from clonal
lines expressing either the full-length (ZO1myc), N-terminal
half (z1-876), or C-terminal half (d67-1033) of
ZO-1 were mixed with F-actin and tested for their ability to cosediment
in the presence (+) and/or absence ( ) of ATP and myosin subfragment-1
(myosin S1) as described in methods. The soluble
(Sup) and actin-bound (Pel) fractions were
resolved by SDS-PAGE and the epitope-tagged proteins detected by
Western blotting with the anti-myc antibody 9E10. B,
Coomassie-stained gel of the same assay. Note that the amount of
F-actin pelleting is unaffected by the addition of lysate, myosin S1,
or ATP.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
- or -catenin (41, 56-58). Both of these proteins have the potential to interact with ZO-1, and target to the plasma membrane at sites of cell-cell contact (41, 57). In addition, the distribution of both ZO1myc and
z1-876 is indistinguishable from that of catenin in very early
cell-cell contacts (data not shown). Thus, the domain between aa 194 and 572, which is required for incorporation into early cell-cell
contacts, may in fact interact with one of the catenins. The sequences
that are required for association with mature cell-cell contacts (aa
573-876) include the 244-aa domain that binds to occludin, raising the
possibility that association with occludin is required for the stable
assembly of ZO-1 into mature tight junctions. However, ZO-1
distribution appears normal in epithelia generated from mice lacking
occludin (59), suggesting that interaction with occludin is not
absolutely required for assembly of ZO-1 into the tight junction.
Understanding assembly will thus ultimately require the further
resolution of these two domains and the identification of the proteins
which interact with them.
-catenin. The
precise location of the actin-binding motif within the C-terminal half
of ZO-1 is currently under investigation.
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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 V. S. Subramanian, J. S. Marchant, D. Ye, T. Y. Ma, and H. M. Said Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1717 - C1726. [Abstract] [Full Text] [PDF]
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P. Zeni, E. Doepker, U. S. Topphoff, S. Huewel, T. Tenenbaum, and H.-J. Galla MMPs contribute to TNF-{alpha}-induced alteration of the blood-cerebrospinal fluid barrier in vitro Am J Physiol Cell Physiol, September 1, 2007; 293(3): C855 - C864. [Abstract] [Full Text] [PDF]
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 G. Gaidos, S. Soni, D. J. Oswald, P. A. Toselli, and K. H. Kirsch Structure and function analysis of the CMS/CIN85 protein family identifies actin-bundling properties and heterotypic-complex formation J. Cell Sci., July 15, 2007; 120(14): 2366 - 2377. [Abstract] [Full Text] [PDF]
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 L. Eiselein, D. W. Wilson, M. W. Lame, and J. C. Rutledge Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2745 - H2753. [Abstract] [Full Text] [PDF]
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 M. Benezra, R. S. Greenberg, and S. K. Masur Localization of ZO-1 in the Nucleolus of Corneal Fibroblasts Invest. Ophthalmol. Vis. Sci., May 1, 2007; 48(5): 2043 - 2049. [Abstract] [Full Text] [PDF]
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 A. T. Blikslager, A. J. Moeser, J. L. Gookin, S. L. Jones, and J. Odle Restoration of Barrier Function in Injured Intestinal Mucosa Physiol Rev, April 1, 2007; 87(2): 545 - 564. [Abstract] [Full Text] [PDF]
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A. S. Fanning, B. P. Little, C. Rahner, D. Utepbergenov, Z. Walther, and J. M. Anderson The Unique-5 and -6 Motifs of ZO-1 Regulate Tight Junction Strand Localization and Scaffolding Properties Mol. Biol. Cell, March 1, 2007; 18(3): 721 - 731. [Abstract] [Full Text] [PDF]
