Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden
Author for correspondence (e-mail: bjorn.obrink{at}cmb.ki.se )
Accepted 13 December 2001
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Summary |
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Key words: CEACAM1, Cell adhesion, Cell polarization, Microvilli, Signal transduction
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Introduction |
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The subcellular location of CEACAM1 exhibits interesting differences
between cell types (Odin et al.,
1988; Sawa et al.,
1994
). In stratified epithelia, CEACAM1 is present on the cell
surface in cell contact areas. In brush-border-carrying simple epithelia, it
is most abundant in the apical surfaces. In the mature liver, it is highly
expressed in bile canalicular membranes. In the vascularizing central nervous
system, it appears in contact areas between endothelial cells and pericytes.
And in non-activated polymorphonuclear neutrophils and platelets, it is
present in intracellular membranes that fuse with the plasma membrane upon
activation.
The expression of CEACAM1 in the apical surfaces of the simple epithelia of
the small intestine and the proximal kidney tubules and in the bile canaliculi
of mature hepatocytes has cast doubts on the adhesive function of CEACAM1 in
these cells, because this location was not expected for a molecule that is
involved in cell-cell adhesion. However, bile canaliculi go through
contraction-relaxation cycles (Phillips et
al., 1982; Watanabe et al.,
1991
), and in the contracted phase, the bile canalicular membranes
are organized in microvillar structures, the membranes of which are in close
contact with each other (Watanabe et al.,
1991
). Also the microvillar membranes of the brush borders of the
apical surfaces of the intestinal and tubular epithelial cells show close
contacts (Copenhaver et al.,
1971
). Therefore, an adhesive function of CEACAM1 cannot be ruled
out in these locations. Another issue that has not yet been satisfactorily
solved is whether CEACAM1 is also present in the lateral membranes of simple
epithelial cells. By immunohistochemistry, a weak staining for CEACAM1 was
observed in rat intestinal epithelial cells
(Hansson et al., 1989
) and in
human mammary epithelial cells (Huang et
al., 1999
). Furthermore, Mowery and Hixson (Mowery and Hixson,
1991), applying fixation and mechanical dissociation of mature rat liver,
demonstrated that CEACAM1 was also present in the lateral membranes between
adjacent hepatocytes, in addition to its expression in the canalicular
membranes. Thus, it seems that CEACAM1 can occur at the lateral surfaces of
polarized epithelial cells but that some of its epitopes may be masked.
The two major isoforms of CEACAM1 are CEACAM1-L and CEACAM1-S, which differ
in their cytoplasmic domains owing to differential splicing of one exon
(Edlund et al., 1993). Both
cytoplasmic domains can be phosphorylated on serine residues
(Odin et al., 1986
;
Edlund et al., 1998
) and can
bind to calmodulin in a calcium-regulated manner
(Edlund et al., 1996
). The
cytoplasmic L domain, which consists of 71 amino acids, has two tyrosine
residues, which upon phosphorylation can recruit and activate src-family
kinases (Brümmer et al.,
1995
) or the protein tyrosine phosphatases SHP-1 and SHP-2
(Huber et al., 1999
). The 10
amino-acid long cytoplasmic S domain lacks these tyrosine residues. CEACAM1-L
and CEACAM1-S are co-expressed in CEACAM1-expressing cells, but the expression
ratios vary between different cell types and between different cellular states
(Baum et al., 1996
;
Singer et al., 2000
). Since
six of the amino acids in the 10 amino-acid long S domain are identical to the
sequence of the L domain, it has been difficult to produce antibodies that
efficiently can distinguish between the two isoforms in situ. Therefore, it is
not known whether they are distributed in an identical fashion in the apical
and lateral surfaces of polarized epithelial cells. This is, however, an
important issue since CEACAM1 participates in signal regulation, and CEACAM1-S
can regulate the signaling activities of CEACAM1-L
(Öbrink, 1997
). Thus, it
is crucial to know the relative expression of each isoform in different parts
of polarized epithelial cells.
