1 Department of Molecular Biology, Yokohama City University School of Medicine,
3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
2 Institute of Cell Biology, ZMBE, University of Muenster, D-48149 Muenster,
Germany
Author for correspondence (e-mail:
ohnos{at}med.yokohama-cu.ac.jp)
Accepted 24 June 2002
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Summary |
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Key words: aPKC, PAR proteins, Epithelial cell polarity, Cell-cell junction, Wound healing
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Introduction |
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PAR (partitioning-defective) proteins, PAR-1 to PAR-6, were first
identified as proteins, mutations of which led to loss of the
anterior-posterior cell polarity of the C. elegans one-cell embryo
(Guo and Kemphues, 1996). The
finding that mammalian PAR-3 (ASIP) specifically binds to atypical PKC (aPKC)
leads us to further reveal that C. elegans aPKC (PKC-3) interacts
with PAR-3 and also plays an indispensable role in cell polarization of the
C. elegans one-cell embryo (Izumi
et al., 1998
; Tabuse et al.,
1998
). We demonstrated recently that in mammalian epithelial
cells, aPKC interacts with not only PAR-3 but also PAR-6 and plays a critical
role in the formation of the apical-basal polarity
(Suzuki et al., 2001
;
Yamanaka et al., 2001
). In
those studies, we overexpressed a dominant-negative mutant of one of the
mammalian aPKC isoforms, aPKC
, in MDCK epithelial cells and found that
the mutant inhibits the development of the tight junction (TJ) structures as
well as the establishment of asymmetric distribution of membrane proteins only
when cell-cell junctions are reset by calcium switch treatment. These results
indicate that aPKC activity is required for the development but not the
maintenance of epithelial polarity, although the molecular targets of aPKC are
still unknown.
One of the interesting features of the cell polarity proteins revealed so
far is that they themselves asymmetrically localize underneath the restricted
regions of the plasma membrane, and this asymmetric submembranous localization
is critical for their function (Bilder,
2001; Ohno, 2001
;
Rose and Kemphues, 1998
). For
example, the localization of aPKC, PAR-3 and PAR-6 is gradually restricted to
the anterior periphery of the C. elegans one-cell embryo in
response to sperm entry, whereas PAR-1 and PAR-2 are restricted to the
posterior periphery during the development of cell polarity
(Rose and Kemphues, 1998
).
Defects in one of the three proteins, aPKC, PAR-3 or PAR-6, result in the
disruption of the asymmetric localization of all other PAR proteins. On the
other hand, during the asymmetric division of neuroblasts in the
Drosophila embryo, basal crescent localization of neuronal
determinants Miranda and Prospero is directed by the apically localized
Inscuteable (Doe and Bowerman,
2001
). Interestingly, recent findings indicate that the correct
localization of Inscuteable depends on the apical localization of the
Drosophila aPKC-PAR complex, which is inherited from the epithelium
from which neuroblast delaminates (Ohno,
2001
; Wodarz et al.,
1999
). Considering that all of these polarity proteins are not
membrane proteins, these results suggest that the establishment of asymmetric
submembranous structures to which these determinants anchor is essential for
cell polarity. However, the molecular basis of this putative submembrane
structure is not known for the C. elegans or Drosophila
embryonic cells. In this sense, the results from mammalian epithelial cells
provided very important clues to address this issue: in these cells, aPKC,
PAR-3 and PAR-6 are asymmetrically localized to TJ, the specialized cell-cell
junctional structure in the most apical region of the basolateral membrane
(Dodane and Kachar, 1996
;
Izumi et al., 1998
).
Furthermore, the cytoplasmic tail of a TJ membrane protein, JAM, interacts
with the first PDZ domain of PAR-3, suggesting that JAM is a strong candidate
for the anchoring partner of the aPKC-PAR complex at TJ
(Ebnet et al., 2001
;
Itoh et al., 2001
). Our recent
results on the inhibitory effect of the dominant-negative mutant of
aPKC
(aPKC
kn) further indicated that the suppression of aPKC
activity resulted in the disruption of the submembranous structure, that is,
TJ to which the aPKC-PAR complex itself is asymmetrically localized
(Suzuki et al., 2001
).