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P. Brun, I. Castagliuolo, V. D. Leo, A. Buda, M. Pinzani, G. Palu, and D. Martines Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis Am J Physiol Gastrointest Liver Physiol, February 1, 2007; 292(2): G518 - G525. [Abstract] [Full Text] [PDF]
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A. Reijerkerk, G. Kooij, S. M. A. van der Pol, S. Khazen, C. D. Dijkstra, and H. E. de Vries Diapedesis of monocytes is associated with MMP-mediated occludin disappearance in brain endothelial cells FASEB J, December 1, 2006; 20(14): 2550 - 2552. [Abstract] [Full Text] [PDF]
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 J. A. Guttman, F. N. Samji, Y. Li, A. W. Vogl, and B. B. Finlay Evidence that Tight Junctions Are Disrupted Due to Intimate Bacterial Contact and Not Inflammation during Attaching and Effacing Pathogen Infection In Vivo Infect. Immun., November 1, 2006; 74(11): 6075 - 6084. [Abstract] [Full Text] [PDF]
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J.-F. Lee, Q. Zeng, H. Ozaki, L. Wang, A. R. Hand, T. Hla, E. Wang, and M.-J. Lee Dual Roles of Tight Junction-associated Protein, Zonula Occludens-1, in Sphingosine 1-Phosphate-mediated Endothelial Chemotaxis and Barrier Integrity J. Biol. Chem., September 29, 2006; 281(39): 29190 - 29200. [Abstract] [Full Text] [PDF]
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D. I. Utepbergenov, A. S. Fanning, and J. M. Anderson Dimerization of the Scaffolding Protein ZO-1 through the Second PDZ Domain J. Biol. Chem., August 25, 2006; 281(34): 24671 - 24677. [Abstract] [Full Text] [PDF]
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Y. Zhang, S. Yeh, B. A. Appleton, H. A. Held, P. J. Kausalya, D. C. Y. Phua, W. Lee Wong, L. A. Lasky, C. Wiesmann, W. Hunziker, et al. Convergent and Divergent Ligand Specificity among PDZ Domains of the LAP and Zonula Occludens (ZO) Families J. Biol. Chem., August 4, 2006; 281(31): 22299 - 22311. [Abstract] [Full Text] [PDF]
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T. Jons, D. Wittschieber, A. Beyer, C. Meier, A. Brune, A. Thomzig, G. Ahnert-Hilger, and R. W. Veh K+-ATP-channel-related protein complexes: potential transducers in the regulation of epithelial tight junction permeability J. Cell Sci., August 1, 2006; 119(15): 3087 - 3097. [Abstract] [Full Text] [PDF]
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 S. Cuzzocrea, G. Nocentini, R. Di Paola, M. Agostini, E. Mazzon, S. Ronchetti, C. Crisafulli, E. Esposito, A. P. Caputi, and C. Riccardi Proinflammatory Role of Glucocorticoid-Induced TNF Receptor-Related Gene in Acute Lung Inflammation J. Immunol., July 1, 2006; 177(1): 631 - 641. [Abstract] [Full Text] [PDF]
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 Y. Persidsky, D. Heilman, J. Haorah, M. Zelivyanskaya, R. Persidsky, G. A. Weber, H. Shimokawa, K. Kaibuchi, and T. Ikezu Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE) Blood, June 15, 2006; 107(12): 4770 - 4780. [Abstract] [Full Text] [PDF]
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L. Shen, E. D. Black, E. D. Witkowski, W. I. Lencer, V. Guerriero, E. E. Schneeberger, and J. R. Turner Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure J. Cell Sci., May 15, 2006; 119(10): 2095 - 2106. [Abstract] [Full Text] [PDF]
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A. Ikari, S. Matsumoto, H. Harada, K. Takagi, H. Hayashi, Y. Suzuki, M. Degawa, and M. Miwa Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions J. Cell Sci., May 1, 2006; 119(9): 1781 - 1789. [Abstract] [Full Text] [PDF]
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 G. A. Tarulli, P. G. Stanton, A. Lerchl, and S. J. Meachem Adult Sertoli Cells Are Not Terminally Differentiated in the Djungarian Hamster: Effect of FSH on Proliferation and Junction Protein Organization Biol Reprod, May 1, 2006; 74(5): 798 - 806. [Abstract] [Full Text] [PDF]
