* Department of Cell Biology, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
Occludin is the only known integral membrane protein localized at the points of membrane- membrane interaction of the tight junction. We have used the Xenopus embryo as an assay system to examine: (a) whether the expression of mutant occludin in embryos will disrupt the barrier function of tight junctions, and (b) whether there are signals within the occludin structure that are required for targeting to the sites of junctional interaction. mRNAs transcribed from a series of COOH-terminally truncated occludin mutants were microinjected into the antero-dorsal blastomere of eight-cell embryos. 8 h after injection, the full-length and the five COOH-terminally truncated proteins were all detected at tight junctions as defined by colocalization with both endogenous occludin and zonula occludens-1 demonstrating that exogenous occludin correctly targeted to the tight junction. Importantly, our data show that tight junctions containing four of the COOH-terminally truncated occludin proteins were leaky; the intercellular spaces between the apical cells were penetrated by sulfosuccinimidyl-6-(biotinamido) Hexanoate (NHS-LC-biotin). In contrast, embryos injected with mRNAs coding for the full-length, the least truncated, or the soluble COOH terminus remained impermeable to the NHS-LC-biotin tracer. The leakage induced by the mutant occludins could be rescued by coinjection with full-length occludin mRNA. Immunoprecipitation analysis of detergent-solubilized embryo membranes revealed that the exogenous occludin was bound to endogenous Xenopus occludin in vivo, indicating that occludin oligomerized during tight junction assembly. Our data demonstrate that the COOH terminus of occludin is required for the correct assembly of tight junction barrier function. We also provide evidence for the first time that occludin forms oligomers during the normal process of tight junction assembly. Our data suggest that mutant occludins target to the tight junction by virtue of their ability to oligomerize with full-length endogenous molecules.
TIGHT junctions, the most apical component of the
junctional complex (Farquhar and Palade, 1963 At least seven proteins (zonula occludens-1 and -2 [ZO-1,
ZO-2], cingulin, 7H6, rab 13, occludin, and symplekin) are
found to be localized at tight junctions (Citi et al., 1988 Three experiments with cell culture systems indicate that
occludin is directly involved in the sealing function of the
tight junction. First, in the experiments outlined above
(Balda et al., 1996 We have investigated the ability of mutant occludin
molecules to assemble in tight junctions during the biogenesis of an epithelium in an intact organism. mRNAs coding
for a series of COOH-terminally truncated chicken occludin molecules have been expressed in the Xenopus embryo. All exogenous proteins were FLAG tagged at the COOH terminus to permit discrimination from endogenous molecules and were seen to target to the tight junction by immunofluorescence. Four of the COOH-terminally truncated mutants caused the disruption of the tight
junction solute seal as assayed by a novel surface biotinylation method. Exogenous and endogenous occludins could be coimmunoprecipitated under nondenaturing conditions
as oligomeric complexes. These data indicate that the
truncated, mutant occludins are targeted to the tight junction in association with intact, endogenous molecules.
Construction of Full-Length and Truncated Occludins
A full-length chicken occludin cDNA (a generous gift from S. Tsukita,
Kyoto University, Japan) was subcloned into the expression vector SP64T
by PCR. The sequence of PCR sense primer was as follows: 5 A nested set of five occludin constructs truncated in the COOH-terminal domain predicted to face the cytoplasm was prepared by endonuclease
digestion of the full-length occludin construct described above. Each truncated occludin was then blunt ligated after the reading frame alignment by
endonuclease digestion of the restriction sites located upstream of FLAG-coding region. In addition, a construct was prepared that coded for the initial 20 amino acids of the NH2 terminus fused to the complete COOH terminus beginning at amino acid 266 and including the FLAG epitope tag.
The full-length construct was sequenced using AmpliCycle Sequencing
Kit (Perkin Elmer Corp., Norwalk, CT) to ensure that the occludin sequence and the FLAG epitope tag were intact and in the correct reading
frame.
Production of Anti-occludin and Anti-ZO-1 Sera
The fusion protein containing glutathione-S-transferase (GST) and the
COOH terminus of chicken occludin (250 amino acids) was constructed
using pGEX-3 vector (Smith and Johnson, 1988 Two rabbits were injected with purified fusion protein to raise polyclonal antibodies (Pocono Rabbit Farm and Laboratory, Canadensis,
PA). The resulting 11350 and 11351 antisera were affinity purified on a
Sepharose 4B column containing 1 mg purified fusion protein. This anti-
chicken occludin antibody cross-reacted with Xenopus occludin both in
immunoblots and in immunohistochemistry.
The anti-ZO-1 antiserum was prepared by immunizing with a GST-
ZO-1 fusion protein. A construct was made consisting of the pGEX-1 expression vector containing 1.8 kb of human ZO-1 sequence spanning the
alternative splice site (the generous gift of J. Anderson, Yale University,
New Haven, CT). The process of preparation and affinity purification of
the antiserum against GST-ZO-1 was the same as described above for the
GST-occludin construct.