In the present work we have addressed four questions: 1) What is the surface location of CEACAM1 in highly polarized epithelial cells that have distinct apical and basolateral surfaces? 2) Is there any difference in the surface location of the two isoforms CEACAM1-L and CEACAM1-S? 3) Do the two CEACAM1 isoforms interact differently with the plasma membrane cortex? 4) Is CEACAM1 involved in adhesive interactions in highly polarized epithelial cells? We demonstrate that the two isoforms localized differently in polarized MDCK cells. CEACAM1-L occurred both on the lateral and the apical surfaces, whereas CEACAM1-S became expressed exclusively on the apical surfaces. The N-domain of the laterally expressed CEACAM1-L was masked, indicating that this isoform at this location participates in homophilic cell-cell adhesion. Furthermore, apically expressed CEACAM1 exhibited both masked and unmasked states of the N-domain, indicating that CEACAM-1 participates in adhesive interactions also in this location.
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Materials and Methods |
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Cell culture and transfection
MDCK II cells were grown in a 5% CO2 humidified athmosphere at
37°C in DMEM supplemented with 10% heat-inactivated fetal bovine serum,
100 U/ml penicillin and 100 mg/ml streptomycin. They were transfected by the
calcium phosphate precipitation method (10 µg of cDNA for CEACAM1-L or
CEACAM1-S in the pRAX vector plus 1 µg of a cDNA coding for the neomycin
resistance gene), selected in 0.5 mg/ml G418 for 14 days and cloned by
limiting dilution as previously described
(Olsson et al., 1995). Cell
polarization was achieved by growth for 3 days on permeable filter inserts
(Falcon, PET filters 0.45 µm pore size, cat. no. 3090) that separated the
apical and basolateral compartments. Formation of tight monolayers was
monitored by 3H-inulin that was added to the upper compartment
(Caplan et al., 1986
). Only
cell layers that permitted passage of less than 1% of the inulin from the
upper to the lower compartment were used for experiments.
Cell solubilization
Cells on tissue culture dishes were solubilized at room temperature with
varying concentrations of Triton X-100 in Buffer P (150 mM NaCl, 25 mM Hepes
pH 7.4, 5 mM EGTA, 10 mM Na4P2O7, 50 mM NaF,
1 mM Na3VO4, 0.5 mM AEBSF, 1000 KIE/ml aprotinin and
1µg/ml leupeptin, 1 µg/ml pepstatin). Supernatants from the solubilized
cells were immunoprecipitated with CC16 at 4°C overnight and
protein A-Sepharose. The immunoprecipitates and the detergent-insoluble cell
residues were solubilized in a 1:1 mixture of Buffer P/2% SDS and 2x SDS
sample buffer, reduced with 50 mM DTT at room temperature for 4 hours, heated
at 100°C for 5 minutes and subjected to SDS-PAGE on 7% polyacrylamide gels
(Laemmli, 1970
).
Biochemical surface localization
Tight monolayers of MDCK cells polarized on permeable insert filters were
incubated for 2 hours at 4°C with CC16 Fab fragments added from
either the upper, apical compartment or the lower, basolateral compartment.
The cell layers were washed three times with PBS/1mM CaCl2/1mM
MgCl2 and were then solubilized in 1% Triton X-100 in Buffer A (150
mM NaCl, 20 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.2% BSA, 1 mM PMSF, 1000 KIE/ml
aprotinin) for 1 hour at 4°C and centrifuged at 14000 g
for 15 minutes. The supernatants were incubated with swine anti-rabbit IgG
(DAKO A/S, Denmark) overnight at 4°C, followed by incubation with protein
A Sepharose for 30 minutes. The beads were collected by centrifugation, washed
once with Buffer A, three times with 1% Triton X-100/0.1% SDS in Buffer A,
three times with 0.5% Triton X-100 in Buffer C (500 mM NaCl, 20 mM Tris-HCl,
pH 8.0, 0.2% BSA) and once in 50 mM Tris-HCl, pH8.0. The immunoprecipitates
were recovered from the washed beads by solubilization in 1x SDS sample
buffer, reduced with 50 mM DTT, heated at 100°C for 5 minutes and analyzed
by SDS-PAGE on 7% polyacrylamide gels and immunoblotting with
CC16.
