1 Department of Molecular Biology, Yokohama City University School of Medicine,
Kanazawa-ku, Yokohama 236-0004, Japan
2 Department of Pathology, Yokohama City University School of Medicine,
Kanazawa-ku, Yokohama 236-0004, Japan
3 Department of Anatomy, Juntendo University School of Medicine, Bunkyo-ku,
Tokyo 113-8421, Japan
* Author for correspondence (e-mail: ohnos{at}med.yokohama-cu.ac.jp )
Accepted 10 April 2002
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Summary |
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Key words: ASIP/PAR-3, Atypical PKC, Epithelial tight junction, Epithelial cell polarity
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Introduction |
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We have previously identified a novel protein with three PDZ domains,
atypical PKC isotype-specific interacting
protein (ASIP), which colocalizes with aPKC at the cell-cell
junctions of confluent fibroblastic and epithelial cells. Immunogold electron
microscopy revealed that ASIP localizes at the TJ of rat intestinal epithelium
(Izumi et al., 1998). In
addition, ASIP/PAR-3 shows significant overall sequence similarity to two
invertebrate polarity proteins, C. elegans PAR-3 and
Drosophila Bazooka, and one of the highly conserved regions in
ASIP/PAR-3 is critical for the interaction with the kinase domain of aPKC
(Izumi et al., 1998
;
Etemad-Moghadam et al., 1995
;
Kuchinke et al., 1998
).
Furthermore, the physical interaction between ASIP/PAR-3 and aPKC is conserved
in C. elegans PAR-3 and PKC-3, as well as in Drosophila
Bazooka and DaPKC (Izumi et al.,
1998
; Tabuse et al.,
1998
; Wodarz et al.,
2000
). Immunofluorescent and genetic analyses of C.
elegans and Drosophila revealed that each pair of proteins,
PAR-3 and PKC-3 or Bazooka and DaPKC, shows asymmetric colocalization in
various cells undergoing asymmetric cell division, and that the two are
mutually dependent on each other for correct localization to regulate spindle
orientation and the distribution of other cell fate determinants
(Tabuse et al., 1998
;
Wodarz et al., 2000
). These
data suggest that an evolutionarily conserved protein complex is involved in
the regulation of various cell polarity and differentiation events; however,
the physiological meaning of the aPKCASIP/PAR-3 interaction in mammals
remains to be clarified.
In the present study, we show that ASIP/PAR-3 distributes to apical
cell-cell junctions of various rat epithelial cells, but in a manner clearly
different from ZO-1, another TJ-associated protein. While ZO-1 staining
patterns show a good correlation with the development level of TJ, ASIP/PAR-3
can be detected in all epithelial cell-cell junctions examined, including
those without highly developed TJ strands. Together with the fact that aPKC is
critical for the establishment of TJs in epithelial cells
(Suzuki et al., 2001), these
data led us to analyze the possibility that ASIP/PAR-3 might play a regulatory
role in TJ formation and/or maintenance, rather than serving only as a
structural element. Indeed, our functional and biochemical analyses revealed
that the overexpression of ASIP/PAR-3, but not its deletion mutant lacking the
aPKC-binding sequence, promotes TJ formation in epithelial MDCK cells.
Furthermore, the aPKC-binding sequence includes two highly conserved PKC
phosphorylation consensus sites [Ser827 and Ser829
(Izumi et al., 1998
)],
suggesting that this function of ASIP/PAR-3 is mediated through
phosphorylation by aPKC. This possibility is further supported by data showing
that ASIP/PAR-3 is phosphorylated at Ser827 and concentrates to the
apical-most cell-cell contacts of MDCK cells during TJ formation. Together,
these results provide the first evidence supporting the involvement of
ASIP/PAR-3 in the promotion of epithelial TJ formation through interaction
with aPKC.
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Materials and Methods |
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pTREHis/L-ASIP WT encodes a splice variant (K.M. and S.O., unpublished) of
the rat ASIP sequence lacking amino acid (a.a.) residues 740-742 and 857-871
fused downstream of the six histidine residues and an 11-a.a. sequence from
the T7 gene 10-leader sequence. pTERHis/L-ASIP PB encodes a T7-tagged
chimera of a splice variant of mouse (N-terminal 989 a.a.) and rat ASIP/PAR-3
(C-terminal 315 a.a.) lacking residues 740-742 and the aPKC-binding sequence
[residues 827-856 (K.M. and S.O., unpublished)]. The expression of both of
these proteins is under the control of the tetracycline repressible
transactivator (Gossen and Bujard,
1992
).