Considering that the formation of the epithelia-specific junctional structures
including TJ, adherens junctions (AJs) and desmosomes, which tightly couple
with the development of asymmetric cytoskeletal structures, represents the
development of submembranous asymmetric structures in epithelial cells
(Denker and Nigam, 1998
;
Yeaman et al., 1999
), these
results suggest an intriguing possibility that aPKC primarily regulates the
development of the epithelia-specific junctional structures of epithelial
cells and thus contributes to the development of the apico-basal polarity.
TJ biogenesis has been suggested to be induced by cell-cell adhesion
mediated by E-cadherin (Gumbiner et al.,
1988), but the molecular mechanism underlying this process is
still unclear. However, by analyzing the wound healing process of a mouse
epithelial cell line, MTD1-A cells, Tsukita and co-workers have provided
evidence that epithelial junctional formation can be dissected into multiple
steps proceeding in a sequential manner during the cell polarization process
(Ando-Akatsuka et al., 1999
;
Yonemura et al., 1995
). On the
basis of immunofluorescence analysis, they demonstrated that at the initial
phase of cell polarization, fibroblastic spot-like AJs containing E-cadherin
as well as ZO-1 are formed as a nascent junctional complex
(Vasioukhin et al., 2000
;
Yonemura et al., 1995
).
Thereafter, as epithelial polarization progresses, ZO-1 dissociates from
E-cadherin, which separately forms the epithelia-specific belt-like AJ, and
gradually colocalizes with occludin at cell-cell contact sites to form TJs
(Ando-Akatsuka et al., 1999
).
Here, by combining the same experimental system and the dominant-negative
mutant of aPKC
used previously
(Suzuki et al., 2001
), we
attempted to clarify how aPKC
kn inhibits TJ formation during
epithelial cell polarization. Our results indicate that aPKC is recruited
after the establishment of the initial spot-like AJ complex to which not only
E-cadherin-catenins but also several TJ components such as JAM, occludin and
claudin-1 are transiently recruited and contributes to the further development
of this premature junctional complex into epithelia-specific structures in
which belt-like AJs, TJs, are asymmetrically segregated.
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Materials and Methods |
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Antibodies
The following monoclonal and polyclonal antibodies (mAb and pAb,
respectively) were used: rabbit anti-PAR-3/ASIP pAb, C2-3
(Izumi et al., 1998); rabbit
anti-aPKC
pAb,
1 (Akimoto
et al., 1994
); rabbit anti-ZO-1 pAb, mouse anti-ZO-1 mAb, rabbit
anti-occludin pAb and rabbit anti-claudin-1 pAb (Zymed); mouse anti-E-cadherin
mAb (Transduction Laboratory); mouse anti-
-catenin mAb (Takara); rat
anti-nectin mAb (Takahashi et al.,
1999
); and rat anti-JAM mAb
(Ebnet et al., 2001
).
Adenovirus infection
The adenovirus vectors carrying cDNA encoding LacZ or aPKCkn have
been described previously (Suzuki et al.,
2001
; Yamanaka et al.,
2001
). For adenovirus infection, MTD1-A cells were seeded on
coverslips in 24-well plates at a density of 1.25x105
cells/cm2 1 day before infection, as described previously
(Suzuki et al., 2001
). After 2
hours of preincubation in low calcium medium containing 5% FCS and 3 µM
Ca2+ (Stuart et al.,
1994
), the cells were incubated for 2 hours with 150 µl of the
appropriate virus solution diluted to 3x108 pfu/ml in LC
medium. Cells were washed two times with PBS and further cultured in normal
growing medium overnight before wounding.
Confocal immunofluorescence microscopy
At an appropriate time after wounding, cells were fixed with 2%
formaldehyde in PBS for 15 minutes at room temperature. After washing twice
with PBS, the cells were permeabilized with 0.5% Triton X-100 in PBS for 10
minutes at room temperature. The cells were then washed and soaked in blocking
solution (PBS containing 10% calf serum) overnight at 4°C. Antibody
incubations were performed at 37°C for 45 minutes in buffer containing 10
mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.01% (v/v) Tween 20 and 0.1% (w/v) BSA.