In Vitro Transcription and Microinjection of mRNA
into Xenopus Oocytes and Embryos
The SP64T vectors containing either wild type or mutant chicken occludins were linearized with BamHI and transcribed in vitro with SP6 RNA
polymerase (Ambion Inc., Austin, TX). Adult female Xenopus laevis
were obtained from a departmental frog facility (Department of Cell Biology, Harvard Medical School, Boston, MA). Oocytes were collected and
defolliculated according to published protocols (Swenson et al., 1989 Fertilized embryos were obtained as described previously (Paul et al.,
1995 Cell Culture and Metabolic Labeling
The A6 Xenopus kidney epithelial cells (kindly provided by B. Gumbiner,
Sloan-Kettering Institute, New York) were grown in 61% Leibovitz L15
medium (GIBCO BRL, Gaithersburg, MD) supplemented with 5% FCS
(Hyclone Laboratories, Logan, UT), 100 U/ml penicillin and 0.1 mg/ml
streptomycin, and 45 mM NaHCO3, pH 7.3, at 27°C in a humidified incubator with 5% CO2. Cells were metabolically labeled for 20 h at 27°C with
150 µCi/ml Tran35S-label ([35S]met/[35S]cys; ICN Radiochemicals, Cleveland, OH) in 70% methionine-free medium (GIBCO BRL) supplemented
with 5% dialyzed FCS (Sigma Chemical Co.). At the end of the labeling
period cells were rinsed three times in ice-cold 0.65× PBS and extracted
with the same immunoprecipitation buffer used for Xenopus embryos
(vide infra).
Immunoprecipitation
RNA-injected Xenopus oocytes or embryos were rinsed three times with
PBS and homogenized on ice by passing 20 times through a 22-gauge needle in lysis buffer (5 mM EDTA, 5 mM EGTA, 5 mM Tris, pH 8.0) with 10 µg/ml each of chymostatin, leupeptin, and pepstatin A, 0.01% diisopropylfluorophosphate, and 20 µM PMSF. The homogenates were centrifuged
at 3,000 g for 10 min at 4°C to pellet yolk granules. The supernatants were
then centrifuged at 100,000 g at 4°C for 30 min. The membrane pellet was
resuspended in lysis buffer plus SDS-PAGE sample buffer to a final concentration of two oocytes per 10 µl for immunoblot. For immunoprecipitation experiments, the membrane pellet was resuspended in IP buffer (1% Triton X-100, 0.5% deoxycholic acid, 0.2% SDS, 150 mM NaCl, 10 mM Hepes, 2 mM EDTA, 10 KIU/ml Trasylol, 10 µg/ml each of chymostatin, leupeptin, and pepstatin A, and 0.01% diisopropylfluorophosphate), incubated on ice for 30 min, and then centrifuged at 100,000 g for
1 h at 4°C. The supernatants were either incubated with primary antibody
directly or mixed with 35S-labeled A6 cell lysates first, and then incubated
with primary antibody overnight at 4°C. The samples were incubated with
either protein A-Sepharose CL-4B or protein G-Sepharose 4B (Sigma
Chemical Co.) for an additional 2 h at 4°C. After three washes in IP
buffer, one in high salt (0.5 M NaCl, 10 mM Tris), and one in 10 mM Tris buffer, the samples were solubilized in SDS-PAGE sample buffer.
SDS Gel Electrophoresis and Immunoblotting
Samples were boiled in SDS-PAGE sample buffer for 3 min and separated on 10 or 12% SDS-polyacrylamide minigels. The proteins were then
transferred onto an Immobilon membrane (Millipore Corp., Bedford,
MA). The membrane was blocked 1 h at room temperature with 5% lowfat milk in TBS plus 0.1% Tween 20, and then incubated at room temperature for 2 h with anti-occludin antibody diluted at 1:1,000 or anti-FLAG
monoclonal antibody used at 10 µg/ml. The primary antibodies were detected by alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega
Corp., Madison, WI), or goat anti-mouse IgG (Boehringer Mannheim
Biochemicals, Indianapolis, IN) used at 1:5,000 or 1:1,000. To reduce the
background the secondary antibodies were preabsorbed with a crude Xenopus oocyte homogenate before reaction with the Immobilon membranes. After 1-h incubation with secondary antibody at room temperature, the blots were washed three times with TBS before the color
reaction was developed according to the manufacturer's instructions.
Surface Biotinylation-Tight Junction
Permeability Assay
6 h after RNA injection, Xenopus embryos were cooled to 10°C for 5 min
before transferring to freshly made 1 mg/ml NHS-LC-Biotin (Pierce
Chemical Co., Rockford, IL) in 0.1 × MMR solution (0.1 M NaCl, 2 mM
KCl, 1 mM MgSO4, 2 mM CaCl2·2H2O, 0.1 mM EDTA, and 5 mM Hepes,
pH 7.8). After 12 min of labeling with biotin at 10°C, the embryos were
washed twice with 0.1 × MMR, and fixed at 4°C overnight in 3% formaldehyde made fresh from paraformaldehyde in 80 mM sodium cacodylate,
pH 7.4. Embryos were rinsed with PBS and embedded in TISSUE-TEK
and cryosectioned. 14-µm frozen sections were incubated in blocking
buffer (1% fish skin gelatin and 1% BSA in PBS) for at least 5 h. Anti-
FLAG M2 antibody diluted 1:200 in blocking buffer was then placed on
the sections and incubated at 4°C overnight. After washing three times
with blocking buffer, sections were then incubated with preadsorbed
FITC-conjugated goat anti-mouse IgG (Boehringer Mannheim Biochemicals) and RITC-avidin (Pierce Chemical Co.), both diluted 1:500 in blocking buffer. Anti-FLAG M2 antibody was used to identify the region of
RNA injection. Control embryos were warmed to 18°C after biotinylation
and allowed to develop to tadpole stages. The 12-min labeling time was
chosen since this was the maximum time at 10°C, which was 100% consonant with normal development of embryos to tadpole stages.