Immunoblotting
Proteins separated by SDS-PAGE were transferred to nitrocellulose
membranes, blocked in 5% defatted milk powder in TBS/0.05% Tween 20, pH 7.4
and incubated with CC16 for 1 hour at room temperature, followed by
horseradish-peroxidase-labeled swine anti-rabbit antibodies and developed by
ECL. The developed films were scanned in a gel documentation equipment
(Herolab) and processed in Photoshop 6 (Adobe).
Confocal microscopy
Tight monolayers of MDCK cells polarized on permeable insert filters were
fixed with 3% paraformaldehyde for 30 minutes at room temperature. The cell
layers were permeabilized with Triton X-100 or methanol (described in detail
in Results), and reactive aldehyde groups were quenched in 50 mM
NH4Cl for 15 minutes at room temperature. In some experiments, the
cells were treated with hyperosmotic sucrose (1 M sucrose in PBS for 30
minutes at 37°C) before fixation and permeabilization. The cells were then
incubated overnight at 4°C with primary antibodies (affinity purified
CC16, Mab 5.4, anti-occludin antibody) followed by secondary antibodies
for 1 hour at room temperature and were examined with a Zeiss LSM 510 scanning
module fitted to an Axiovert 100 M microscope using a 63x oil immersion
objective. Routinely, 0.35 µm thick focal planes were scanned. The data
files were processed with the LSM software and transferred to PowerPoint
(Microsoft).
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Results |
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Solubility of surface-localized CEACAM1
Immunofluorescence of transfected cells grown on regular Petri dishes
revealed a striking difference in the extractability of CEACAM1-L and
CEACAM1-S (data not shown). Thus, treatment of cells that had been fixed with
2% paraformaldehyde for 30 minutes and with 0.1% Triton X-100 for 30 minutes
resulted in a complete loss of immunoreactive staining for CEACAM1-S, whereas
the staining for CEACAM1-L remained to a large extent, particularly at the
lateral cell surfaces. This result indicated that a portion of CEACAM1-L was
associated with the cell cortex, that is the plasma membrane and the
underlying actin filament network, in a different way from CEACAM1-S.
The finding that detergent treatment caused loss of CEACAM1 even after fixation, and that this affected the two isoforms differently, prompted a detailed investigation of the fixation and permeabilization conditions in order to find a procedure in which no CEACAM1 of either isoform was lost, while at the same time maximal antibody binding for efficient staining was preserved. To that end we used combinations of paraformaldehyde as fixative and Triton X-100 or methanol as permeabilizing agents and varied both the concentrations and time of treatment for both the fixative and the permeabilizing agents. The solubilization of the two CEACAM1 isoforms was analyzed by immunoblotting. When unfixed cells were treated with different concentrations of Triton X-100, we found that CEACAM1-L was more firmly bound in the cell cortex than CEACAM1-S. Thus, incubation at room temperature with 0.5% Triton X-100 for 10 minutes solubilized all of CEACAM1-S, whereas a significant portion of CEACAM1-L remained insoluble after incubation with 1% Triton X-100 (Fig. 2A). Both isoforms could be solubilized to some extent even after fixation with 3% paraformaldehyde, and CEACAM1-S was still more easily solubilized than CEACAM1-L (Fig. 2A). In order to completely prevent CEACAM1-S solubilization the cells had to be fixed with 4% paraformaldehyde for 60 minutes (data not shown). However, when methanol was used for permeabilization instead of Triton X-100, it was enough to fix the cells with 3% paraformaldehyde for 30 minutes to prevent CEACAM1 solubilization (Fig. 2A). The different fixation and permeabilization conditions were also applied to polarized cells, which demonstrated that the most efficient immunostaining was obtained by permeabilization with methanol for 2 minutes at room temperature (Fig. 2B).