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E. McNeil, C. T. Capaldo, and I. G. Macara Zonula Occludens-1 Function in the Assembly of Tight Junctions in Madin-Darby Canine Kidney Epithelial Cells Mol. Biol. Cell, April 1, 2006; 17(4): 1922 - 1932. [Abstract] [Full Text] [PDF]
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M. Anitei, M. Ifrim, M.-A. Ewart, A. E. Cowan, J. H. Carson, R. Bansal, and S. E. Pfeiffer A role for Sec8 in oligodendrocyte morphological differentiation J. Cell Sci., March 1, 2006; 119(5): 807 - 818. [Abstract] [Full Text] [PDF]
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L. DeMaio, M. Rouhanizadeh, S. Reddy, A. Sevanian, J. Hwang, and T. K. Hsiai Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H674 - H683. [Abstract] [Full Text] [PDF]
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D. Mehta and A. B. Malik Signaling Mechanisms Regulating Endothelial Permeability Physiol Rev, January 1, 2006; 86(1): 279 - 367. [Abstract] [Full Text] [PDF]
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M. E. Osler, M. S. Chang, and D. M. Bader Bves modulates epithelial integrity through an interaction at the tight junction J. Cell Sci., October 15, 2005; 118(20): 4667 - 4678. [Abstract] [Full Text] [PDF]
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M. Utech, A. I. Ivanov, S. N. Samarin, M. Bruewer, J. R. Turner, R. J. Mrsny, C. A. Parkos, and A. Nusrat Mechanism of IFN-{gamma}-induced Endocytosis of Tight Junction Proteins: Myosin II-dependent Vacuolarization of the Apical Plasma Membrane Mol. Biol. Cell, October 1, 2005; 16(10): 5040 - 5052. [Abstract] [Full Text] [PDF]
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M. M. Walsh-Reitz, E. F. Huang, M. W. Musch, E. B. Chang, T. E. Martin, S. Kartha, and F. G. Toback AMP-18 protects barrier function of colonic epithelial cells: role of tight junction proteins Am J Physiol Gastrointest Liver Physiol, July 1, 2005; 289(1): G163 - G171. [Abstract] [Full Text] [PDF]
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M. D. Luyer, W. A. Buurman, M. Hadfoune, G. Speelmans, J. Knol, J. A. Jacobs, C. H. C. Dejong, A. J. M. Vriesema, and J. W. M. Greve Strain-Specific Effects of Probiotics on Gut Barrier Integrity following Hemorrhagic Shock Infect. Immun., June 1, 2005; 73(6): 3686 - 3692. [Abstract] [Full Text] [PDF]
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A. Kantardzhieva, I. Gosens, S. Alexeeva, I. M. Punte, I. Versteeg, E. Krieger, C. A. Neefjes-Mol, A. I. den Hollander, S. J. F. Letteboer, J. Klooster, et al. MPP5 Recruits MPP4 to the CRB1 Complex in Photoreceptors Invest. Ophthalmol. Vis. Sci., June 1, 2005; 46(6): 2192 - 2201. [Abstract] [Full Text] [PDF]
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M. Bruewer, M. Utech, A. I. Ivanov, A. M. Hopkins, C. A. Parkos, and A. Nusrat Interferon-{gamma} induces internalization of epithelial tight junction proteins via a macropinocytosis-like process FASEB J, June 1, 2005; 19(8): 923 - 933. [Abstract] [Full Text] [PDF]
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 B. T. Hawkins and T. P. Davis The Blood-Brain Barrier/Neurovascular Unit in Health and Disease Pharmacol. Rev., June 1, 2005; 57(2): 173 - 185. [Abstract] [Full Text] [PDF]
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J. G. Laing, B. C. Chou, and T. H. Steinberg ZO-1 alters the plasma membrane localization and function of Cx43 in osteoblastic cells J. Cell Sci., May 15, 2005; 118(10): 2167 - 2176. [Abstract] [Full Text] [PDF]
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K. S.M. Smalley, P. Brafford, N. K. Haass, J. M. Brandner, E. Brown, and M. Herlyn Up-Regulated Expression of Zonula Occludens Protein-1 in Human Melanoma Associates with N-Cadherin and Contributes to Invasion and Adhesion Am. J. Pathol., May 1, 2005; 166(5): 1541 - 1554. [Abstract] [Full Text] [PDF]
