1 Department of Biological Chemistry
2 Howard Hughes Medical Institute
3 Department of Internal Medicine, University of Michigan Medical School, Ann
Arbor, MI 48109-0650, USA
Author for correspondence (e-mail:
bmargoli{at}umich.edu)
Accepted 24 March 2003
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Summary |
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Key words: Crumbs3, Pals1, PDZ domain, Tight junction, Polarity
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Introduction |
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The apical and basolateral membranes are demarcated by the tight junction
(TJ), a specialized site of cell contact at the apical aspect of the lateral
membrane. TJs are composed of a complex network of integral and peripheral
membrane proteins. Claudins, occludin, and junctional adhesion molecule
mediate cell adhesions at this site. In turn, these proteins associate with a
diverse collection of cytoplasmic proteins that serve a variety of functions
(Zahraoui et al., 2000). For
instance, Zona occludens-1 (ZO-1), Zona occludens-2, and Zona occludens-3 link
the above proteins to the underlying actin cytoskeleton
(Fanning et al., 1998
;
Fanning et al., 2002
). A subset
of TJ-associated proteins (Rab3b, Rab8, Rab13, Sec6, Sec8, etc.) are involved
in trafficking and docking of vesicles
(Zahraoui et al., 2000
).
Recently, an increasing number of studies have elucidated an intimate
functional relationship between the TJ and the establishment of apico-basal
polarity. The majority of these reports have focused on an evolutionarily
conserved signaling complex composed of aPKC and the PDZ domain containing
proteins, Par6 and Par3/ASIP (Joberty et
al., 2000
; Lin et al.,
2000
). DaPKC, D-Par6, and Bazooka represent the orthologues of
these proteins in Drosophila, respectively. In Drosophila,
this complex has been shown to play important roles in establishing asymmetry
in epithelia and delaminating neuroblasts during embryogenesis
(Petronczki and Knoblich,
2001
; Wodarz et al.,
2000
). In mammalian epithelia, the Par6-ASIP-aPKC complex, in
association with the monomeric GTPase CDC42, has been shown to regulate the
assembly of TJs (Gao et al.,
2002
; Hirose et al.,
2002
; Izumi et al.,
1998
; Joberty et al.,
2000
; Suzuki et al.,
2001
; Yamanaka et al.,
2001
).
We and others have recently characterized another evolutionarily conserved
TJ complex composed of the orthologues of Drosophila Crumbs (CRB),
Stardust (Sdt), and Discs lost (Dlt): Crumbs 3 (CRB3), Pals1, and Pals1
associated TJ protein (PATJ), respectively
(Lemmers et al., 2002;
Makarova et al., 2003
;
Roh et al., 2002b
). CRB and
CRB3 are transmembrane proteins whereas the other proteins are cytoplasmic
scaffolding proteins. Sdt and Pals1 are membrane associated guanylate kinase
(Maguk) proteins each containing a single PDZ domain
(Bachmann et al., 2001
;
Hong et al., 2001
;
Kamberov et al., 2000
). In
contrast, Dlt and PATJ bear multiple PDZ domains
(Tepass, 2002
). In mammalian
epithelia, Pals1 acts as an adaptor mediating the indirect interaction between
Crumbs and PATJ (Roh et al.,
2002b
). In Drosophila, Sdt is predicted to be involved in
similar types of interactions with CRB and Dlt
(Tepass, 2002
).
Fly embryos lacking CRB and Sdt expression exhibit similar phenotypes
failure to form a zonula adherens and disruption of apico-basal
polarity (Knust et al., 1993;
Tepass and Knust, 1993
). The
CRB null phenotype can be rescued by exogenously expressing full-length or
only the transmembrane and intracellular portion of CRB
(Klebes and Knust, 2000
).
Furthermore, CRB overexpression in a wild-type background also leads to
polarity defects (Wodarz et al.,
1995
). These two phenomena specifically require the extreme
C-terminus of CRB, which binds to the PDZ domain of Sdt
(Klebes and Knust, 2000
;
Roh, 2002b
). These results
demonstrate the intimate relationship between CRB and Sdt during the
regulation of apico-basal polarization in Drosophila epithelial
cells. Although it has been shown that Sdt contains multiple protein-protein
interaction domains, the identities of Sdt binding partners that are important
for epithelial polarity are less understood.
Although Drosophila epithelia lack TJs, the CRB-Sdt-Dlt and
CRB3-Pals1-PATJ complexes both target to the apical aspect of cell contacts in
Drosophila and mammalian epithelia, respectively
(Tepass, 2002). To date, the
physiological roles of the CRB3-Pals1-PATJ complex in mammalian epithelia are
poorly understood. However, the similarity in the subcellular localization of
these complexes suggests that they perform analogous functions. In the present
study, we investigate the functional relationship between CRB3 and mammalian
epithelial cell polarity. We demonstrate the importance of the CRB3-Pals1
interaction in the regulation of TJ biogenesis and apico-basal
polarization.