Immunofluorescence
Frozen embryos were sectioned on a cryostat HM 500 OM (MICROM;
Carl Zeiss, Inc., Thornwood, NY) at 10 µm. The tissues were fixed in
100% methanol for 3 min at Full-Length and Truncated Chicken Occludins Can Be
Expressed in Xenopus Oocytes
Full-length chicken occludin, a nested set of five COOH-terminally truncated occludins, and a soluble construct
consisting of 20 NH2-terminal amino acids fused to the occludin COOH terminus were subcloned into the SP64T
vector. These constructs are diagrammed in Fig. 1 (504, 486, 385, 336 320, 266, and CT). The nomenclature indicates the deletion site in each protein and CT represents COOH terminus. The constructs have FLAG epitope tags
attached to the COOH terminus of the molecule to distinguish exogenous chicken occludin from endogenous Xenopus occludin, since our polyclonal anti-chicken occludin
cross-reacted with Xenopus occludin. The mRNAs transcribed from these seven constructs were microinjected into Xenopus oocytes and the results analyzed by immunoblots shown in Fig. 2. The data in Fig. 2 a were obtained
by immunoblotting with anti-occludin antibody 11350, and
in Fig. 2 b with anti-FLAG antibody M2. Fig. 2 demonstrates that all proteins were equivalently expressed in Xenopus oocytes. The asterisks at the right side of each lane
in both a and b indicate the predicted molecular weight of
each protein. In addition, all occludin molecules that contained the four membrane-spanning domains formed presumed dimers in SDS gel sample buffer under reducing
conditions as marked by arrowheads in both a and b. The
aggregation of occludin in SDS is not an indication of the
state of association between occludin proteins in vivo. This
SDS-induced aggregation is a phenomenon shared with
some of the members of the gap junction connexin family of proteins, particularly connexin 32 (Hertzberg and Gilula, 1979
Disruption of Tight Junction Permeability Barrier by
Four COOH-terminally Truncated Occludins
We used a novel surface biotinylation method to examine
the permeability of tight junctions in the embryo. During
the normal development of Xenopus embryos, biotin molecules are blocked from entry to restricted intercellular
spaces beginning at the two-cell stage, suggesting that the
formation of functional tight junctions and formation of
the blastocoele begin at the first cleavage (Slack and
Warner, 1973
These dominant-negative effects of the truncated occludin mutants were sensitive to dilution and to competition
from wild-type protein. Leaks between blastomeres were
observed when truncated occludin mRNAs were diluted 1:
10 but not 1:50 (data not shown). The induced tight-junctional leakage could be rescued by coinjection of an equal
amount of mRNA from construct 504 (full-length) together with mRNA from construct 266. Fig. 4 shows the
biotin staining of Xenopus embryos 6 h after injection of
mRNAs from construct 504 (a), 266 (b), and 266 with 504 together (c). Coinjection of 504 and 266 mRNAs together
resulted in no detectable leak between the blastomeres
(Fig. 4 c).
Targeting of Exogenous Chicken Occludin to Tight
Junctions in Early Xenopus Embryos
Full-length occludin, as well as all truncated mutants,
could be localized to tight junctions in early Xenopus embryos except for the soluble CT. Fig. 5 shows an example
of exogenous truncated occludin protein colocalized with
endogenous full-length Xenopus occludin. Frozen sections
of embryos injected with construct 266 mRNA (the most
COOH-terminally truncated one) were double stained with anti-occludin polyclonal antibody 11350 (Fig. 5 a) and
anti-FLAG monoclonal antibody M2 (Fig. 5 b). In Fig. 5,
the extraembryonic space is oriented toward the top of the
figure and the blastocoele is oriented toward the bottom.
As the apical regions of the blastomeres remained in the
plane of section for variable distances, the linear tight
junction staining (Fig. 5, a and b, arrowheads) was sampled such that the staining appeared as single or branching slender threads outlining those portions of the apical cellular
surfaces contained in the section. Since the anti-occludin
11350 did not recognize 266 mutant occludin (Fig. 2 a, lane
266), the signal in Fig. 5 a came from only endogenous full-length occludin, while the signal in Fig. 5 b localized the
most COOH-terminally truncated exogenous occludin. In
addition, all expressed exogenous proteins contained cytoplasmic staining. The colocalization of anti-occludin (Fig.
5 a) and anti-FLAG (Fig. 5 b) demonstrated that a fraction of the expressed exogenous occludin had been correctly
targeted to tight junctions.