Rabbit anti-ASIP/PAR-3 antibodies were raised against a GST fusion protein
of the aPKC-binding region (C2-3; residues 712-936), the third PDZ domain
(P2-2; residues 584-708), and the C-terminal region (A2-2; residues 1124-1337)
as described (Izumi et al.,
1998). Antibodies against the specific C-terminal tail of the
splice variant of ASIP/PAR-3 and ASIP/PAR-3 phosphorylated at Ser827
(anti-S827-P) were raised against synthetic peptides corresponding to residues
1023-1034 (NH2-MFSLAKLKPEKR-COOH) and residues 822-832
phosphorylated at Ser827 (NH2-CGFGRQpSMSEKR-COOH), respectively.
Each rabbit antibody produced against a KLH-coupled antigen was affinity
purified (Y.T.-N. and S.O., unpublished). The mouse anti-ZO-1 monoclonal
antibody was kindly provided by S. Tsukita (Kyoto University, Kyoto, Japan) or
purchased from Zymed Laboratories (South San Francisco, CA). Rabbit
anti-occludin polyclonal antibody, rabbit anti-T7 polyclonal antibody (Omni
probe), mouse anti-T7 monoclonal antibody, FITC-conjugated secondary
antibodies and Cy3-conjugated secondary antibodies were purchased from Zymed
Laboratories, Santa Cruz Biotechnology (Santa Cruz, CA), Novagen (Madison,
WI), E. Y. Laboratories (San Mateo, CA) and Amersham Life Science (Arlington
Heights, IL), respectively.
Gel electrophoresis and western blot analysis
Samples of various rat organs and intestinal epithelial cell scrapings
(Saxon et al., 1994) were
subjected to SDS-PAGE (Laemmli,
1970
) and electrotransferred to a polyvinylidene difluoride
membrane, which was then soaked in 5% nonfat milk and 10% calf serum in PBS
(137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl and 1.47 mM
KH2PO4). The membrane was incubated first with affinity
purified anti-ASIP antibody (C2-3AP, A2-2AP or Yap) and then with horseradish
peroxidase-conjugated secondary antibody. Antibodies were detected by a
chemiluminescence ECL plus system (Amersham Life Science).
Immunofluorescence microscopy
Adult rat forestomach (Izumi et al.,
1998) and duodenum (Saxon et
al., 1994
) were prepared as described. A kidney from a 12-week-old
rat was rinsed in ice-cold PBS, cut into small blocks and immersed in
paraformaldeyde-lysine-periodate fixative
(McLean and Nakane, 1974
) for
30 minutes at 4°C. After fixation, the tissue was washed three times with
PBS containing 50 mM NH4Cl for 15 minutes at 4°C, and
cryoprotected in 30% (w/v) sucrose in PBS for 18 hours at 4°C. The tissue
blocks were embedded and frozen in Tissue Tek OCT compound. The frozen
specimens were cut in a cryostat to a thickness of about 4 µm, mounted on
glass slides, and air-dired. The sections were permeabilized in PBS containing
0.2% Triton X-100 for 5 minutes at room temperature, and then the nonspecific
sites were blocked with PBS containing 10% calf serum for 30 minutes at room
temperature. The sections were incubated for 45 minutes at 37°C with
primary antibodies diluted in TBST (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05%
Tween-20) containing 0.1% bovine serum albumin, and washed three times for 5
minutes with TBST. After the first incubation, the sections were incubated for
45 minutes at 37°C with secondary antibodies (FITC-conjugated goat
anti-rabbit and Cy3-conjugated anti-mouse antibodies) and washed three times
for 5 minutes with TBST.
MDCK cells grown on 1.0 cm2 Transwell-ClearTM filters (#3460, Corning Coster, Cambridge, MA) were fixed in 2% formaldehyde in PBS for 10 minutes at room temperature, washed twice in PBS, permeabilized in PBS containing 0.5% Triton X-100 and 100 mM glycine for 5 minutes at room temperature, and subjected to immunofluorescence detection as described above.
The samples were mounted in PBS containing 50% VECTASHIELD mounting medium (Vector Labs, Burlingame, CA) and examined under a fluorescence microscope (BX40, Olympus) equipped with a CCD camera (Princeton Instruments, Trenton, NJ) or a confocal microscope system (Nikon E-600 microscope equipped with BioRad µ-Radiance). Images were arranged and labeled using Adobe PhotoShop (Adobe Systems, San Jose, CA).