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Materials and methods |
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DNA constructs
The DNA encoding CRB3, excluding the signal peptide sequence, was PCR
amplified and cloned into the pSecTag2B vector (Invitrogen, Carlsbad, CA). The
pSecTag2B-MycCRB3 construct has been described previously
(Makarova et al., 2003). This
construct was used as a template during PCR amplification of the sequence
encoding the cytoplasmic 37 residues of CRB3. This DNA was subsequently cloned
into the pGSTag vector and the resulting construct used to express the
GST-CRB3 fusion protein in bacterial cells. Point and deletion mutagenesis in
these constructs was carried out as previously described
(Makarova et al., 2000
).
The pRK5-Myc-Lin-2 construct has also been described elsewhere
(Lee et al., 2002). The PDZ
domain was removed via deletion mutagenesis to yield the
pRK5-Myc-Lin-2
PDZ construct. To design the Myc-Lin-2Pals1 PDZ
chimeric construct, the encoding sequence of the Lin-2 PDZ domain was replaced
with the DNA encoding the Pals1 PDZ domain. First, the DNA encoding the Pals1
PDZ domain was PCR amplified using the following primers:
5'-CCAGAGTT-CGGCTGGTACAGTTTGAAAAGGCTCGGGATATT-3'
(forward) and
5'-GGACGAAGACTGAGTGCGGTACTGTTGACTAGGAATCAGAAC-3'
(reverse). The underlined flanking bases correspond to the sequences flanking
the DNA encoding the PDZ domain of Lin-2. The bases shown in bold represent
the start and end of the DNA sequence encoding the Pals1 PDZ domain. Next, the
sense and antisense strands of the resulting PCR product were utilized as
primers in a mutagenesis reaction using the pRK5-Myc-Lin-2 template and Pfu
turbo polymerase (Stratagene, Cedar Creek, TX). All constructs were verified
by automated sequencing at the University of Michigan DNA Sequencing Core.
Cell culture and transfection
MDCK cells and HEK293 cells were transfected and cultured as described
previously (Roh et al.,
2002b). MDCK cell cysts were grown in three-dimensional collagen
gels as previously described (O'Brien et
al., 2001
; Pollack et al.,
1998
). Essentially, MDCK cells were trypsinized, triturated into a
single cell suspension, and then mixed into an ice-cold solution containing 2
mg/ml calf skin type I collagen (Sigma), 1x DMEM, 20 mM Hepes pH 7.4,
and 5% FBS. The resulting suspensions were added to 10 mm diameter Transwell
membrane filters (0.4 µm pore size; Corning Costar) and allowed to form a
solidified gel at 37°C prior to addition of culture media. The media was
replaced every two days and the cysts were allowed to develop over 5-6
days.
Immunoprecipitation and immunoblotting
Lysates were prepared from MDCK and HEK293 cell lines as previously
described (Roh et al., 2002b).
Immunoprecipitation, GST pulldown, and immunoblotting experiments were
performed according to previously published protocols
(Kamberov et al., 2000
).
Calcium switch experiments
Approximately 5x105 MDCK cells were seeded onto 10 mm
diameter Transwell membrane filters and cultured overnight in normal calcium
media (DMEM, 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate,
and 2 mM L-glutamine) containing 1.8 mM Ca2+ allowing for the
formation of a fully confluent monolayer. Subsequently, the monolayers were
washed five times with PBS and grown in low calcium media containing 5%
dialyzed FBS and 5 µM Ca2+ overnight to dissociate cell-cell
contacts. The next day, the low calcium media was replaced with pre-warmed
normal calcium media and this was designated as the t=0 timepoint.
Immunostaining and confocal microscopy
MDCK monolayers and cysts were processed and immunostained as described
previously (O'Brien et al.,
2001; Roh et al.,
2002b
). For staining of cysts, collagen gels were detached from
the filter supports, washed with PBS, and incubated in PBS supplemented with
100 U/ml collagenase VII (Sigma) for 15 minutes at room temperature.
Subsequently, gels were fixed in 4% paraformaldehyde/PBS for 30 minutes,
permeabilized in 0.25% Triton X-100/PBS for 30 minutes, and then incubated in
2% goat serum/PBS for 1 hour. Next, gels were incubated with primary
antibodies in 2% goat serum/PBS for two days at 4°C under constant
agitation. Gels were then washed extensively with 2% goat serum/PBS and then
soaked in the 2% goat serum/PBS supplemented with the appropriate
fluorochrome-conjugated secondary antibodies overnight at 4°C. Staining of
actin microfilaments was achieved by adding fluorochrome-conjugated phalloidin
(Molecular Probes, Eugene, OR) to the secondary antibody solution. Finally,
gels were washed extensively in PBS and mounted onto glass coverslips using
ProLong antifade reagent (Molecular Probes). Cysts were visualized using a
ZEISS LSM510 Axiovert 100M inverted confocal laser-scanning microscope. Images
were analyzed using the LSM Image Examiner and Adobe Photoshop software.