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The surface location of CEACAM1-L and CEACAM1-S differs in polarized
MDCK cells
The surface location of CEACAM1-L and CEACAM1-S was analyzed by confocal
microscopy of fully polarized MDCK cells. Even if it was possible to
immunostain cell surface molecules both on the apical and the basolateral
surfaces by applying antibodies from either side, we found that the staining
was more efficient if the cells were permeabilized before addition of the
antibodies, since we used filters with low pore density, which restricted the
diffusion of the antibodies through the filters. In order to prevent loss of
CEACAM1 by the permeabilization procedure, we routinely fixed the cells with
3% paraformaldehyde for 30 minutes and permeabilized them by treatment with
methanol for 2 minutes at room temperature.
Antibody staining of confluent unpermeabilized cell monolayers utilizing polyclonal antibodies showed a strong expression of both CEACAM1-L and CEACAM1-S on the apical surfaces (Fig. 3). However, when the cell monolayers were permeabilized with methanol to allow efficient access of the antibodies to beneath the tight junctions, CEACAM1-L-expressing cells but not CEACAM1-S-expressing cells were strongly stained on their lateral surfaces (Fig. 2B, Fig. 3).
In order to obtain further proof of the different localization patterns of
the two CEACAM1 isoforms seen by confocal microscopy, we designed a
biochemical labelling technique. Viable, polarized and tight MDCK cell layers
were incubated with CC16 Fab fragments that were added either from the
apical compartment or from the basolateral compartment. Fab fragments instead
of intact immunoglobulins were used to facilitate rapid penetration through
the filters and between the cells. After washing and solubilization of the
cells, cell-bound Fab fragments were retrieved with anti-Fab antibodies and
protein A-Sepharose, and Fab-complexed CEACAM1 was detected by immunoblotting.
Using this procedure, CEACAM1-S was only retrieved from the apical surface,
whereas CEACAM1-L could be retrieved both from the apical and the basolateral
surfaces (Fig. 4). Thus, in
agreement with the results of the confocal microscopy, this analysis also
demonstrated that CEACAM1-L was expressed both on the lateral and the apical
cell surfaces, whereas CEACAM1-S was localized exclusively to the apical
surfaces. In addition, this experiment demonstrated that both isoforms were
indeed exposed on the extracellular face of the plasma membrane.
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The apical staining of both CEACAM1-L and CEACAM1-S appeared in a layer
with a vertical height of 1-2 µm (Fig.
2B, Figs 3,
5,6,7).
Polarized MDCK cells have a dense layer of uniform 1-2 µm long microvilli
on their apical surfaces (Butor and
Davoust, 1992). Thus, the confocal microscopical data demonstrated
that both CEACAM1-L and CEACAM1-S were localized along the entire length of
the apical microvilli.
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Lateral localization of CEACAM1-L requires cell-cell contact between
CEACAM1-L-expressing cells
CEACAM1 can appear both as monomers and parallel dimers in the plane of the
membrane (Hunter et al., 1996)
and the N-terminal Ig-domain mediates homophilic adhesion by a reciprocal
binding to an N-terminal Ig-domain presented by an adjacent membrane
(Wikström et al., 1996
).
Therefore, it was of interest to use a monoclonal antibody specifically
recognizing the N-terminal Ig-domain in the localization studies by confocal
microscopy. Using Mab 5.4 we found a staining pattern that was strikingly
different from that obtained with the polyclonal antibody
CC16.