Immunoelectron microscopy
Rat kidneys were perfused with 1% paraformaldehyde fixative buffered with
0.1 M sodium phosphate buffer (PB, pH 7.4) and immersed in the same fixative
for 30 minutes at 4°C. The samples were rinsed with 5% sucrose for 30
minutes at 4°C. Tissue samples were then infiltrated with 40%
polyvinylpyrrolidone (Sigma)/2.3 M sucrose buffered with 0.1 M PB, embedded on
nails, and frozen quickly in liquid nitrogen. Ultrathin cryosections were cut
with a Leica Ultracut UCT equipped with a Leica EM FCS cryoattachment (Wien,
Austria) at -110°C. Sections were transferred to Formvarcoated nickel
grids (150 mesh). Subsequent incubation steps were carried out by floating the
grids on droplets of the filtered solution. Free aldehyde groups were quenched
with PBS-0.01 M glycine, and the sections were incubated overnight with PBS
containing 10% fetal bovine serum (FBS) and affinity purified rabbit anti-ASIP
antibody (C2-3AP, 1:50 dilution). Next, the grids were incubated with
antirabbit IgG coupled to 5 nm gold (diluted 1:100, British BioCell, Cardiff,
UK) for 1 hour. Double immunogold staining with ASIP and ZO-1 antibody was
also carried out. In brief, ultrathin cryosections of aldehyde-fixed kidney
were cut as described above. The sections were incubated with
affinity-purified rabbit anti-ASIP antibody (C2-3AP, 1:50 dilution with PBS
containing 10% FBS) and mouse monoclonal anti-ZO-1 antibody (Zymed
Laboratories, 1:100 dilution) and then incubated with 10 nm gold-conjugated
goat anti-rabbit IgG (diluted 1:100, British BioCell) and 5 nm gold-conjugated
goat anti-mouse IgG (diluted 1:100, British BioCell). After immunostaining,
the samples were fixed in 2.5% glutaraldehyde buffered with 0.1 M PB (pH 7.4).
The sections were then contrasted with 2% uranyl acetate solution for 20
minutes, and absorption-stained with 3% polyvinyl alcohol containing 0.2%
uranyl acetate for 20 minutes. All sections were observed with a JEOL 1230-EX
electron microscope.
Cell cultures
Parental MDCK Tet-Off cells [(Barth et
al., 1997) #C3017-1, Clontech, Palo Alto, CA) and established
stable clones were grown in Dulbecco's modified Eagle's medium (DMEM;
GibcoBRL, Rockville, MD) containing 10% FBS, penicillin, and streptomycin at
37°C in an air/5% CO2 atmosphere at constant humidity. The MDCK
cell lines were cultured with or without 10 ng/ml of DC for at least 3 days
before experiments.
Selection of MDCK cell lines stably expressing mutant ASIPs
The parental MDCK Tet-Off cells were co-transfected with expression
plasmids for ASIP/PAR-3 (pTREHis/L-ASIP WT or pTERHis/L-ASIP PB) and
pTK-Hyg. The overall procedures followed mainly the previously described
method (Jou and Nelson, 1998
).
9 µg ASIP/PAR-3 plasmid, 3 µg pTK-Hyg, and 40 µl lipofectamine PLUS
reagent (GibcoBRL, Rockville, MD) were mixed in 1160 µl of serumfree DMEM
containing 50 µM Ca2+ (SF-LCM) at room temperature for 30
minutes; 1.2 ml of SF-LCM containing 5% lipofectamine reagent (GibcoBRL) was
then added and the mixture was incubated for an additional 15 minutes. The
mixture was diluted with 9.6 ml of SF-LCM and added to the
1.5x106 cells plated in each of two 10 cm dishes; the cells
were in log phase growth and had been maintained in 2 µg/ml of TC. After 6
hours at 37°C, the medium was replaced with DMEM containing 10% FBS and 2
µg/ml of TC, and the cells were incubated for a further 24 hours and
passaged to fifteen 10 cm dishes in medium containing 200 µg/ml hygromycin
B (Wako, Osaka, Japan). After selection for 10 days, the surviving colonies
were isolated using cloning rings, and exogenous ASIP/PAR-3 expression was
assessed in each colony by western blotting 48 hours after the removal of TC.
Positive clones were expanded in the presence of TC, divided into aliquots and
stored in liquid nitrogen.
Measurement of transepithelial electric resistance (TER) and cell
growth
Cells were trypsinized, suspended in DMEM containing 10% FBS with or
without 10 ng/ml of DC, and plated on 1.0 cm2
Transwell-ClearTM (#3460) filters at the indicated cell densities
and on P-60 dishes. TER was measured using a Millicell-ERS (Millipore Corp.),
and cell growth was assessed by counting the number of cells on the P-60
dishes at different times after plating. TER values were calculated by
subtracting the values of blank Transwell-ClearTM filters, and
normalized to the area of the monolayers. Every point is the mean
±s.e.m of three groups of cells on independent filters.