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A. Nusrat, G. T. Brown, J. Tom, A. Drake, T. T.T. Bui, C. Quan, and R. J. Mrsny Multiple Protein Interactions Involving Proposed Extracellular Loop Domains of the Tight Junction Protein Occludin Mol. Biol. Cell, April 1, 2005; 16(4): 1725 - 1734. [Abstract] [Full Text] [PDF]
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S. L. Muller, M. Portwich, A. Schmidt, D. I. Utepbergenov, O. Huber, I. E. Blasig, and G. Krause The Tight Junction Protein Occludin and the Adherens Junction Protein {alpha}-Catenin Share a Common Interaction Mechanism with ZO-1 J. Biol. Chem., February 4, 2005; 280(5): 3747 - 3756. [Abstract] [Full Text] [PDF]
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R. Vogelmann and W. J. Nelson Fractionation of the Epithelial Apical Junctional Complex: Reassessment of Protein Distributions in Different Substructures Mol. Biol. Cell, February 1, 2005; 16(2): 701 - 716. [Abstract] [Full Text] [PDF]
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L. Taliana, M. Benezra, R. S. Greenberg, S. K. Masur, and A. M. Bernstein ZO-1: Lamellipodial Localization in a Corneal Fibroblast Wound Model Invest. Ophthalmol. Vis. Sci., January 1, 2005; 46(1): 96 - 103. [Abstract] [Full Text] [PDF]
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 C. Graham and N. L. Simmons Functional organization of the bovine rumen epithelium Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2005; 288(1): R173 - R181. [Abstract] [Full Text] [PDF]
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 C. M. Van Itallie and J. M. Anderson The Molecular Physiology of Tight Junction Pores Physiology, December 1, 2004; 19(6): 331 - 338. [Abstract] [Full Text] [PDF]
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P. J. Kausalya, D. C.Y. Phua, and W. Hunziker Association of ARVCF with Zonula Occludens (ZO)-1 and ZO-2: Binding to PDZ-Domain Proteins and Cell-Cell Adhesion Regulate Plasma Membrane and Nuclear Localization of ARVCF Mol. Biol. Cell, December 1, 2004; 15(12): 5503 - 5515. [Abstract] [Full Text] [PDF]
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 M. M. Thi, J. M. Tarbell, S. Weinbaum, and D. C. Spray The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A "bumper-car" model PNAS, November 23, 2004; 101(47): 16483 - 16488. [Abstract] [Full Text] [PDF]
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L. Fraemohs, R. R. Koenen, G. Ostermann, B. Heinemann, and C. Weber The Functional Interaction of the {beta}2 Integrin Lymphocyte Function-Associated Antigen-1 with Junctional Adhesion Molecule-A Is Mediated by the I Domain J. Immunol., November 15, 2004; 173(10): 6259 - 6264. [Abstract] [Full Text] [PDF]
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 K. Kawkitinarong, L. Linz-McGillem, K. G. Birukov, and J. G. N. Garcia Differential Regulation of Human Lung Epithelial and Endothelial Barrier Function by Thrombin Am. J. Respir. Cell Mol. Biol., November 1, 2004; 31(5): 517 - 527. [Abstract] [Full Text] [PDF]
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K. Umeda, T. Matsui, M. Nakayama, K. Furuse, H. Sasaki, M. Furuse, and S. Tsukita Establishment and Characterization of Cultured Epithelial Cells Lacking Expression of ZO-1 J. Biol. Chem., October 22, 2004; 279(43): 44785 - 44794. [Abstract] [Full Text] [PDF]
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L. Guillemot, E. Hammar, C. Kaister, J. Ritz, D. Caille, L. Jond, C. Bauer, P. Meda, and S. Citi Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression J. Cell Sci., October 15, 2004; 117(22): 5245 - 5256. [Abstract] [Full Text] [PDF]
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H. Wen, D. D. Watry, M. C. G. Marcondes, and H. S. Fox Selective Decrease in Paracellular Conductance of Tight Junctions: Role of the First Extracellular Domain of Claudin-5 Mol. Cell. Biol., October 1, 2004; 24(19): 8408 - 8417. [Abstract] [Full Text] [PDF]