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Results |
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We first investigated this possibility by isolating MDCK stable cell lines that vastly overexpressed human CRB3, Myc-CRB3, or moderately overexpressed Myc-CRB3 (Fig. 1A). In monolayers overexpressing CRB3 cultured on filter supports in normal calcium media (1.8 mM Ca2+) for 12 hours, a fraction of CRB3 displayed an apical distribution (Fig. 1B,C). However, significant amounts of CRB3 localized to the lateral membrane. The amount of CRB3 present at the lateral surface seemed to correlate with the degree of CRB3 overexpression. Specifically, in MDCK cells vastly overexpressing Myc-CRB3, a significant fraction of Myc-CRB3 targeted laterally (Fig. 1D). In the moderately overexpressing Myc-CRB3 cell line, lateral Myc-CRB3 was also observed but to a slightly lesser extent. It should be noted that CRB3 expression was above endogenous levels in all of these cell lines (Fig. 1A).
|
To assess the degree of apical expansion respective to apical marker proteins, we immunostained these cell lines with anti-gp135 and anti-ezrin at 12 hours post-calcium switch. In monolayers overexpressing CRB3 and Myc-CRB3, a fraction of ezrin and gp135 was observed to target laterally (Fig. 1C, data not shown). These effects were not observed in the parental MDCK cells cultured in a similar manner (Fig. 1E). Collectively, these observations suggest that CRB3 overexpression results in the expansion of the apical membrane in agreement with Drosophila studies on Crumbs overexpression.
CRB3 overexpression negatively regulates tight junction biogenesis in
a Pals1-dependent manner
In MDCK cells vastly overexpressing CRB3, CRB3 and apical surface markers
were localized to both the apical and lateral surface suggesting that CRB3
overexpression can result in apical membrane expansion. Because a
CRB3-Pals1-PATJ complex probably exists at TJs
(Makarova et al., 2003), we
wondered if CRB3 could regulate TJ assembly. Therefore, we analyzed TJ
formation in MDCK cells overexpressing Myc-CRB3. We also generated specific
mutations in Myc-CRB3 (summarized in Fig.
2A) and transfected the mutant Myc-CRB3 constructs into MDCK
cells. The cytoplasmic tail of 37 amino acids contains two conserved regions:
a putative juxtamembrane protein 4.1/ezrin/radixin/moesin (FERM)-binding
domain and the extreme C-terminal PDZ-binding motif
(Izaddoost et al., 2002
;
Medina et al., 2002b
). Three
highly conserved residues in the FERM-binding region were all replaced with
alanine to yield the Myc-CRB3 FERMmut construct. The PDZ-binding sequence,
ERLI, was also deleted (Myc-CRB3
ERLI). The Myc-CRB3N
D mutation
has been described previously (Makarova et
al., 2003
).
|
MDCK stable cell lines expressing each of these mutant constructs were
isolated. Each cell line expressed the various Myc-CRB3 proteins above
endogenous CRB3 levels (data not shown). Immunostaining these cells with
anti-Myc antibody revealed that Myc-CRB3 and each of the Myc-CRB3 mutant
proteins predominantly localize to the apical surface
(Fig. 2B). Next, we studied the
ability of these four Myc-CRB3 proteins to bind Pals1. We prepared lysates
from the MDCK cell lines expressing the various Myc-CRB3 proteins, and
anti-Myc immunoprecipitations were performed. The presence of Pals1 in the
immunoprecipitates was examined by anti-Pals1 immunoblot. We found that the
extreme C-terminal ERLI sequence of CRB3 is necessary to bind Pals1 since
Myc-CRB3ERLI was the only protein that failed to co-immunoprecipitate
Pals1 (Fig. 2C). These results
are consistent with previous studies on the interactions between Crumbs
proteins and Pals1 (Makarova et al.,
2003
; Roh et al.,
2002b
). It should be noted in
Fig. 2C that the amount of
Myc-CRB3
ERLI expression is underestimated since the anti-CRB3 antibody
was raised against the last 20 residues of CRB3 encompassing the ERLI
sequence. As predicted, this antibody still detects CRB3 tail harboring the
ERLI deletion but with lower affinity with respect to full length CRB3 tail
(Fig. 2D). Unfortunately, our
attempts to more accurately quantify relative expression of the various
Myc-CRB3 proteins via anti-Myc immunoblot were unsuccessful.