Calcium switch and occludin fractionation
MDCK stable clones were plated on 3.9 cm2
Transwell-ClearTM (#3450) filters at a density of
2x105 cells/cm2, incubated for 20 hours in normal
calcium medium, rinsed once with PBS, and changed from medium to low calcium
medium [LCM; DMEM supplemented with 5 µM CaCl2 and 5% FBS that
had been dialyzed against PBS (Gumbiner
and Simons, 1986)]. After a 20 hour incubation in LCM, the level
of CaCl2 was adjusted back to 1.8 mM for calcium switch. Cells were
harvested with PBS at different times after switching, and the cell pellets
were frozen at -80°C. NP-40-insoluble fractions were prepared by following
the method described previously
(Sakakibara et al., 1997
).
Briefly, the cells were lysed in 700 µl of ice-cold NP-40-IP buffer (25 mM
Hepes/NaOH, pH 7.4, 150 mM NaCl, 1% NP-40, 50 mM NaF, 1 mM
Na3VO4, 4 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, 2
µg/ml aprotinin, 0.5 mM benzamidine) by 30-minute rotation at 4°C.
After centrifugation (10,000 g for 30 minutes) the pellet was
redissolved in 87.5 µl of 2x SDS-sample buffer
(Laemmli, 1970
), and then
subjected to western blotting analyses with anti-ASIP antibody (C2-3AP) and
anti-occludin pAb (Zymed). The levels of NP-40-insoluble occludin were
detected by chemiluminescence ECL (Amersham Life Science), quantified directly
with an LAS-1000 plus system (FUJI Photo Film, Tokyo, Japan), and then
normalized to the amount of tubulin in each lane.
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Results |
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We next examined the distribution of ASIP/PAR-3 in a variety of rat
epithelial tissues in comparison with another TJ-associated protein, ZO-1
(Stevenson et al., 1986). The
specificity of ASIP/PAR-3 staining was confirmed by the abolishment of the
signal by the corresponding antigen, or by the identity of the staining
pattern with another affinity-purified antibody against the third PDZ domain
of ASIP/PAR-3 (P2-2AP), or both (data not shown). One of the most striking
differences between the distribution of ASIP/PAR-3 and ZO-1 is the lack of
ASIP/PAR-3 staining in the cell-cell contact region of endothelial cells in
every epithelial tissue examined (forestomach, small intestine, renal cortex)
where ZO-1 is highly expressed (Fig.
2B,F, arrowheads) (Stevenson
et al., 1986
).
|
In epithelial cells, the ASIP/PAR-3 staining pattern is similar to that of
ZO-1. In general, ASIP/PAR-3 (Fig.
2, green) distributes to the subapical domain of every epithelial
cell-cell junction examined. Furthermore, from the basal to the granular
layers of the stratified squamous epithelium in forestomach, where
characteristic TJ are not established, the stainings of both ASIP/PAR-3 and
ZO-1 are mostly detected as non-continuous patterns in the cell-cell contact
regions and appear as punctate patterns in the cytoplasm
(Fig. 2A-C). These results are
reminiscent of previous observations in MDCKII cells where ASIP/PAR-3 and ZO-1
show cytoplasmic punctate distributions during the reconstitution of TJ
(Izumi et al., 1998;
Rajasekaran et al., 1996
).
However, a close comparison of the staining patterns reveals an intriguing
difference between the distribution of ASIP/PAR-3 and ZO-1 in epithelial
cells. Although ASIP/PAR-3 signals can be detected throughout the epithelial
layer, their levels are slightly lower in the basal layer and the most
superficial zone of the granular layer. In contrast, ZO-1 signal level is very
low in the basal layer and increase from the spinous to the granular layer
(Fig. 2C), as reported
previously (Morita et al.,
1998
). Considering the correlation between the levels of
epithelial cell differentiation and the depth of the epithelial layer
(Fig. 2D), these results
suggest that ASIP/PAR-3 staining level is relatively higher in cells
undergoing differentiation, whereas ZO-1 staining increases with epithelial
cell differentiation in the forestomach. In agreement with this observation, a
merged view of ASIP/PAR-3-ZO-1 staining in the small intestine demonstrates
yellow signals in the crypts (Fig.