The secondary antibodies used were Alexa488-conjugated goat anti-rabbit IgG
(Molecular Probes Inc., Eugene, OR), Cy3-conjugated goat anti-mouse IgG,
Cy3-conjugated goat anti-rabbit IgG or Cy3-conjugated goat anti-rat IgG
(Amersham Corp., Arlington Heights, IL). To stain F-actin,
rhodamine-phalloidin (Molecular Probes) was used in place of the secondary
antibodies. Coverslips were mounted using Vectashield (Vector Laboratories,
Burlingame, CA) and examined under a fluorescence microscope equipped with a
confocal system (µRadiance, Bio-Rad Laboratories, Hercules, CA). An
oil-immersion objective lens (Plan APOCHROMAT x63, NA 1.40) (Nikon), and
argon and red diode lasers were used. Conventional images were composed of
512x512 pixels. Usually, about 30 optical sections covering the basal to
the apical region of cells were taken with an interval of 0.4 µm, and all
images were projected unless indicated otherwise.
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Results |
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aPKC is recruited last to TJs
The TJ membrane proteins occludin and JAM colocalize with ZO-1 and
E-cadherin in the spot-like AJs prior to forming continuous TJ structures
(Ando-Akatsuka et al., 1999;
Ebnet et al., 2001
). Therefore,
the results in Fig. 1 suggest
that aPKC and PAR-3 are recruited into cell-contact regions later than these
TJ membrane proteins. In fact, we demonstrated by double staining analysis
that many JAM-positive spot-like AJs observed in intermediate stages of the
wound healing of MTD1-A cells were negative for PAR-3
(Ebnet et al., 2001
). Again,
owing to the lack of appropriate antibodies, we could not perform similar
experiments for the other TJ membrane proteins, occludin as well as claudin-1.
However, during indirect comparison between these TJ proteins and PAR-3/aPKC
with respect to junctional recruitment, we unexpectedly found that claudin-1
is recruited to the junctions considerably later than JAM and occludin and as
late as aPKC
and PAR-3. As demonstrated previously, JAM almost
completely colocalized with the spot-like AJs positive for ZO-1 formed at the
very early stage of wound healing (Fig.
2A top panels). The occludin signal was also detected in most of
the nascent spot-like AJs, although its signal on the ZO-1-positive dot-like
AJs tended to be weaker than that of JAM (arrows in
Fig. 2A, middle panels). On the
other hand, many ZO-1-positive dot-like or fragmental structures at nascent
cell-cell contacts showed a weak or negative signal for claudin-1 (arrows in
Fig. 2A, bottom panels). Even
relatively developed junctions more than 3 µm long were frequently negative
for claudin-1 staining. These results suggest that claudin-1 is recruited into
the junctional area after the establishment of the dot-like AJs. This was
further confirmed by the direct comparison between doubly stained JAM and
claudin-1 (Fig. 2B, top
panels); again, many JAM-positive junctional structures of 3 µm length were
often negative for claudin-1 staining. Considering the similarity of the
immunostaining pattern of claudin-1 to those of PAR-3 and aPKC
in
nascent junctional areas (Fig.
1, Fig. 2B bottom
panels), these results indicate that TJ components can be subdivided into at
least two groups in terms of junctional recruitment, and the polarity
proteins, aPKC and PAR-3, as well as claudin-1 belong to the same group whose
recruitments is rather late.
|
Overexpression of a dominant-negative mutant of aPKC inhibits
the development of spot-like AJs into epithelia-specific belt-like AJs
Previously, by using the adenovirus gene-transfer technique, we
demonstrated that overexpression of a kinase-negative mutant of aPKC
or
(aPKCkn) disturbs the junctional formation of MDCK II cells observed
in the re-polarization process after a calcium switch treatment through their
dominant-negative activity against endogenous aPKC (
or
) kinase
activity (Suzuki et al.,
2001
). To address the question of which steps of the epithelial
junctional formation pathway are blocked by aPKCkn, we applied this
aPKC
mutant to the wound-healing assay using MTD-1A. When a confluent
monolayer of MTD1A cells (2.5x105 cells/cm2) was
infected with adenovirus vector at MOI 600, aPKC
kn was expressed in
approximately 90% of the cells showing heterogeneous expression levels
(Fig. 3A, lower left panel).