To understand the effects of overexpressing Crumbs3 on TJ formation, we performed calcium switch experiments using the MDCK stable cell line expressing Myc-CRB3. MDCK cells grown in low calcium media usually exhibit a rounded morphology and lack TJs as well as adherens junctions. Upon re-addition of 1.8 mM calcium, parental MDCK cells form intact adherens junctions as well as TJs within six hours (Fig. 3A). In MDCK cells expressing Myc-CRB3, the ZO-1 staining pattern was fragmented within the first six hours post-calcium switch (Fig. 3B). In contrast, adherens junction formation was not significantly affected. The ZO-1 distribution still did not circumscribe the entire circumference of every cell in the monolayer even 12-24 hours after calcium switch; however, TJs seemed to be completely assembled by 48 hours (data not shown). Defects in TJ biogenesis were observed in stable cell lines expressing non-tagged CRB3 or moderately expressing Myc-CRB3 above endogenous CRB3 levels (data not shown).
|
We next sought to determine the moiety of CRB3 that was important for
inhibiting TJ formation. Therefore, we performed calcium switch experiments
with the MDCK stable cell lines that expressed the three mutant Myc-CRB3
proteins described above. We found that the ability of overexpressed Myc-CRB3
to delay TJ formation required the Pals1 PDZ-binding motif. Specifically, ZO-1
staining was observed to fully circumscribe the cell periphery of MDCK cells
overexpressing Myc-CRB3ERLI by 6 hours after calcium re-addition as
observed in parental MDCK cells (Fig.
3A,B). In contrast, moderate overexpression of Myc-CRB3N
D
also inhibited TJ biogenesis (Fig.
3B). Cells overexpressing Myc-CRB3 FERMmut also exhibited delayed
TJ assembly (not shown).
The inhibition of TJ formation by overexpressed Myc-CRB3 correlates with
its ability to bind Pals1. In light of these results, we next investigated the
localization of endogenous Pals1 in cell lines expressing Myc-CRB3 and
Myc-CRB3ERLI at six hours post-calcium switch. In the former, we found
that Pals1 was enriched at the fragmented TJs along with ZO-1
(Fig. 3C). In the
Myc-CRB3
ERLI cell line, Pals1 and ZO-1 staining pattern was observed to
co-localize to TJs.
CRB3 overexpression disrupts apico-basal polarity in MDCK cells
Overexpression of CRB3 in MDCK cell monolayers results in delayed TJ
formation. This effect is dependent on the last four residues of CRB3, the
binding site for Pals1. Studies using Drosophila, an organism that
lacks TJs, have shown that Crumbs overexpression leads to a disruption of
epithelial morphogenesis. Thus, we wanted to investigate the global effect of
CRB3 overexpression on apico-basal polarization of mammalian epithelia. In
MDCK cells overexpressing CRB3, the majority of gp135 and ezrin distributed to
the apical membrane. In the cell lines examined in Figs
2 and
3, the wild-type and mutant
Myc-CRB3 proteins mainly localized apically. These results suggest that
overexpression of CRB3 and Myc-CRB3 do not result in a significant disruption
of apico-basal polarity in MDCK cell monolayers grown on synthetic tissue
culture supports.
Recently, it has been demonstrated that MDCK cysts, grown in
three-dimensional collagen gels, represent a sensitive model system to detect
perturbations in apico-basal polarity
(O'Brien et al., 2001).
Consequently, we cultured each of the MDCK stable cell lines expressing the
various Myc-CRB3 constructs in collagen. In parallel, we also cultured the
parental MDCK cell line in the same manner. When grown in collagen for six
days wild-type MDCK cells develop into cysts in which the epithelial cells
surround a central lumen (Fig.
4A). The apical membranes of the cells face the lumen and stains
positive for the apical marker gp135. PATJ and ZO-1 were localized to TJs,
which demarcate the apical surface from the basolateral membrane (visualized
by E-cadherin). The cortical actin cytoskeleton was localized along the entire
plasma membrane of each epithelial cell; however, it was clearly concentrated
at the apical membrane.
|
In contrast, when MDCK cells overexpressing Myc-CRB3, Myc-CRB3ND, or
Myc-CRB3 FERMmut were cultured in collagen, a multicellular aggregate of cells
formed (Fig. 4B, data not
shown). These aggregates did not contain any recognizable lumina. In addition,
the subcellular distribution of ZO-1 and gp135 staining exhibited a
disorganized pattern suggestive of TJ and apical surface defects
(Fig. 4C). Furthermore,
E-cadherin still remained localized to the plasma membrane at sites of cell
contacts. MDCK cells expressing Myc-CRB3
ERLI formed relatively normal
cysts as the apical marker gp135 clearly outlined a central lumen. The
distribution of ZO-1 was similar to that in wild-type MDCK cysts as well
suggesting normal TJ formation (data not shown). Staining these cysts with
anti-Myc antibodies revealed that Myc-CRB3
ERLI was localized all along
the plasma membrane.