2G, unfilled arrowheads) and orange signals in the villus
(asterisk), indicating that the signal intensity of ASIP/PAR-3 (green) in
immature epithelial cells is relatively higher than that of ZO-1 (red). Thus,
ASIP/PAR-3 exists in cell-cell junctions at relatively higher levels than ZO-1
in differentiating epithelial cells with less developed TJ strands in the
forestomach and small intestine (Marcial
et al., 1984
).
A difference between ASIP/PAR-3 and ZO-1 is also observed in epithelial
cells in renal tubules (Fig.
2I-K). There is no significant difference between the signal
intensities of ASIP/PAR-3 in the proximal and distal renal tubules
(Fig. 2I). In contrast, the
signal intensity of ZO-1 is much higher in distal rather than proximal renal
tubules (Fig. 2J), consistent
with a previous report that ZO-1 signals correlate with the number of TJ
strands (Fig. 2L)
(Schnabel et al., 1990).
In order to clarify this characteristic distribution of ASIP/PAR-3 at the
ultrastructural level, the localization of ASIP/PAR-3 and ZO-1 was
investigated in renal tubule epithelial cells by immunogold electron
microscopy (Fig. 3). In both
epithelial cells with deep TJ in the distal tubules
(Fig. 3A) and those with
relatively shallow TJ in the proximal tubules
(Fig. 3B), ASIP/PAR-3 localizes
at the cytoplasmic surfaces of TJ. Intriguingly, not all of the TJ are
decorated with gold particles for ASIP/PAR-3: ASIP/PAR-3 is always detected at
the upper edges of TJ (Fig.
3A-D) and is frequently observed at the lower edges of TJ
(Fig. 3A,B). By double-label
immunogold electron microscopy, this characteristic localization of ASIP/PAR-3
contrasts sharply with that of ZO-1, which distributes alongside TJs as
reported previously (Kurihara et al.,
1992) (Fig. 3C and
data not shown). Therefore, the amount and distribution of ASIP/PAR-3 do not
necessarily correlate with the level of TJ development in the epithelial cells
examined here, whereas those of ZO-1 depend closely on the number of TJ
strands, the structural basis of TJ. Together with the fact that the kinase
activity of aPKC, the binding partner of ASIP/PAR-3, is indispensable for the
development of TJs in epithelial cells
(Suzuki et al., 2001
), our
observations led us to investigate the possibility that ASIP/PAR-3 might play
a regulatory role in establishing and/or maintaining TJ structure, rather than
serving only as a structural component of TJ.
|
Induced overexpression of ASIP/PAR-3 in MDCK cells promotes the
development of transepithelial electrical resistance (TER) after plating
To examine the possibility that ASIP/PAR-3 regulates epithelial TJ
formation, we evaluated the effect of the overexpression of ASIP/PAR-3 or its
mutant on the formation and function of TJ. For this purpose, we employed
stable transformants in which ASIP/PAR-3 could be induced by tetracycline or
doxycycline deprivation (Gossen and
Bujard, 1992; Barth et al.,
1997
). We prepared two different T7-tagged expression plasmids for
L-ASIPs; L-ASIP WT has the aPKC-binding sequence, whereas L-ASIP
PB
does not (Fig. 4A). The
regulation of the expression of each protein in the established cell line was
evaluated by western blotting and immunofluorescence with the antibody against
the T7-tag, and we found that the expression of L-ASIP WT or
PB was
completely repressed to under the detectable level by 1.0 µg/ml of
tetracycline (TC) (Fig. 4B,E)
or 10 ng/ml of doxycycline (DC) (Fig.
5G). We also assessed the distribution of each T7-tagged
ASIP/PAR-3 and the homogeneity of its expression level by immunofluorescence.
In MDCK cell lines overexpressing L-ASIP WT or
PB, even though the type
of exogenously expressed ASIP/PAR-3 is different, both T7-tagged ASIPs
preferentially concentrate at cell-cell contacts as endogenous ASIP/PAR-3, and
there is no significant difference in homogeneity of protein expression
between the two MDCK cell lines overexpressing L-ASIP
(Fig. 4C,D).
|
|
We analyzed the development of transepithelial electrical resistance (TER)
using these cell lines. Since TER essentially reflects paracellular resistance
regulated by TJ, de novo formation of TJ can be followed through the
development of TER after plating (Cereijido
et al., 1998). The TER of MDCK cell monolayers initially increases
with time after plating and begins to decrease after the cells become
confluent (Jou et al., 1998
).
First, since cell proliferation is a key factor affecting TER development, we
examined whether the overexpression of ASIP/PAR-3 can alter cell
proliferation; however, we found no significant difference between cells
cultured in the presence or absence of the repressor (10 ng/ml of DC) in any
cell line examined (Fig. 5A-C).