Similar to the situation shown for MDCK II cells in previous work
(Suzuki et al., 2001
),
junctional structures monitored by ZO-1 staining were hardly affected by the
expression of aPKC
kn unless the infected cells were subjected to
regeneration of cell-cell adhesion by a calcium switch treatment (data not
shown) (see ZO-1 staining at cell-cell boundaries in non-wounded regions of
aPKC
kn-expressing cells in Fig.
3A, lower right panel). When monolayers of MTD1-A cells expressing
aPKC
kn or LacZ were wounded, they showed apparently normal wound
closure within 6-10 hours under phase-contrast microscope observation (data
not shown). In LacZ-expressing cells, continuous ZO-1 staining was completely
restored in the healed regions (Fig.
3A, upper right panel). This was also the case when nPKC
kn
was used as a negative control instead of LacZ (data not shown). However, in
cells expressing aPKC
kn, the completion of TJ formation monitored by
ZO-1 was significantly inhibited in cells burying the wound
(Fig. 3A, lower panels). Closer
inspection demonstrated that in cells that participated in burying the wound,
ZO-1 staining was broadly observed in dot-like structures or in very short
fragments at cell-cell borders, and this staining pattern did not change even
after the wound was completely healed (30 hours after wounding,
Fig. 3B). These cells also
exhibited aberrant E-cadherin staining that revealed dot-like discontinuous
structures instead of belt-like AJs as observed in LacZ-expressing cells.
Comparison of ZO-1 and E-cadherin staining in a single confocal plane
confirmed that many ZO-1-positive dot-like AJs, if not all, are also positive
for E-cadherin (Fig. 4, top
panels). It was further demonstrated that
-catenin and nectin also
showed colocalization with ZO-1 in these structures with higher frequency
(Fig. 4, middle and bottom
panels). Together with the fact that many F-actin bundles are running into
these dot-like structures (see below), these results indicate that the
ZO-1-positive dot-like structures induced by aPKC
kn expression are
structurally identical to the spot-like AJs observed during the normal wound
healing process, which appear only in newly formed cell-cell contacts between
encountering cells in the first row of the wound margin. Taken together, the
results in Figs 3 and
4 suggest that the inhibition
of aPKC
kinase activity does not suppress the formation of the
primordial spot-like AJ complexes; rather, it blocks their development into
the epithelia-specific belt-like AJs.
|
|
|
Interestingly, confocal z-sectional analysis revealed that
aPKCkn-expressing cells burying the wound also develop a columnar
shape with a height comparable to that of surrounding cells, and the spot-like
AJs reside at the apical tip of the lateral membranes (small arrowhead in
Fig. 5B). In fact, besides
being distributed on the dot-like AJs, E-cadherin and
-catenin also
showed broad distribution on the lateral membrane (small arrowheads in
Fig. 4, top and middle panels),
suggesting that asymmetric domain formation in the lateral membrane occurred
even in aPKCkn-expressing cells lacking mature belt-like AJs. In these cells,
the premature cortical bundle structures linking the spot-like AJs were formed
underneath the apical membrane independently of the basal stress fibers
(Fig. 4B,C). Therefore, these
results indicate that in aPKC
kn-expressing cells, F-actin
reorganization to restore cortical bundles and the epithelia-specific columnar
shape proceeds to some extent during the re-epithelialization process, but the
final step involving connection of the F-actin cortical bundles closely to
fused belt-like AJs is inhibited completely in these cells.
TJ membrane proteins and PAR-3 are trapped in the persistent
spot-like AJs in aPKCkn-expressing cells
As shown in Fig. 6A, almost
all of the ZO-1-positive spot-like AJs in aPKCkn-expressing
wound-healing cells were positive for JAM and occludin. Interestingly,
claudin-1 also showed almost complete colocalization with ZO-1 in the dot-like
structures induced by aPKC
kn, although during the normal wound healing
process, it is recruited into the junctional area rather late and is thus
hardly detected in the dot-like AJs (Fig.