While the anti-FLAG antibody revealed threadlike staining patterns with all constructs (data not shown), double
labeling with anti-FLAG and anti-occludin to demonstrate
colocalization was ambiguous since the anti-occludin reagent recognized both exogenous and endogenous molecules except for construct 266 (shown in Fig. 5). For this
reason, exogenous occludin mutants were colocalized with
another tight junction marker ZO-1. Fig. 6 shows immunofluorescence photographs of frozen sections of an embryo
injected with construct 266 mRNA, double stained with
anti-ZO-1, an independent marker for the position of the
tight junction (Stevenson et al., 1986
Immunoprecipitation Revealed
Interactions between Endogenous Xenopus Occludin
and Exogenous Chicken Occludin In Vivo
The fact that our polyclonal antiserum was unable to recognize construct 266, which lacked most of the cytoplasmic
COOH terminus (see Fig. 2), offered an opportunity to investigate possible interactions between endogenous occludin and the mutant 266 by immunoprecipitation of detergent extracts. Embryos injected with mRNA from the
construct 266 were detergent solubilized and immunoprecipitated with anti-occludin 11350 or anti-FLAG M2. After SDS-PAGE and electrophoretic transfer, the immunoprecipitates were then immunoblotted with anti-FLAG M2 or anti-occludin antibodies to probe for potential interactions between the native and truncated molecules. In
Fig. 7, a and b, left lanes are immunoblots of immunoprecipitates from embryos injected with 266 mRNA; right
lanes are samples from embryos without mRNA injection.
Fig. 7 a (left lane) shows a 11350 immunoprecipitate blotted with anti-FLAG M2, and Fig. 7 b (left lane) shows an
anti-FLAG immunoprecipitate blotted with 11350. In each
case, immunoblotting reveals that each immunoprecipitate
contains the alternate antigen, demonstrating that the endogenous and truncated occludins were both contained in
a common detergent-soluble structure. Samples incubated
with rabbit preimmune serum or with only protein A-Sepharose beads showed no specific signal. Similar coimmunoprecipitation results were obtained from oocytes injected
with 266 mRNA (data not shown), indicating that the association of the mutant and endogenous occludin could
occur in the absence of tight junction formation.
To demonstrate that the association of Xenopus occludin with chicken truncated occludin did not arise by subunit exchange between the detergent-solubilized forms
of occludin during experimental manipulations, we performed an experiment in which radiolabeled, detergent-solubilized occludin from A6 cells was mixed and incubated in vitro with solubilized occludin from embryos (Fig.
8). After solubilization in detergent, the high speed supernatants from embryos injected with construct 266 mRNA
were mixed with similarly solubilized [35S]methionine-
labeled A6 cell lysates to test whether the radiolabeled occludin could be immunoprecipitated with the anti-FLAG
antibody. The embryo and A6 detergent extracts were
mixed, incubated, and then immunoprecipitated with either anti-occludin antibody (Fig. 8, left lane), anti-FLAG
M2 (Fig. 8, center lane), or rabbit preimmune serum (Fig.
8, right lane). The immunoprecipitates were examined by
SDS-PAGE and autoradiography. The 35S-occludin band
was detectable only in the left lane (arrowhead), not in the
middle or right lanes, indicating that radiolabeled occludin
did not exchange between solubilized structures in detergent solutions. The top band in all three lanes was nonspecific. Therefore, the interaction of Xenopus full-length occludin with chicken truncated occludin was not an artifact of
subunit exchange under detergent-solubilization conditions.
We demonstrate in the present study that expression of
occludin mutants in the early Xenopus embryo resulted in
the disruption of the transepithelial permeability barrier
to solutes. Embryos injected with mRNAs encoding full-length occludin or the soluble occludin COOH terminus
remained impermeable to a biotin tracer. Removal of the
ultimate 18 amino acids of the occludin molecule was not
sufficient to cause the leaky phenotype; only mutants which were truncated by 119 amino acids or more had the
disruptive activity. The leaky phenotype could be rescued
by coinjection of full-length and truncated occludin
mRNA, indicating that the disruption of the permeability
seal was not a nonspecific effect of exogenous protein expression. We also showed that the full-length and a nested
set of five COOH-terminally truncated occludins were all
localized at tight junctions as defined by colocalization with endogenous occludin and ZO-1. By immunoprecipitating detergent-solubilized membranes under nondenaturing conditions, we report here for the first time that exogenous truncated chicken occludin interacted with
endogenous Xenopus occludin in vivo to form an oligomer
during the tight junction assembly.
The mechanism by which the full-length occludin rescued the tight junction phenotype was not clear. The activity of mutant 266 was sensitive to a dilution of 1:50 but not
1:10, suggesting a competitive inhibition of the endogenous molecules. However, the oligomeric state of occludin
is not known, nor is the number of copies of occludin required per oligomer to disrupt activity. Since chicken occludin may have different binding constants for amphibian
molecules, it is not possible to predict the stoichiometries required for inhibition. The rescue data do demonstrate
that the effects of the truncated molecules were not due to
cytotoxicity or other nonspecific effects, since coexpression of full-length occludin protein can rescue the mutant
phenotype.