By contrast, the induced overexpression of L-ASIP WT reproducibly results in
the promotion of the early phase of TER development after cell plating at
subconfluent density (1.5x105 cells/cm2;
Fig. 5D, asterisks). Although
the two types of ASIP/PAR-3 are expressed in virtually the same way and remain
constant during cell proliferation (Fig.
5G), the overexpression of L-ASIP
PB has no effect on TER
development (Fig. 5E). In
addition, we confirmed that the depletion of DC itself does not promote but
rather suppresses TER development in the parental cell line
(Fig. 5F). Even if each cell
line is plated at lower density, 1.0x105
cells/cm2, the differences between the three cell lines are still
observed; however, it takes more time for each TER to reach a peak (data not
shown). Therefore, these data indicate that the overexpression of L-ASIP WT,
but not its mutant lacking the aPKC-binding sequence, promotes TJ formation
after plating without changing the rate of MDCK cell proliferation.
Induced overexpression of ASIP/PAR-3 in MDCK cells promotes
insolubilization of occludin after calcium switch
Besides the development of TER, the phosphorylation and enhanced resistance
to NP-40 extraction of occludin are also closely related to epithelial TJ
formation in various cultured cells
(Sakakibara et al., 1997). If
L-ASIP WT does promote TJ formation, the biochemical behavior of occludin can
be expected to be affected in MDCK cells expressing L-ASIP WT. To evaluate
this possibility, we examined occludin insolubilization after Ca2+
switch (Gumbiner and Simons,
1986
) in MDCK cells expressing L-ASIP WT or
PB. Culture in
low (5 µM) Ca2+ medium disrupts cell-cell junctional complexes,
and switching to normal (1.8 mM) Ca2+ medium triggers
cadherin-mediated cell-cell contacts and a series of molecular events,
including occludin insolubilization, to reconstitute junctional complexes.
Confluent monolayers of MDCK cell lines were cultured in low Ca2+
medium for 20 hours, and then CaCl2 was added back to restore the
Ca2+ level (Ca2+ switch). Each cell line cultured in the
presence or absence of 10 ng/ml of DC was subjected to Ca2+ switch.
At different times after Ca2+ switch, total cell lysates were
fractionated into NP-40-soluble and -insoluble fractions, and the amount of
insoluble occludin in each cell line was evaluated by western blotting with
anti-occludin antibody. The amount of protein applied to each lane for each
cell line were normalized to CBB-stained tubulin levels.
As reported previously, phosphorylated and slowly migrating occludin
collects preferentially in the NP-40-insoluble fraction, and there was no
significant change in the total amount of occludin in any cell line examined
throughout the time course (data not shown)
(Sakakibara et al., 1997). In
MDCK cell lines expressing L-ASIP WT, the amount of NP-40-insoluble occludin
starts to increase rapidly to about 1.7-fold within 30 minutes after
Ca2+ switch, and finally reaches about twice the level before
Ca2+ switch (Fig.
6A, left panel; Fig.
6C, filled circles). By contrast, in cells without the induced
overexpression of L-ASIP WT (DC=10 ng/ml), it takes 60 minutes to reach the
same level (Fig. 6A, right
panel; Fig. 6C, unfilled
circles). Furthermore, the induced overexpression of L-ASIP
PB has no
significant effect on the insolubilization of occludin after Ca2+
switch (Fig. 6B;
Fig. 6D, filled and unfilled
squares). Consistent with the above results of TER development, these data
indicate that only L-ASIP WT promotes TJ formation in MDCK cells.
|
Taken together, these independent results evaluating TJ formation by
functional and biochemical analyses clearly indicate that the overexpression
of L-ASIP WT promotes TJ formation in MDCK cells. Furthermore, only L-ASIP WT,
and not L-ASIP PB, has such an effect, suggesting that the interaction
between ASIP/PAR-3 and aPKC may be involved in the promotion of TJ
formation.
ASIP/PAR-3 phosphorylated at Ser827 concentrates at the most apical
tip of cell-cell contacts during the restoration of cell-cell junctions
The aPKC-binding sequence in ASIP/PAR-3 includes two highly conserved
serine residues (827 and 829) within the PKC phosphorylation consensus
sequence (Izumi et al., 1998).
Furthermore, we have recently identified Ser827 in ASIP/PAR-3 as being
phosphorylated by aPKC in vivo and in vitro (Y.T.-N. and S.O., unpublished).