2). These results strongly reinforce the idea that the dot-like
AJs are intermediate structures for epithelial junctional development to which
TJ components including claudin-1 can be transiently recruited. Furthermore,
they also indicate that aPKC
kinase activity is not required for this
translocation. Rather, aPKC
plays a critical role in the subsequent
segregation step of the junctional proteins in these primordial AJ structures
into mature belt-like AJs and TJs.
|
We next examined the localization of PAR-3 in aPKCkn-expressing
wound-healing cells and found that a substantial part of the persistent
dot-like AJs was positive for PAR-3 (Fig.
6B). Therefore, PAR-3 translocation to the spot-like AJs can also
occur without aPKC
kn activity. This is consistent with the indirect
observation that PAR-3 translocates to the spot-like AJs before the
recruitment of aPKC into the junctional regions
(Fig. 1). However, it should
also be noted that in contrast with other TJ components, many ZO-1-positive
spot-like AJs show weak or negative staining for PAR-3 even 30 hours after
wounding (see small arrows in Fig.
6B, right panel). This may indicate that the translocation or
stability of PAR-3 at the spot-like AJs partially depends on aPKC
activity.
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Discussion |
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In this work, we reinforced this idea by finding that the overexpression of
aPKCkn results in the blockage of development of the epithelial
junctional structure at the formation of spot-like AJs. The present results
indicate that aPKC activity is not required for the first step of junctional
development, that is, the recruitment of junctional proteins into contact
sites, but is indispensable for the second step to reconstruct the nascent
junctional complex into epithelia-specific mature junctional structures.
Significantly, the persistent spot-like AJs in aPKC
kn-expressing cells
contain multiple junctional membrane proteins, such as E-cadherin, nectin,
JAM, occludin and claudin-1, confirming that the spot-like AJs are structures
in which many junctional proteins gather together prior to their subsequent
segregation. These membrane proteins are considered to assemble in the
structures by interacting directly or indirectly (through peripheral protein
such as catenins for E-cadherin) with various peripheral scaffold proteins
containing PDZ domains, such as ZO-1, afadin and MUPP1, which can associate
with the cytoplasmic regions of the multiple membrane proteins
(Ebnet et al., 2000
; Hamazaki
et al., 2001; Itoh et al.,
1999
; Itoh et al.,
2001
). Therefore, these results raise an intriguing possibility
that aPKC is responsible for modification of the interactions between
junctional membrane proteins and scaffold proteins or between membrane
proteins by themselves, thereby promoting the transition of junctional
structures from the first step to the second one. This is consistent with
another observation in this study that aPKC is one of the later TJ components
with respect to junctional recruitment.
Of course, in addition to modifying junctional proteins, aPKC may affect
junctional formation by interfering with other events required for the
development of belt-like AJs and TJs. In this sense, it should be noted that
Vasioukhin et al. have suggested that an antiparallel pair of filopodia, at
the tip of which the spot-like AJs (or puncta) are formed, physically draws
the two cell surfaces together, thereby extending the zone of cell-cell
contact (Vasioukhin et al.,
2000). They further demonstrated that actin polymerization at the
tip of the protrusions plays an active role in extending the cell-cell contact
area. Therefore, the regulation of F-actin polymerization and reorganization
may be another target of aPKC phosphorylation to promote belt-like AJ
formation. In fact, we observed that cortical bundle formation is blocked in
aPKC
kn-expressing cells at an intermediate stage
(Fig. 5) (Yonemura et al., 1995
).