In our studies, the FLAG signal could be detected in the
tight junctions in frozen sections 4 h after RNA injection.
This is consistent with the results obtained by McCarthy et
al. (1996) A previous study showed that chicken occludin with 120 amino acids deleted from the COOH terminus failed to localize at tight junctions in transfected MDBK cells (Furuse
et al., 1994 Protein oligomerization is a common step in the assembly of integral membrane proteins in cells, which usually
occurs in the endoplasmic reticulum (Hurtley and Helenius, 1989),
form selective permeability barriers along the paracellular pathways of epithelial and endothelial cells (Diamond, 1977
; Gumbiner, 1990
; Reuss, 1989
). Tight junctions also act as a "fence" between the apical and lateral
plasma membrane domains to prevent the mixing of membrane lipids and proteins between these two compartments (van Meer et al., 1986
; Cereijido et al., 1989
;
Schneeberger and Lynch, 1992
). In response to different stimuli, tight junctions rapidly change their permeability
and functional properties, permitting dynamic fluxes of
ions and solutes as well as transepithelial passage of whole
cells (Claude and Goodenough, 1973
; Duffey et al., 1981
;
Kachar and Pinto da Silva, 1981
; Mazariegos et al., 1984
;
Milks et al., 1986
; Madara and Pappenheimer, 1987
; Pappenheimer, 1987
, 1990
; Pappenheimer and Reiss, 1987
).
;
Gumbiner et al., 1991
; Furuse et al., 1993
; Zhong et al.,
1993
; Jesaitis and Goodenough, 1994
; Zahraoui et al.,
1994
; Ando-Akatsuka et al., 1996
; Keon et al., 1996
).
Among these proteins, occludin is the only integral membrane protein localized at the points of membrane-membrane interaction of tight junctions as revealed by immunogold labeling of thin-sections and freeze-fracture replicas (Furuse et al., 1993
; Fujimoto, 1995
). Hydropathy analysis
predicts that occludin has four transmembrane domains,
two extracellular loops, and a long COOH-terminal cytoplasmic tail consisting of 255 amino acids (Furuse et al.,
1993
; Ando-Akatsuka et al., 1996
). By transfection of various deletion mutants of chicken occludin into Madin-Darby bovine kidney (MDBK) cells, Furuse et al. (1994)
showed that the COOH-terminal ~150 amino acids (domain E358/504) were necessary for the localization of occludin at tight junction. Their in vitro binding assay also indicated that domain E358/504 directly associated with
ZO-1. However, recent data reported by Balda et al. (1996)
showed that COOH-terminally truncated chicken occludin
localized efficiently to the tight junction in transfected MDCK cells. The discrepancy between these experiments
raises the question of whether COOH terminus of occludin is required for targeting.
), expression of a COOH-terminally
truncated occludin resulted in an electrically tighter paracellular pathway that paradoxically had an increased paracellular flux of solutes. Second, McCarthy et al. (1996)
transfected MDCK cells with chicken occludin cDNA in a
Lac-inducible expression vector. Isopropyl-
-D-thiogalactoside (IPTG)1-induced expression of chicken occludin increased transepithelial resistance (TER) by 30-40%, which
declined back to uninduced states after removal of IPTG
from the culture medium. Freeze fracture showed an increase in the mean number and complexity of branching of
tight junctional strands, together with a concomitant increase in the apical-basal width of the tight junction
network. McCarthy et al. (1996)
also observed the paradoxical transepithelial mannitol flux which progressively
increases as TER increases. Third, Wong and Gumbiner
(1997)
found that a synthetic peptide (OCC2), corresponding to the second extracellular domain of occludin, reversibly disrupted the transepithelial permeability barrier
when added to Xenopus kidney epithelial A6 cell monolayers. In these experiments, OCC2 decreased TER and
increased the paracellular flux of tracers.
Materials and Methods
-TGG GCC
ACC ATG TTC AGC AAG AAG-3
, which corresponded to the NH2-terminal 15 codons of chicken occludin plus 9 bp preceding the start
codon. The antisense primer corresponded to the last 15 codons of the
COOH terminus of occludin plus the BglII site and three restriction sites,
which formed a linker between the occludin and the FLAG epitope: 5
-GCC TAC GAC AAG GTG CGG GAG ATC TTC GCG ATA TCA
AGG CCT GAC TAC AAG GAC GAC GAT GAC AAG TAA-3
. These restriction sites permitted placing the FLAG-coding sequence in
frame with each truncation. Using these primers, a 1.6-kb occludin cDNA
with the FLAG epitope tag was amplified by Vent polymerase (New England Biolabs Inc., Beverly, MA) with full-length occludin cDNA in
pBluescript SK(
) as a template. The PCR products were phosphorylated, separated by agarose gel electrophoresis, and purified by Gel Extraction Kit (QIAGEN Inc., Chatsworth, CA). The purified occludin
DNA was cloned into the BglII site of the expression vector SP64T (Krieg
and Melton, 1984
).