Therefore, the above results suggesting the significance of the aPKC-binding
sequence of ASIP/PAR-3 in the promotion of TJ formation imply that the
phosphorylation of Ser827 is involved in this ASIP/PAR-3 function. To assess
this possibility, we observed the localization of the Ser827-phosphorylated
form of ASIP/PAR-3 in MDCK cells overexpressing L-ASIP WT after
Ca2+ switch using an antibody that specifically recognizes this
phosphorylated form of ASIP/PAR-3 (Fig.
7L,M). When mature cell-cell junctions are established, the
immunoreactivities of Ser827-phosphorylated ASIP/PAR-3
(Fig. 7A,C, red) completely
overlap overexpressed L-ASIP WT in apical cell-cell junctions
(Fig. 7A-C, arrows). By
contrast, 1 hour after Ca2+ switch, when cell-cell junctions are
developing, the Ser827-phosphorylated ASIP/PAR-3 signals
(Fig. 7D) are concentrated at
the most apical tip of cell-cell contacts
(Fig. 7F,G, filled arrowhead),
while overexpressed L-ASIP WT (Fig.
7E) is still detected predominantly in a region slightly basal to
the apical tip (Fig. 7F,H,
unfilled arrowhead). These results imply a relationship between the
localization of ASIP/PAR-3 and the phosphorylation at Ser827 during TJ
formation.
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![]() |
Discussion |
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We have previously identified ASIP, the mammalian homolog of C.
elegans polarity protein PAR-3, as an epithelial TJ-associated peripheral
protein (Izumi et al., 1998);
however, the physiological functions of mammalian ASIP/PAR-3 remain to be
clarified. In this study, we provide two lines of evidence suggesting that
ASIP/PAR-3 is involved in the early phase of epithelial TJ formation. First,
immunofluorescence analysis of various rat epithelial tissues shows that, in
contrast to ZO-1, ASIP/PAR-3 distributes to the apical junctional complex
regions of epithelial cells that do not have well-developed TJ strands
(Fig. 2), and a higher level of
ASIP/PAR-3 is observed in some epithelial cells with immature TJs
(Fig. 2C, forestomach;
Fig. 2G, small intestine). In
addition, immunogold electron microscopy of epithelial cells in renal tubules
reveals that ASIP/PAR-3 concentrates exclusively in the cytoplasm of the
apical edge of TJ, whereas ZO-1 distributes alongside the TJ
(Fig. 3). These observations
indicate that, unlike ZO-1, the distribution patterns of ASIP/PAR-3 do not
necessarily correlate with the level of TJ development in epithelial cells.
Thus, these findings do not favor the possibility that ASIP/PAR-3 serves
simply as a structural element of TJs. Second, we observed that the
overexpression of L-ASIP WT in MDCK cells promotes the initial phase of TJ
formation as evaluated by functional (Fig.
5; TER development measurement) as well as biochemical
(Fig. 6; occludin
insolubilization) analyses. Intriguingly, the promotion of TJ formation by
ASIP/PAR-3 is observed in MDCK cells overexpressing L-ASIP WT, but not those
overexpressing L-ASIP
PB, which lacks the aPKC-binding sequence,
implying that this function of ASIP/PAR-3 is mediated through the interaction
with aPKC. This conclusion is further supported by our demonstration that aPKC
activity is indispensable for the formation but not the maintenance of TJs in
mammalian epithelial cells (Suzuki et al.,
2001
). Furthermore, the significance of the interaction between
ASIP/PAR-3 and aPKC for TJ formation is consistent with previous reports that
C. elegans PAR-3 and aPKC/PKC-3, or their Drosophila
homologs, are mutually dependent on each other for their proper functions in
the establishment of cell polarity (Tabuse
et al., 1998
; Wodarz et al.,
2000
). Taken together, our results strengthen the possibility that
the evolutionarily conserved aPKCASIP/PAR-3 complex is one of the
regulatory elements in mammalian epithelial TJs, and is required to promote
the initial phase of TJ formation rather than serving only as a structural
component.
The ultrastructural analysis of ASIP/PAR-3 localization described here is
the first example of a protein that concentrates at the apical edge of TJs
(Fig. 3). With respect to
mammalian epithelial polarity, TJs are the borders of the apical and lateral
membrane domains. In epithelial cells of arthropods, which lack TJs, the
boundary between the apical and lateral membrane domains is marked by the
`marginal zone', the edge of the apical membrane domain characterized by the
specific accumulation of key regulators of apical polarity such as Crumbs and
Discs Lost (Tepass, 1997;
Tanentzapf et al., 2000
).
Although it has not been shown that mammalian epithelial cells possess such a
structure, the characteristic localization of ASIP/PAR-3 is reminiscent of
that of Crumbs and Discs Lost. Taken together with the possible function of
ASIP/PAR-3, our data suggest the intriguing hypothesis that the apical edge of
TJs might be a functional analog of the marginal zone to specify membrane
polarity in mammalian epithelial cells.