The ability of overexpressed Myc-CRB3 to disrupt apico-basal polarity in three-dimensional MDCK cysts again correlated with its ability to bind Pals1. We therefore sought to determine the distribution of Pals1 in these cysts; however, our attempts to immunostain endogenous Pals1 in wild-type MDCK cysts or multicellular aggregates overexpressing Myc-CRB3 were unsuccessful because of relatively high background staining. Alternatively, we were able to immunostain an endogenous Pals1-binding partner, PATJ, in these cysts and aggregates. PATJ co-localized with ZO-1 to TJs in wild-type MDCK cysts (Fig. 4A). In Myc-CRB3 overexpressing cell aggregates, however, PATJ exhibited a disorganized localization pattern that partially, but not completely, overlapped with the overexpressed Myc-CRB3 (Fig. 4C).
Disruption of the association between endogenous CRB3 and Pals1
perturbs tight junction formation and apical polarity
Overexpression of CRB3 leads to disruption of TJs and apico-basal polarity
in mammalian epithelial cells. These events require the extreme C-terminus of
CRB3 suggesting that Pals1 is an important element of CRB3 function. We
previously demonstrated that the PDZ domain of Pals1 directly interacts with
the extreme C-terminal ERLI motif of mammalian Crumbs proteins
(Makarova et al., 2003;
Roh et al., 2002b
). We next
wanted to examine the role of the CRB3-Pals1 association during the
establishment of TJs and polarity. We sought to disrupt this endogenous
interaction using a dominant-negative approach. Therefore, a construct
encoding a Myc-Lin-2Pals1 PDZ chimeric protein in which the mLin-2/CASK
PDZ domain was replaced with the Pals1 PDZ domain
(Fig. 5A) was designed.
|
Recently, we demonstrated that Myc-Pals1 associates with a GST fusion
protein containing the last 20 amino acids of CRB3
(Makarova et al., 2003). Here,
we observed that the Myc-Lin-2Pals1 PDZ chimera, expressed in HEK293
cells, also associates with GST-CRB3 tail
(Fig. 5B). In contrast,
wild-type Myc-Lin-2 failed to associate with GST-CRB3. Myc-Lin2 missing the
PDZ domain (Myc-Lin2
PDZ) also did not bind GST-CRB3 (not shown). We
next transfected MDCK cells with constructs encoding Myc-Lin-2Pals1 PDZ
and Myc-Lin-2
PDZ (negative control) and isolated MDCK stable cell lines
expressing comparable levels of these proteins
(Fig. 5C). Anti-Myc
immunoprecipitations were performed using lysates derived from these two cell
lines. We observed that endogenous CRB3 co-precipitated with the
Myc-Lin-2Pals1 PDZ chimera but not with the Myc-Lin-2
PDZ
suggesting that the chimeric protein could compete with endogenous Pals1 for
binding CRB3 (Fig. 5D).
To investigate the effects of expressing the Myc-Lin-2Pals1 PDZ
chimera on TJ formation in MDCK cells, we performed a calcium switch
experiment similar to that shown in Fig.
3. We compared the assembly of cell-cell contacts in MDCK cells
expressing the chimera and those expressing Myc-Lin-2PDZ. Junctional
assembly was monitored 0, 3, 6, and 96 hours after calcium switch
(Fig. 6). The formation of TJs
proceeded normally in the Myc-Lin-2
PDZ expressing cell line as ZO-1
staining labeled the entire circumference of each cell at cell contacts by six
hours after re-addition of calcium (Fig.
6A). In contrast, TJ assembly in the chimera expressing cell line
was significantly delayed and was not complete even at 96 hours post-calcium
switch. This effect was also observed but to a lesser extent in another stable
cell line expressing lower amounts of the chimeric protein. For the
Myc-Lin-2Pals1 PDZ cell line, adherens junctions were visualized at the
same timepoints as in Fig. 6A
by staining with anti-E-cadherin antibody
(Fig. 6B). Within the first
three hours after the calcium switch, E-cadherin was observed to target to the
lateral surface in both the Myc-Lin-2Pals1 PDZ and the
Myc-Lin-2
PDZ cell lines. Strikingly, however, some E-cadherin was seen
to localize to the apical membrane in a fraction of the MDCK cells expressing
the Myc-Lin-2/Pals1 PDZ chimera. This was especially evident within the first
24 hours post-calcium switch (Fig.