). An overnight bacterial
culture was diluted in Luria-Bertani medium with 0.01% ampicillin, and
grown for 2-3 h at 37°C. The synthesis of the fusion protein was induced
by adding 0.2 mM IPTG. After 4-5 h of induction, cells were collected by
centrifugation at 2,100 g for 10 min, and resuspended in PBS. The cell suspension was sonicated 1 min on ice with 1% Triton X-100, and centrifuged at 5,100 g for 15 min. The supernatant was incubated with glutathione- agarose beads (Sigma Chemical Co., St. Louis, MO) for 30 min at 4°C,
washed three times with PBS, and then the bound fusion proteins were released by adding 5 mM glutathione. The full-length fusion protein was
separated on 12% SDS-polyacrylamide gel and purified by electroelution.
).
Oocytes were microinjected in their vegetal hemispheres with 40 nl (~100
pg) RNA or water, and incubated at 17°C overnight for immunoblot or
immunoprecipitation experiments.
). The anterior, dorsal blastomere of eight-cell embryos were injected
with 4 nl (~10 pg) of FLAG-tagged, full-length or truncated chicken occludin RNA. 6-8 h after injection, embryos were either directly embedded
in TISSUE-TEK embedding medium (Miles Inc., Elkhart, IN) without
fixation and quickly frozen in liquid propane or used for surface biotinylation experiments (see below).
20°C, and blocked with 1% fish skin gelatin
plus 1% BSA in PBS for at least 5 h. The sections were double-stained
with the affinity-purified anti-occludin antibody 11350 at 1:500 dilution
and anti-FLAG monoclonal antibody M2 diluted at 1:200 (Eastman Kodak
Co., Rochester, NY) 1 h at room temperature or 4°C overnight. Sections
were washed three times, 10 min each in blocking medium, and then incubated for 1 h at room temperature with FITC-conjugated goat anti-rabbit
IgG (Boehringer Mannheim Biochemicals) diluted 1:500 and rhodamine-conjugated goat anti-mouse IgG (Cappel Laboratories, Malvern, PA) diluted 1:500, which both were preabsorbed with a crude Xenopus oocyte
homogenate. Sections were examined by epifluorescence using a Zeiss
Axioskop (Carl Zeiss, Inc.) and photographed with TMAX-400 film
(Eastman Kodak Co.).
Results
; Goodenough et al., 1988
). Proteins 385 and 320 migrated as doublets whose identity was not investigated.
It is possible that doublets were caused by posttranslational modifications in certain deletion constructs or were
caused by degradation. All preparations were prepared
from oocyte membrane fractions except CT, which was
obtained from soluble supernatant fractions. A small
amount of CT protein could be detected in membrane
fractions, although full-length protein could not be detected in supernatants (data not shown). Fig. 2 also shows
that all expressed proteins were recognized by both the
11350 antiserum and the anti-FLAG M2 monoclonal antibody, except 266, which lacked almost the entire COOH
terminus and was not recognized by 11350.
Fig. 1.
Schematic drawing of full-length (504) and six truncated chick occludin proteins that were subcloned into expression
vector SP64T. Each molecule has a FLAG epitope tag (black
box) at the end of COOH terminus to distinguish it from Xenopus occludin. The shaded half of the molecule is predicted to
traverse the membrane four times, and contains an NH2 terminus
facing the cytoplasm, two extracellular loop domains, and one cytoplasmic loop domain. The unshaded half of the protein is predicted to face the cytoplasm. The numbers on the right indicate
the total number of amino acids in each protein. CT, soluble
NH2-terminal 20 amino acids fused to the predicted cytoplasmic
tail; N, NH2 terminus; C, COOH terminus.
[View Larger Version of this Image (18K GIF file)]
Fig. 2.
Western blot analysis of Xenopus oocytes injected with
mRNAs corresponding to the occludin proteins diagrammed in
Fig. 1. mRNAs were microinjected in the vegetal hemisphere of
stage VI oocytes, and the injected oocytes were incubated at 17°C
overnight. Oocyte homogenates were separated on 12% SDS-PAGE, transferred to Immobilon membranes, and then immunoblotted with either anti-occludin antibody 11350 (a) or with anti-FLAG monoclonal antibody M2 (b). The number on the top of
each lane identifies each protein as named in Fig. 1. The asterisks
in a and b indicate the predicted position of each expressed protein. With the exception of the soluble CT, the expressed proteins
formed dimers as indicated by the arrowheads. Note that protein
266, with almost the entire COOH terminus deleted, was not recognized by 11350 antibody. H2O, control oocytes injected with
water. Molecular weight markers (from top to bottom): 97.4, 66, 45, and 31 kD.