Accumulating experimental evidence favors the possibility of multiple steps
in epithelial TJ formation. Our present data imply that the
aPKCASIP/PAR-3 complex positively regulates epithelial TJ formation,
but for which step is this complex responsible? Several lines of evidence
suggest that the E-cadherin adhesion system mediates the initial organization
of TJ components, including ZO-1, into primordial adherence junctions
(Gumbiner et al., 1988;
Rajasekaran et al., 1996
;
Ando-Akatsuka et al., 1999
). In
the next step, vinculin associated with the cadherin
E-catenin
complex serves to assemble apical actin bundles
(Watabe-Uchida et al., 1998
).
Moreover, since there are data to indicate that ZO-1, ZO-2 and ZO-3 bind
directly to occludin and claudins, which are the primary components of TJ
strands, and actin filaments, it appears reasonable to speculate that ZO-1,
ZO-2 and ZO-3 may be recruited from cadherin-based primordial adherens
junctions to crosslink the assembled apical actin bundles and claudin-based TJ
strands (Furuse et al., 1994
;
Haskins et al., 1998
;
Itoh et al., 1999
;
Wittchen et al., 1999
). The
aPKCASIP/PAR-3 complex, including activated aPKC, and its concentration
at apical cell-cell contacts might be critical for this segregation of ZO-1 to
the TJ structure from cadherin-based primordial adherens junctions. This idea
is based on our previous observations and the results presented here. First,
the overexpression of an aPKC
mutant lacking kinase activity not only
affects proper ASIP/PAR-3 localization, but also significantly perturbs ZO-1
localization to cell-cell junctions in MDCK cells during TJ formation without
severely disturbing the localization of E-cadherin or ß-catenin
(Suzuki et al., 2001
). Second,
we demonstrate here that a relatively higher amount of ASIP/PAR-3 concentrates
to cell-cell junctions in the immature epithelia of forestomach and small
intestine where relatively low amounts of ZO-1 are concentrated
(Fig. 2A-H). Third, we show
that ASIP/PAR-3 phosphorylated at Ser827 is more concentrated at the apical
tip of developing cell-cell contacts than another form of ASIP/PAR-3 without
phosphorylated Ser827 (Fig.
7D-H). This Ser827 is phosphorylated by aPKC in vitro and in vivo
in polarized MDCK cells (Y.T.-N. and S.O., unpublished). Lastly, our recent
observations have revealed that the aPKCASIP/PAR-3 complex exists in
the cytoplasm even in the absence of cell-cell contacts, and that it
translocates to the apical cell-cell contact region very early after
calcium-triggered cell-cell adhesion
(Yamanaka et al., 2001
).
Therefore, our results, together with previously published data, allow us to
speculate about the following possibility: the de novo formation of cell-cell
contacts initiates the translocation of pre-existing aPKCASIP/PAR-3
complexes to primordial adherens junctions, and aPKC might phosphorylate
ASIP/PAR-3 at Ser827 to concentrate this complex at developing apical
cell-cell junctions; consequently, aPKCASIP/PAR-3 could promote the
segregation of TJ-associating proteins, including ZO-1, from primordial
adherens junctions.
Besides the peripheral proteins of TJ, occludin, claudins and junctional
adhesion molecules have been identified as integral TJ membrane proteins
(Furuse et al., 1993;
Furuse et al., 1998a
;
Martin-Padura et al., 1998
).
Previous experimental evidence suggests that highly phosphorylated occludin
copolymerizes into claudin-based TJ strands at a relatively later phase of TJ
formation, whereas non- or less-phosphorylated occludin distributes to the
basolateral membrane (Sakakibara et al.,
1997
; Furuse et al.,
1998b
; Morita et al.,
1998
). Our present data indicate that the overexpression of
ASIP/PAR-3 accelerates occludin insolubilization, which is presumably due to
phosphorylation during the formation of TJ in MDCK cells
(Fig. 6C). In addition, we
demonstrate here the frequent localization of ASIP/PAR-3 at the basal edges of
TJ as visualized by immunoelectron microscopy
(Fig. 3A,B). Therefore, we
propose that ASIP/PAR-3 may participate in the translocation of occludin from
the basolateral membrane domain into TJ strands to establish mature TJs.
Further analyses, including the targeted disruption of the aPKC and/or
ASIP/PAR-3 gene, will make it possible to elucidate the hierarchy of stages in
the formation of epithelial TJ and the development of epithelial polarity.
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