6B,C). In contrast, this effect was not observed in the
Myc-Lin-2
PDZ control cell line.
|
The above observations prompted us to examine the apical membrane of these
two cell lines. Therefore, we co-stained the MDCK cell monolayers expressing
Myc-Lin-2/Pals1 PDZ and Myc-Lin-2PDZ with antibodies directed against
CRB3 and gp135 (Fig. 6C). At 24
hours, all of the Myc-Lin-2
PDZ expressing cells exhibited apical CRB3
and gp135 staining. In contrast, these proteins were absent from the apical
surface in a fraction of cells expressing the chimera. The absence of these
apical proteins seemed to correlate with positive E-cadherin staining at the
apical surface.
These results suggest that perturbing the endogenous Pals1/CRB3 interaction
leads to apical membrane defects. To further assess the effect of expressing
Myc-Lin-2/Pals1 PDZ on apico-basal polarity, we cultured cells expressing the
chimera and Myc-Lin-2PDZ (control) in collagen gels. The latter cells
developed into normal cysts (Fig.
7A) similar to parental MDCK cell cysts
(Fig. 4A). Cells expressing the
chimera, however, formed disordered multicellular aggregates that lacked
single continuous lumina. Specifically, gp135 appeared in discontinuous
patches (Fig. 7B), which is
consistent with the previous observation that a fraction of cells in
monolayers expressing the chimera lack gp135-positive plasma membranes.
Collectively, these results highlight the functional importance of the
CRB3/Pals1 association in apical surface determination in mammalian epithelial
cells.
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Discussion |
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Fly embryos lacking Crumbs, Stardust, and Discs lost all exhibit defects in
epithelial polarity. It has also been shown that Crumbs and Stardust are
essential for zonula adherens formation as embryos missing either protein fail
to form this structure from spot adherens junctions during early epithelial
morphogenesis (Grawe et al.,
1996; Klebes and Knust,
2000
). Since the zonula adherens represents the most apical cell
junction, it could be inferred that Crumbs and Stardust play important roles
in demarcating the apical surface and determining the final localization of
the zonula adherens. Strikingly, overexpression of Crumbs also leads to a
disruption of the zonula adherens, expansion of the apical membrane, induction
of multilayered epithelia, and embryonic lethality
(Klebes and Knust, 2000
;
Wodarz et al., 1995
). These
data suggest that Crumbs is an apical polarity determinant and that the
regulation of its expression is crucial for proper establishment of epithelial
polarity.
The first reported mammalian homologue of Crumbs was Crumbs1 (CRB1). CRB1
is expressed highest in the retina and neuronal tissues
(den Hollander et al., 2002;
den Hollander et al., 1999
).
Interestingly, when CRB1 was exogenously expressed in MDCK renal epithelial
cells, it co-localized with Pals1 and PATJ at TJs thereby illustrating for the
first time that a conserved Crumbs complex could exist in mammalian epithelia
(Roh et al., 2002b
). With the
sequencing of the human genome, two other putative Crumbs genes were
recognized and designated as CRB2 and CRB3
(Medina et al., 2002a
;
Tepass et al., 2001
).
Recently, we reported the cloning of CRB3 and its native expression
in mammalian epithelial cells (Makarova et
al., 2003
). Unlike the distribution of exogenously expressed CRB1,
endogenous CRB3 localizes not only to TJs but also to the apical plasma
membrane. On the basis of its subcellular distribution, it seemed likely that
CRB3 could serve as an apical polarity determinant and the functional Crumbs
homologue in mammalian epithelial cells.
We initially studied CRB3 function by isolating and examining MDCK cells
that expressed high levels of CRB3 relative to endogenous levels. In these
cells, the localization of two apical markers extended to the basolateral
surface domain indicative of apical surface expansion. This is in agreement
with previous studies on Drosophila Crumbs overexpression. Next, we
sought to determine the effects of CRB3 overexpression on the formation of
cell junctions. Mammalian epithelial cell lines represent a useful model
system to examine the sequential assembly of adherens junctions and TJs using
a calcium switch assay (Rajasekaran et
al., 1996). We observed that in wild-type and CRB3 overexpressing
MDCK cells, adherens junctions were formed at relatively similar rates. In
contrast, TJ biogenesis was significantly delayed in the cells overexpressing
CRB3. The ability of overexpressed CRB3 to inhibit TJ formation depended only
on the last four residues (ERLI). This motif is responsible for binding the
PDZ domain of Pals1, and MDCK cells expressing elevated levels of CRB3 missing
this sequence formed TJs in a timely manner. Thus, high Crumbs expression in
mammalian and fly epithelia seems to negatively regulate the formation of the
cell junction positioned closest to the apical surface.