However, we also observed that even the cells expressing aPKC
kn
lacking continuous junctional structures increased their heights to a level
(>10 µm) comparable to those of cells in non-wounded areas. The
premature spot-like AJs and the premature cortical bundles are localized at
the apical tip of the lateral membranes, at which neighboring cells are in
close contact. Therefore, these results indicate that the cells expressing
aPKC
kn can reorganize the actin filament architecture to some extent
and physically draw neighboring cell surfaces together to assume a thick
epithelia-specific columnar shape. Therefore, the incomplete cortical bundle
formation of F-actin may be a secondary consequence of the inhibition of the
fusion of the spot-like AJs into belt-like ones.
aPKC plays an indispensable role in the establishment of cell polarity not
only in the C. elegans and Drosophila early embryo but also
in mammalian epithelial cells by forming an evolutionarily conserved protein
complex with PAR-3 and PAR-6. We have previously demonstrated that PAR-6 may
mediate signals from Rac1/Cdc42 to aPKC by interacting with both proteins and
activating aPKC in a GTP-dependent manner
(Yamanaka et al., 2001). Since
we observed that, as in the case of MDCK, overexpression of a PAR-6 mutant
lacking aPKC-binding domain also showed effects similar to those induced by
aPKCKn during wound healing of MTD1A cells (data not shown)
(Yamanaka et al., 2001
), aPKC
is thought to function as a component of the aPKC-PAR complex in promoting
belt-like AJ formation. Here, we also found that an aPKC-binding scaffold
protein with three PDZ domains, PAR-3, is substantially but not completely
recruited into the premature spot-like AJ complex induced by aPKC
kn,
suggesting that it can translocate to the structure without aPKC activity.
These results indicate that PAR-3 works as a scaffold protein to recruit aPKC
and PAR-6, which can act together to stabilize the complex at the junctional
area. In fact, although indirectly, we observed that PAR-3 is recruited faster
than aPKC into cell-cell contact regions during the normal wound healing
process (Fig. 1). This is
consistent with the observation that in the C. elegans one-cell
embryo, PAR-3 is transiently present at the cell periphery even in the absence
of either PKC-3 or PAR-6, although both PKC-3 and PAR-6 absolutely require
PAR-3 (Hung and Kemphues,
1999
; Tabuse et al.,
1998
; Watts et al.,
1996
). Considering the fact that the cytoplasmic region of JAM
binds to the first PDZ domain of PAR-3, JAM, which is recruited into the
junctional region as early as ZO-1, may be a membrane target of PAR-3
(Ebnet et al., 2001
;
Itoh et al., 2001
,). Then,
aPKC may target PAR-3 anchoring to JAM. We previously suggested the
possibility that aPKC translocates to the junctional region as a ternary
complex with PAR-3 and PAR-6 in MDCKII cells after a calcium switch, since a
considerable amount of the ternary complex was detected even in depolarized
MDCK II cells cultured in low calcium medium
(Yamanaka et al., 2001
). We do
not know the precise reason for the apparent discrepancy between the present
results and the previous data. However, it is possible that the asynchronous
and rapid polarization of MDCK II cells after a calcium switch made it
difficult to detect subtle differences between the recruitment of aPKC and
that of PAR-3. It may also reflect differences between the depolarization
states of cells induced by calcium depletion or mechanical wounding.
In summary, the present work reveals that aPKC is required at the step
where the immature cell-cell junctional complex differentiates into
epithelia-specific asymmetric junctional structures. This finding supports our
hypothesis that the aPKC-PAR system plays an indispensable role in the
establishment of cell polarity by primarily regulating the formation of an
asymmetric submembranous structure to which it anchors. Interestingly, recent
progress in the genetic and molecular analysis of Drosophila or
C. elegans embryos also revealed that epithelial junctions are
assembled in a two-step process, that is, the formation of spot-like AJs along
the lateral membrane rather randomly and their accumulation into the apical
tip of the membrane and development into belt-like AJs
(Michaux et al., 2001;
Tepass et al., 2001
). Many
mutants of polarity proteins including BAZOOKA, a Drosophila
homologue of PAR-3, have been reported to block the transition between these
two steps, indicating that this transition, which is indispensable for
asymmetric membrane domain differentiation is the critical step for epithelial
cell polarization. Many polarity proteins are expected to interact in a
complex fashion during this step. There is no doubt that the identification of
the molecular target of aPKC is one of the important steps to resolve the
molecular basis for this epithelial cell polarization process.
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Acknowledgments |
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Footnotes |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, C. L., Chen, Y. T., Smith, S. J. and Nelson, W. J.