[View Larger Version of this Image (43K GIF file)]
; Merzdorf, C., Y.-H. Chen, and D.A. Goodenough, manuscript in preparation). When mRNAs from
constructs 504 (full-length), 486 (the least COOH-terminally truncated), and CT (COOH terminus only) were injected into eight-cell stage embryos, the results were identical to embryos injected with only water: tight junctions were impermeable to the biotin tracer (Fig. 3, a and b; data
not shown for CT). NHS-LC-biotin labeled the vitelline
envelope and apical blastomere membranes, which appear
together as a single thick line at the surface of the embryo
(Fig. 3, a and b). No signal was detectable between the
blastomeres in the intercellular spaces. However, if the
embryos were injected with mRNAs from the four
COOH-terminally truncated constructs 385, 336, 320, and 266, the intercellular spaces between epithelial cells were
penetrated by biotin (Fig. 3, c-f), indicating that there is a
critical region between residues 486 and 385 that is required for correct assembly critical for the tight junctional
seal. Fig. 3 c (arrowhead) reveals that the membranes of
internal cells beneath those at the embryonic surface have
also been biotinylated, indicating that the tracer penetrated beyond the first tier of cells. This observation indicates that the truncated occludin molecules must have altered the permeability function of the tight junctions.
Double labeling of sections with both the avidin and anti-
FLAG M2 antibody revealed that the leaks occurred only
between blastomeres expressing the truncated occludin proteins; the tight junctions between the blastomeres unlabeled by M2 within the same embryo were well sealed
(data not shown).
Fig. 3.
Functional assay
of tight junctions by surface
biotinylation in Xenopus embryos injected with chicken
full-length or truncated occludins. 6 h after mRNA injection (2,000 cell blastula),
the embryos were labeled by
incubation in 1 mg/ml NHS-LC-biotin for 12 min at 10°C,
washed, and then fixed in 3%
formaldehyde in 80 mM sodium cacodylate. Frozen sections were stained with RITC- avidin and observed by
fluorescence microscopy. In all
embryos, NHS-LC-biotin
reacts with molecules in the
vitelline envelope, the subvitelline space, and the apical
plasma membranes of the blastomeres, which together
appear as a thick, continuous
line at the surface of the embryo. The tight junctions in
the embryos injected with
504 (a, the full-length) or 486 (b, the least COOH-terminally truncated) occludin
mRNAs deny the biotin access to the intercellular spaces,
and the basolateral membranes of the blastomeres are
not stained. In the embryos
injected with 385 (c), 336 (d)
320 (e), and 266 (f) (four
COOH-terminally truncated
occludin mRNAs), the biotin
molecules penetrated into intercellular spaces demonstrating the disruption of the tight
junction seal. Bar, 10 µm.
[View Larger Version of this Image (106K GIF file)]
Fig. 4.
Coinjection of the full-length (504) occludin mRNA
with the most COOH-terminally truncated occludin mRNA
(266) rescued the tight junction leakage caused by injection of
construct 266 alone. The experimental procedure was same as in
Fig. 3. The frozen sections from the embryos injected with ~2 pg
of 504 (a), 266 (b), or 504 and 266 (c) construct mRNA were
stained with RITC-avidin. The extraembryonic space is toward
the top of figure in a, and left in b and c. No leak was seen in the
embryo infected with 504 mRNA (a). Biotin molecules clearly
penetrated through the tight junctions and labeled the intercellular spaces between the apical cells of embryo injected with 266 mRNA (b). However, there was no detectable leak in the embryo
injected with both 504 and 266 mRNA (c). Bar, 15 µm.
[View Larger Version of this Image (68K GIF file)]
Fig. 5.
Double labeling of frozen sections of Xenopus embryos
with anti-occludin 11350 (a) and anti-FLAG M2 (b). 4 nl (~10
pg) of mutant 266 mRNA were microinjected into the anterior,
dorsal blastomere of eight-cell stage embryo and incubated for 8 h
at room temperature before freezing. The arrowheads indicate
the colocalization of Xenopus full-length occludin and chicken
truncated occludin at the junctional complexes. Bar, 10 µm.
[View Larger Version of this Image (111K GIF file)]
), and anti-FLAG M2.
The staining revealed a colocalization of the most COOH-terminally truncated exogenous occludin with endogenous ZO-1, indicating the targeting of mutant occludin to the
junctional complex, even though it lacked almost the entire COOH terminus, which contains the binding domain
to ZO-1. The full-length and the other four COOH-terminally truncated chicken occludin proteins were all localized at tight junctions in a similar pattern as shown in Figs.
5 and 6 (data not shown). For construct CT, which encodes
a soluble COOH-terminal peptide, the anti-FLAG signal was in the cytoplasm and was not detectable at the tight
junction (data not shown). By sampling embryos at different developmental stages, FLAG-tagged chicken occludins were detectable at the tight junctions by 4 h after injection. By 20 h after injection, immunofluorescence signals
for the FLAG epitope were significantly decreased, which may be due to the degradation or dilution of the injected
mRNA.
Fig. 6.
Immunocolocalization of Xenopus ZO-1 with the protein directed by mutant 266, the most truncated occludin in Xenopus embryos. Frozen sections were made of embryos injected
with mutant 266 mRNA and stained with either anti-ZO-1 (a) or
with anti-FLAG M2 (b). The arrowheads indicate the colocalization of both proteins at the junctional complexes. Bar, 10 µm.
[View Larger Version of this Image (85K GIF file)]
Fig. 7.