Although TJs were disrupted, the overall polarity of MDCK monolayers
overexpressing CRB3 was not dramatically affected. Specifically, the majority
of CRB3, ezrin, and gp135 were localized apically, whereas E-cadherin was
still present at the lateral membrane. Thus, in spite of apical surface
expansion, overall apico-basal asymmetry was maintained. This could be because
of the presence of a free surface and cell-substratum/cell-cell contacts
providing sufficient cues to establish polarity in the monolayer independent
of the Crumbs complex (Yeaman et al.,
1999b).
The establishment and maintenance of mammalian epithelial cell polarity has
also been addressed from a different experimental angle the growth of
MDCK cysts in a collagen matrix. The utility of three-dimensional culture as a
sensitive system to examine molecular polarity signals is becoming
increasingly appreciated. For instance, the expression of dominant-negative
Rac1 (Rac1-N17) does not affect polarity of MDCK cell monolayers; however,
cysts derived from these cells in collagen gels exhibit an inversion of
polarity (O'Brien et al.,
2001). MDCK cells overexpressing CRB3 do not develop into
polarized cysts; instead, they form non-polarized multicellular aggregates.
This is in contrast to cysts derived from parental MDCK cells or cells
expressing CRB3 missing the Pals1 PDZ-binding motif; these cysts exhibit
relatively normal apico-basal polarity. These results suggest that Pals1 is an
important downstream mediator of the CRB3 overexpression phenotypes. This is
in agreement with previous studies in Drosophila that established
that crumbs functions upstream of stardust (the
Drosophila Pals1 orthologue) in a common genetic pathway
(Tepass and Knust, 1993
).
Two other mutant versions of CRB3 were exogenously expressed above
endogenous levels in this study: CRB3ND and CRB3FERMmut. MDCK cells
overexpressing either of these two proteins exhibited similar degrees of TJ
assembly and polarity disruption as those overexpressing wild-type CRB3. This
suggests that N-glycosylation of the extracellular domain or presence of an
intact intracytoplasmic FERM-binding region do not play significant roles in
the CRB3 overexpression phenotype. This is in agreement with studies using
Myc-Crumbs-intra, a truncated version of Crumbs in which the large
extracellular portion of Crumbs is replaced with a Myc epitope tag, and
Myc-Crumbs-intra harboring point mutations in the FERM domain. Flies
expressing high levels of either of these proteins in a wild-type background
exhibit similar polarity phenotypes as those overexpressing full length Crumbs
(Klebes and Knust, 2000
).
The exact role of the N-glycosylation of CRB3 is not known; however, it was
recently reported that the FERM domain of Crumbs binds D-moesin in flies
(Medina et al., 2002b). Crumbs
overexpression in flies leads to D-moesin redistribution suggesting that
Crumbs could be linked to the apical cortical cytoskeleton. In our study, we
observed that high CRB3 expression leads to the redistribution of ezrin
raising the possibility that the FERM domain of CRB3 could bind ezrin.
However, attempts to demonstrate a physical interaction between these two
proteins via co-immunoprecipitation assays have not been successful (data not
shown). Consequently, the identity of the binding partner for the CRB3 FERM
domain remains to be elucidated. In Drosophila lacking Crumbs,
exogenous Crumbs expression is able to rescue the embryonic phenotype. Here,
both the FERM domain and the extreme C-terminal PDZ-binding motif are
required. Hence, it can be inferred that the FERM domain, in concert with the
Pals1 PDZ-binding sequence, could play some role during the establishment
and/or maintenance of mammalian epithelial polarity.
Another issue that remains unresolved is the moiety of CRB3 that mediates
its trafficking to the apical membrane. Myc-CRB3 and the three mutants tested
in this study all targeted to the apical surface. It is somewhat surprising
that the Myc-CRB3ERLI protein, which does not associate with Pals1,
still distributes apically suggesting that a Pals1-independent mechanism
underlying CRB3 targeting exists. In Drosophila expressing Crumbs
missing the last 23 residues (crb8F105 gene product), this
mutant protein displays a diffuse cytoplasmic localization pattern in some
ectodermally derived epithelia such as the epidermis and pharynx. However, in
other epithelia including those found in the salivary glands and Malpighian
tubules, this truncated Crumbs is expressed exclusively to the apical membrane
(Knust et al., 1993
).