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 D. D. Mruk and C. Y. Cheng Sertoli-Sertoli and Sertoli-Germ Cell Interactions and Their Significance in Germ Cell Movement in the Seminiferous Epithelium during Spermatogenesis Endocr. Rev., October 1, 2004; 25(5): 747 - 806. [Abstract] [Full Text] [PDF]
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M. Cereijido, R. G. Contreras, and L. Shoshani Cell Adhesion, Polarity, and Epithelia in the Dawn of Metazoans Physiol Rev, October 1, 2004; 84(4): 1229 - 1262. [Abstract] [Full Text] [PDF]
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M. Bruewer, A. M. Hopkins, M. E. Hobert, A. Nusrat, and J. L. Madara RhoA, Rac1, and Cdc42 exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and F-actin Am J Physiol Cell Physiol, August 1, 2004; 287(2): C327 - C335. [Abstract] [Full Text] [PDF]
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N. P.Y. Lee and C. Y. Cheng Adaptors, Junction Dynamics, and Spermatogenesis Biol Reprod, August 1, 2004; 71(2): 392 - 404. [Abstract] [Full Text] [PDF]
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G. Bazzoni and E. Dejana Endothelial Cell-to-Cell Junctions: Molecular Organization and Role in Vascular Homeostasis Physiol Rev, July 1, 2004; 84(3): 869 - 901. [Abstract] [Full Text] [PDF]
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E. E. Schneeberger and R. D. Lynch The tight junction: a multifunctional complex Am J Physiol Cell Physiol, June 1, 2004; 286(6): C1213 - C1228. [Abstract] [Full Text] [PDF]
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T. Y. Ma, G. K. Iwamoto, N. T. Hoa, V. Akotia, A. Pedram, M. A. Boivin, and H. M. Said TNF-{alpha}-induced increase in intestinal epithelial tight junction permeability requires NF-{kappa}B activation Am J Physiol Gastrointest Liver Physiol, March 1, 2004; 286(3): G367 - G376. [Abstract] [Full Text] [PDF]
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X. Han, M. P. Fink, T. Uchiyama, R. Yang, and R. L. Delude Increased iNOS activity is essential for pulmonary epithelial tight junction dysfunction in endotoxemic mice Am J Physiol Lung Cell Mol Physiol, February 1, 2004; 286(2): L259 - L267. [Abstract] [Full Text] [PDF]
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A. B. Singh and R. C. Harris Epidermal Growth Factor Receptor Activation Differentially Regulates Claudin Expression and Enhances Transepithelial Resistance in Madin-Darby Canine Kidney Cells J. Biol. Chem., January 30, 2004; 279(5): 3543 - 3552. [Abstract] [Full Text] [PDF]
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E. Raschperger, U. Engstrom, R. F. Pettersson, and J. Fuxe CLMP, a Novel Member of the CTX Family and a New Component of Epithelial Tight Junctions J. Biol. Chem., January 2, 2004; 279(1): 796 - 804. [Abstract] [Full Text] [PDF]
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K. Ebnet, A. Suzuki, S. Ohno, and D. Vestweber Junctional adhesion molecules (JAMs): more molecules with dual functions? J. Cell Sci., January 1, 2004; 117(1): 19 - 29. [Abstract] [Full Text] [PDF]
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X. Han, M. P. Fink, T. Uchiyama, R. Yang, and R. L. Delude Increased iNOS activity is essential for hepatic epithelial tight junction dysfunction in endotoxemic mice Am J Physiol Gastrointest Liver Physiol, January 1, 2004; 286(1): G126 - G136. [Abstract] [Full Text] [PDF]
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B.-H. Peng, J. C. Lee, and G. A. Campbell In Vitro Protein Complex Formation with Cytoskeleton-anchoring Domain of Occludin Identified by Limited Proteolysis J. Biol. Chem., December 5, 2003; 278(49): 49644 - 49651. [Abstract] [Full Text] [PDF]
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 K. Ichimura, H. Kurihara, and T. Sakai Actin Filament Organization of Foot Processes in Rat Podocytes J. Histochem. Cytochem., December 1, 2003; 51(12): 1589 - 1600. [Abstract] [Full Text] [PDF]