The interaction of
Xenopus occludin with
chicken occludin in vivo assayed by immunoprecipitation. The embryos were microinjected with the most COOH-terminally truncated occludin
mRNA (266; a and b, left lanes) or water (C; a and b, right lanes)
and incubated at room temperature for 10 h. After homogenization, the samples were centrifuged at 100,000 g for 30 min at 4°C.
The membrane pellet was solubilized in modified RIPA buffer
(see Materials and Methods) and centrifuged at 100,000 g for 1 h
at 4°C. The resulting supernatant was immunoprecipitated with
anti-occludin 11350 (a) or anti-FLAG M2 (b) overnight at 4°C.
Immunoprecipitates were immunoblotted with anti-FLAG M2
(a) or anti-occludin 11350 (b). A specific band corresponding to
the chicken truncated occludin (compare with Fig. 2 b, lane 266)
was present in a (left lane) after immunoprecipitated with anti-occludin 11350. The Xenopus full-length occludin was observed
at b (left lane) after immunoprecipitated with anti-FLAG M2.
The two thick bands in b were IgG heavy chain.
[View Larger Version of this Image (23K GIF file)]
Fig. 8.
Coimmunoprecipitation of the mutant with endogenous occludin was not
the result of exchange of
monomers between detergent-solubilized oligomeric assemblies. Confluent A6 cell monolayers were metabolically labeled with 150 µCi/ml [35S]methionine/cysteine for 20 h at 27°C, solubilized under nondenaturing conditions, and then mixed with similarly detergent-solubilized supernatant from Xenopus embryos injected with 266 construct
mRNA at the two-cell stage and incubated for 10 h at room temperature. After immunoprecipitation with anti-occludin 11350 (left lane), anti-FLAG M2 (middle lane), or preimmune rabbit
serum (right lane), samples were separated on 12% SDS-PAGE
and autoradiographed. 35S-labeled occludin could not be immunoprecipitated with anti-FLAG M2 (middle lane), demonstrating
that mixing of the mutant with wild-type molecules after extraction did not result in detergent-mediated exchange of protein
monomers. The bar above the arrowhead indicates the molecular weight marker 66 kD.
[View Larger Version of this Image (44K GIF file)]
Discussion
, who found chicken occludin staining in tight
junctions of MDCK cells 4 h after addition of IPTG to the
Lac-inducible system. Electron microscopy of Xenopus
blastomeres expressing truncated occludins showed no evidence of structural perturbation of the tight junctions
(data not shown), which was consistent with previously published studies in MDCK cells (Balda et al., 1996
). Injected embryonic blastomeres were lineage marked by
coinjection of 15 nm colloidal gold together with the mutant occludin 266 mRNA. We observed no cell death either grossly (Paul et al., 1995
), or by electron microscopy
(data not shown), indicating that the truncated proteins
were not cytotoxic, and that the induced paracellular leaks
could be tolerated by the developing embryos.
). In contrast, Balda et al. (1996)
reported that
in transfected MDCK cells, a chicken occludin lacking almost the entire COOH-terminal domain was efficiently
transported to tight junctions. Their data indicate that this
truncated occludin causes a discontinuous junctional distribution of transfected and endogenous occludin. Our results were partially consistent with Balda et al. (1996)
in
that COOH-terminally truncated occludin could be delivered to the tight junctions. We have not observed the discontinuous distribution pattern of mutant and endogenous
occludin in Xenopus embryos. This difference could be
due to the differences in the constructs used for study or in
the expression systems used.
) but may sometimes occur in the trans-Golgi
(Musil and Goodenough, 1993
). In the present study, our
experimental data provide evidence that occludin formed
an oligomeric complex in vivo during tight junction assembly, a finding consistent with the resolution of tight junction freeze-fracture strands as closely apposed 6-10-nm
particles (Raviola et al., 1980
). An intact COOH terminus
on all copies of occludin in the oligomer is not required for
this oligomerization process, since a truncated occludin
will cooligomerize with full-length. While the intracellular
location is not known, occludin oligomerization occurred
in oocytes before tight junction assembly. In addition to its
association with endogenous occludin, truncated occludin colocalized with ZO-1 by double immunofluorescence labeling, although it lacked a binding site for ZO-1 (Furuse
et al., 1994
). Taken together, these data indicate that the
oligomerization of occludin occurred before the assembly
of the tight junction, and that the presence of full-length
endogenous occludin in the oligomeric assemblies provided the required signals for both ZO-1 binding and
membrane targeting.
Received for publication 30 April 1997 and in revised form 8 July 1997.
Please address all correspondence to Daniel A. Goodenough, Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. Tel.: (617) 432-1652. Fax: (617) 432-2955. e-mail: dgoody{at}warren.med.harvard.eduWe would like to thank Drs. J. Goliger, J. Jiang, and A. Simon for their technical support and the members of the Paul/Goodenough laboratory for critical reading of the manuscript.
This work was supported by grant No. GM18974 from the National Institutes of Health.
CT, COOH terminus;
GST, glutathione-S-transferase;
IPTG, isopropyl--D-thiogalactoside;
NHS-LC-biotin, sulfosuccinimidyl isopropyl-6-(biotinamido) Hexanoate;
TER, transepithelial resistance;
ZO, zonula occludens.
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