Coincidentally, in stardust mutant flies apical Crumbs expression is
maintained in these same tissues (Tepass
and Knust, 1993
). Therefore, further mutations need to be made in
CRB3 to determine the exact residue(s) involved in CRB3 membrane trafficking
in mammalian epithelia.
CRB3 overexpression studies have illustrated a functional link between CRB3
and Pals1. To confirm the importance of the CRB3-Pals1 association during
epithelial polarization, we next sought to disrupt this interaction in MDCK
cells. We initially addressed this task by attempting to express only the PDZ
domain of Pals1. However, we were not able to isolate stable clones expressing
the Pals1 PDZ domain. Thus, we employed an alternative strategy in which the
Pals1 PDZ domain would be expressed as a part of a more stably expressed
protein. Thus, we decided to express a Myc-Lin-2/Pals1 PDZ chimera as
expression of exogenous Myc-Lin-2 or its various deletion mutants that do not
disrupt apico-basal polarity (Lee et al.,
2002). This chimera, in contrast to Myc-Lin-2 or Myc-Lin-2 missing
the PDZ domain, was able to co-immunoprecipitate CRB3 confirming that CRB3 can
associate with the PDZ domain of Pals1. Expression of this chimera resulted in
a disruption of TJs in MDCK monolayers as the ZO-1 staining pattern was
fragmented. In addition, these cells exhibited apical membrane defects in a
cell autonomous fashion, a phenomenon that correlated with the presence of the
lateral membrane marker, E-cadherin, to the apical surface. In
stardust mutant flies, apical surface defects are also detected.
Specifically, the apical marker, Stranded at Second, is absent from cells
lacking Stardust expression (Bachmann et
al., 2001
). Collectively, these results suggest that the
CRB3/Pals1 complex and other proteins associated with it could be important
for establishing apical membrane identity, influencing the distribution of
lateral proteins, and the coordinated assembly of continuous TJ fibrils at
apico-lateral membrane thereby demarcating the apical and basolateral
surfaces.
What are the exact mechanisms through which the CRB3/Pals1 interaction
influences the above processes during epithelial cell polarization? In MDCK
cells overexpressing CRB3 or the Lin-2Pals1 PDZ chimeric protein,
endogenous Pals1 localized predominantly to the fragmented TJs
(Fig. 3C, data not shown)
suggesting that Pals1 mislocalization or sequestration alone cannot account
for the phenotypic effects observed throughout this study. Pals1 consists of
multiple protein-protein interaction domains and some of their binding
partners are known. For instance, Pals1 contains two Lin-2/Lin-7 (L27)
heterodimerization domains, L27N and L27C. These domains bind to the L27
domains of PATJ and mLin-7, respectively
(Kamberov et al., 2000;
Roh et al., 2002b
). We
recently demonstrated that the N-terminal portion of Pals1, probably the U1
domain, binds to the Par6-Par3-aPKC polarity complex through the direct
interaction with Par6 (Hurd et al.,
2002
). It is possible that the CRB3-Pals1 association influences
the binding of Pals1 to its partners (e.g. PATJ and/or Par6) and that these
dynamic interactions regulate various stages of polarized membrane
trafficking, junction formation, and demarcation of apical and basolateral
membrane subdomains. Furthermore, although a CRB3-Pals1-PATJ complex localizes
to TJs of fully polarized epithelial cells
(Makarova et al., 2003
), it is
not absolutely certain where the functionally relevant CRB3-Pals1-containing
complexes exist.
Polarity proteins acting downstream of Stardust are not well known seeing
that Discs lost is the only known binding partner of Stardust besides Crumbs.
We have partially addressed this issue in mammalian epithelia by analyzing
MDCK cells expressing a Myc-PATJ (1-238) dominant-negative protein. We were
able to show in these cells that endogenous Pals1 and aPKC were
mislocalized away from TJs (Hurd et al.,
2002
). Interestingly, TJ biogenesis was also disrupted suggesting
that the Par6/Par3/aPKC complex could represent important effectors that lie
downstream of CRB3/Pals1. This is supported by studies demonstrating the
intimate relationship between Par6/Par3/aPKC and TJ formation
(Gao et al., 2002
;
Hirose et al., 2002
;
Izumi et al., 1998
;
Joberty et al., 2000
;
Ohno, 2001
;
Yamanaka et al., 2001
).
Further experiments will have to be performed to determine the relative
contributions of Par6 and PATJ containing complexes during TJ biogenesis and
the establishment of polarity in epithelial cells. Nonetheless, the results of
this study provide initial insight into the importance of the CRB3 and its
associated proteins in mammalian epithelial cell biology.
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Acknowledgments |
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Footnotes |
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References |
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