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M. Bruewer, A. Luegering, T. Kucharzik, C. A. Parkos, J. L. Madara, A. M. Hopkins, and A. Nusrat Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms J. Immunol., December 1, 2003; 171(11): 6164 - 6172. [Abstract] [Full Text] [PDF]
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A. K. Rajasekaran and S. A. Rajasekaran Role of Na-K-ATPase in the assembly of tight junctions Am J Physiol Renal Physiol, September 1, 2003; 285(3): F388 - F396. [Abstract] [Full Text] [PDF]
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R. Yang, X. Han, T. Uchiyama, S. K. Watkins, A. Yaguchi, R. L. Delude, and M. P. Fink IL-6 is essential for development of gut barrier dysfunction after hemorrhagic shock and resuscitation in mice Am J Physiol Gastrointest Liver Physiol, August 8, 2003; 285(3): G621 - G629. [Abstract] [Full Text] [PDF]
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M. H. Roh, S. Fan, C.-J. Liu, and B. Margolis The Crumbs3-Pals1 complex participates in the establishment of polarity in mammalian epithelial cells J. Cell Sci., July 15, 2003; 116(14): 2895 - 2906. [Abstract] [Full Text] [PDF]
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P. A. Nielsen, A. Baruch, V. I. Shestopalov, B. N.G. Giepmans, I. Dunia, E. L. Benedetti, and N. M. Kumar Lens Connexins {alpha}3Cx46 and {alpha}8Cx50 Interact with Zonula Occludens Protein-1 (ZO-1) Mol. Biol. Cell, June 1, 2003; 14(6): 2470 - 2481. [Abstract] [Full Text] [PDF]
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 M. R. Ghassemifar, J. J. Eckert, F. D. Houghton, H. M. Picton, H. J. Leese, and T. P. Fleming Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro Mol. Hum. Reprod., May 1, 2003; 9(5): 245 - 252. [Abstract] [Full Text] [PDF]
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 C. B. Coyne, C. M. P. Ribeiro, R. C. Boucher, and L. G. Johnson Acute Mechanism of Medium Chain Fatty Acid-Induced Enhancement of Airway Epithelial Permeability J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 440 - 450. [Abstract] [Full Text] [PDF]
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W.-Y. Lui, D. Mruk, W. M Lee, and C. Y. Cheng Sertoli Cell Tight Junction Dynamics: Their Regulation During Spermatogenesis Biol Reprod, April 1, 2003; 68(4): 1087 - 1097. [Abstract] [Full Text] [PDF]
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N. M. Rubenstein, Y. Guan, P. L. Woo, and G. L. Firestone Glucocorticoid Down-regulation of RhoA Is Required for the Steroid-induced Organization of the Junctional Complex and Tight Junction Formation in Rat Mammary Epithelial Tumor Cells J. Biol. Chem., March 14, 2003; 278(12): 10353 - 10360. [Abstract] [Full Text] [PDF]
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G. Benais-Pont, A. Punn, C. Flores-Maldonado, J. Eckert, G. Raposo, T. P. Fleming, M. Cereijido, M. S. Balda, and K. Matter Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability J. Cell Biol., March 3, 2003; 160(5): 729 - 740. [Abstract] [Full Text] [PDF]
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A. Traweger, R. Fuchs, I. A. Krizbai, T. M. Weiger, H.-C. Bauer, and H. Bauer The Tight Junction Protein ZO-2 Localizes to the Nucleus and Interacts with the Heterogeneous Nuclear Ribonucleoprotein Scaffold Attachment Factor-B J. Biol. Chem., January 17, 2003; 278(4): 2692 - 2700. [Abstract] [Full Text] [PDF]
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A. Pedram, M. Razandi, and E. R. Levin Deciphering Vascular Endothelial Cell Growth Factor/Vascular Permeability Factor Signaling to Vascular Permeability. INHIBITION BY ATRIAL NATRIURETIC PEPTIDE J. Biol. Chem., November 8, 2002; 277(46): 44385 - 44398. [Abstract] [Full Text] [PDF]
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