(1998). Mechanisms of epithelial cell-cell adhesion and cell
compaction revealed by high-resolution tracking of E-cadherin-green
fluorescent protein. J. Cell Biol.
142,1105
-1119.
Akimoto, K., Mizuno, K., Osada, S., Hirai, S., Tanuma, S.,
Suzuki, K. and Ohno, S. (1994). A new member of the third
class in the protein kinase C family, PKC lambda, expressed dominantly in an
undifferentiated mouse embryonal carcinoma cell line and also in many tissues
and cells. J. Biol. Chem.
269,12677
-12683.
Ando-Akatsuka, Y., Yonemura, S., Itoh, M., Furuse, M. and Tsukita, S. (1999). Differential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity. J. Cell Physiol. 179,115 -125.[CrossRef][Medline]
Bilder, D. (2001). Cell polarity: squaring the circle. Curr. Biol. 11,R132 -R135.[CrossRef][Medline]
Denker, B. M. and Nigam, S. K. (1998). Molecular structure and assembly of the tight junction. Am. J. Physiol. 274,F1 -F9.[Medline]
Dodane, V. and Kachar, B. (1996). Identification of isoforms of G proteins and PKC that colocalize with tight junctions. J. Membr. Biol. 149,199 -209.[CrossRef][Medline]
Doe, C. Q. and Bowerman, B. (2001). Asymmetric cell division: fly neuroblast meets worm zygote. Curr. Opin. Cell Biol. 13,68 -75.[CrossRef][Medline]
Ebnet, K., Schulz, C. U., Meyer Zu Brickwedde, M. K., Pendl, G.
G. and Vestweber, D. (2000). Junctional adhesion molecule
interacts with the PDZ domain-containing proteins AF-6 and ZO-1. J.
Biol. Chem. 275,27979
-27988.
Ebnet, K., Suzuki, A., Horikoshi, Y., Hirose, T., Meyer Zu
Brickwedde, M. K., Ohno, S. and Vestweber, D. (2001). The
cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion
molecule (JAM). EMBO J.
20,3738
-3748.
Enami, J., Enami, S. and Koga, M. (1984). Isolation of an insulin-responsive preadipose cell line and a mammary tumor virus-producing, dome-forming epithelial cell line from a mouse mammary tumor. Dev. Growth Differ. 26,223 -234.[CrossRef]
Gumbiner, B., Stevenson, B. and Grimaldi, A. (1988). The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex. J. Cell Biol. 107,1575 -1587.[Abstract]
Guo, S. and Kemphues, K. J. (1996). Molecular genetics of asymmetric cleavage in the early Caenorhabditis elegans embryo. Curr. Opin. Genet. Dev. 6, 408-415.[CrossRef][Medline]
Hamazaki, Y., Itoh, M., Sasaki, H., Furuse, M. and Tsukita,
S. (2002). Multi-PDZ-containing protein 1 (MUPP1) is
concentrated at tight junctions through its possible interaction with
claudin-1 and junctional adhesion molecule. J. Biol.
Chem. 277,455
-461.
Hirano, S., Nose, A., Hatta, K., Kawakami, A. and Takeichi, M. (1987). Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles. J. Cell Biol. 105,2501 -2510.[Abstract]
Hung, T. J. and Kemphues, K. J. (1999). PAR-6
is a conserved PDZ domain-containing protein that colocalizes with PAR-3 in
Caenorhabditis elegans embryos. Development
126,127
-135.
Itoh, M., Furuse, M., Morita, K., Kubota, K., Saitou, M. and
Tsukita, S. (1999). Direct binding of three tight
junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of
claudins. J. Cell Biol.
147,1351
-1363.
Itoh, M., Sasaki, H., Furuse, M., Ozaki, H., Kita, T. and
Tsukita, S. (2001). Junctional adhesion molecule (JAM) binds
to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight
junctions. J. Cell Biol.
154,491
-497.
Izumi, Y., Hirose, T., Tamai, Y., Hirai, S., Nagashima, Y.,
Fujimoto, T., Tabuse, Y., Kemphues, K. J. and Ohno, S.
(1998). An atypical PKC directly associates and colocalizes at
the epithelial tight junction with ASIP, a mammalian homologue of
Caenorhabditis elegans polarity protein PAR-3. J. Cell
Biol. 143,95
-106.
Michaux, G., Legouis, R. and Labouesse, M. (2001). Epithelial biology: lessons from Caenorhabditis elegans. Gene 277,83 -100.[CrossRef][Medline]
Ohno, S. (2001). Intercellular junctions and cellular polarity: the PAR-aPKC complex, a conserved core cassette playing fundamental roles in cell polarity. Curr. Opin. Cell Biol. 13,641 -648.[CrossRef][Medline]
Rose, L. S. and Kemphues, K. J. (1998). Early patterning of the C. elegans embryo. Annu. Rev. Genet. 32,521 -545.[CrossRef][Medline]
Stuart, R. O., Sun, A., Panichas, M., Hebert, S. C., Brenner, B. M. and Nigam, S. K. (1994). Critical role for intracellular calcium in tight junction biogenesis. J. Cell Physiol. 159,423 -433.[Medline]
Suzuki, A., Yamanaka, T., Hirose, T., Manabe, N., Mizuno, K.,
Shimizu, M., Akimoto, K., Izumi, Y., Ohnishi, T. and Ohno, S.
(2001). Atypical protein kinase C is involved in the
evolutionarily conserved par protein complex and plays a critical role in
establishing epithelia-specific junctional structures. J. Cell
Biol. 152,1183
-1196.
Tabuse, Y., Izumi, Y., Piano, F., Kemphues, K. J., Miwa, J. and
Ohno, S. (1998). Atypical protein kinase C cooperates with
PAR-3 to establish embryonic polarity in Caenorhabditis elegans.Development 125,3607
-3614.
Takahashi, K., Nakanishi, H., Miyahara, M., Mandai, K., Satoh,
K., Satoh, A., Nishioka, H., Aoki, J., Nomoto, A., Mizoguchi, A. et al.
(1999). Nectin/PRR: an immunoglobulin-like cell adhesion molecule
recruited to cadherin-based adherens junctions through interaction with
afadin, a PDZ domain-containing protein. J. Cell Biol.
145,539
-549.
Tepass, U., Tanentzapf, G., Ward, R. and Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35,747 -784.[CrossRef][Medline]
Vasioukhin, V., Bauer, C., Yin, M. and Fuchs, E. (2000). Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell 100,209 -219.[Medline]
Watts, J. L., Etemad-Moghadam, B., Guo, S., Boyd, L., Draper, B.
W., Mello, C. C., Priess, J. R. and Kemphues, K. J. (1996).
par-6, a gene involved in the establishment of asymmetry in early C.
elegans embryos, mediates the asymmetric localization of PAR-3.
Development 122,3133
-3140.
Wodarz, A., Ramrath, A., Grimm, A. and Knust, E.
(2000). Drosophila atypical protein kinase C associates
with Bazooka and controls polarity of epithelia and neuroblasts. J.
Cell Biol. 150,1361
-1374.
Wodarz, A., Ramrath, A., Kuchinke, U. and Knust, E. (1999). Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts. Nature 402,544 -547.[CrossRef][Medline]
Yamanaka, T., Horikoshi, Y., Suzuki, A., Sugiyama, Y., Kitamura,
K., Maniwa, R., Nagai, Y., Yamashita, A., Hirose, T., Ishikawa, H. et al.
(2001). PAR-6 regulates aPKC activity in a novel way and mediates
cell-cell contact-induced formation of the epithelial junctional complex.
Genes Cells 6,721
-731.
Yeaman, C., Grindstaff, K. K. and Nelson, W. J.
(1999). New perspectives on mechanisms involved in generating
epithelial cell polarity. Physiol. Rev.
79, 73-98.
Yonemura, S., Itoh, M., Nagafuchi, A. and Tsukita, S.
(1995). Cell-to-cell adherens junction formation and actin
filament organization: similarities and differences between non-polarized
fibroblasts and polarized epithelial cells. J. Cell
Sci. 108,127
-142.