Although a significant portion of CEACAM1-L-expressing cells were stained on
their apical surfaces, there was no lateral surface staining after
methanol-permeabilization of the polarized cells
(Fig. 5). Polarized
CEACAM1-S-expressing cells were hardly stained at all
(Fig. 5). Thus, the epitope
recognized by Mab 5.4 was masked to a large extent under these conditions. In
an attempt to investigate whether this might be due to homophilic binding
between the N-terminal Ig-domains, we subjected confluent polarized cells to
osmotic shrinkage by treatment with hyperosmotic sucrose, with the aim of
physically breaking lateral cell-cell contacts. This resulted in a strong
staining of CEACAM1-L with Mab 5.4 on the lateral surfaces
(Fig. 5). Staining for occludin
after sucrose treatment showed that the tight junctions were still intact
(Fig. 6), and therefore the
lateral staining of CEACAM1-L by Mab 5.4 was only detected after methanol
permeabilization. Since, the tight junctions were intact there would be no
re-localization of apical CEACAM1-L, but the hyperosmotic treatment of
confluent cells caused an unmasking of the N-domain epitope on laterally
localized CEACAM1-L.
The expression of CEACAM1-L in the lateral domain of polarized MDCK cells in a state in which the N-domain epitope was blocked and its unmasking by hyperosmotic sucrose suggested that this reflected involvement of CEACAM1-L in cell-cell adhesion. The question was raised whether this was caused by homophilic binding between the N-domains of CEACAM1-L molecules on opposing cell surfaces or whether CEACAM1-L bound to some other ligand presented by the adjacent cell. To answer this question we made confluent, polarized cell layers from a 50:50 mixture of CEACAM1-L transfected and untransfected cells. This resulted in a monolayer composed of islands of CEACAM1-L-expressing and non-expressing cells that were in contact and joined by tight junctions evident from the occludin staining pattern (Fig. 7). CEACAM1-L-expressing and non-expressing cells could clearly be distinguished because the strong apical staining for CEACAM1 was completely missing in the nonexpressing cells. As before, lateral localization of CEACAM1-L was seen between two CEACAM1-L-expressing cells (Fig. 7). However, the borders between CEACAM1-L-expressing and non-expressing cells were completely free of CEACAM1-L, as judged by the lack of staining with either the polyclonal or the monoclonal antibodies, even after sucrose treatment (Fig. 7). Thus, it appears that CEACAM1-L did not bind to any other cell surface molecule on adjacent MDCK cells and that homophilic binding between opposing CEACAM1-L molecules was necessary for their maintenance at the lateral cell borders. This result also demonstrated that rat CEACAM1-L did not recognize and bind to endogenous canine CEACAM1.
Similar experiments with a mixture of CEACAM1-S-transfected and untransfected cells showed no lateral staining between expressing and non-expressing cells under any conditions (Fig. 7).
CEACAM1-L appears in two different conformational or supramolecular
states
Apically localized CEACAM1-L in polarized cells was stained by Mab 5.4, but
significantly fewer cells were stained by 5.4 than by CC16. In
addition, the 5.4 staining was weaker and more patchy than the more
homogeneous staining seen with
CC16 (Figs
5,
7). Taken together with the
masking of the N-terminal epitope in the lateral location, this indicates that
CEACAM1-L occurs in at least two different conformational or supramolecular
organizational states in polarized MDCK cells. In order to analyze the
topographical relationship between these two states, we performed
double-staining with the two antibodies, Mab 5.4 and
CC16. The cells
were first stained with the monoclonal antibody and then with the polyclonal
antibody. In this double-staining procedure the monoclonal antibody showed the
unmasked N-terminal epitope (denoted N-epitope), whereas the polyclonal
antibody detected all other epitopes (denoted O-epitopes) except the
N-terminal one. (If the order of staining was reversed, i.e. the cells were
first stained with
CC16 and then with 5.4, no staining with the
monoclonal antibody was observed, because the polyclonal
CC16 contained
N-domain-recognizing antibodies that blocked binding of the monoclonal
antibody.) When the two staining patterns were superimposed it became apparent
that the majority of the apically localized CEACAM1-L exhibited O-epitopes
(Fig. 8A), a minor portion of
CEACAM1-L exhibited only the N-epitope, and in several locations there was an
apparent colocalization between the N-and the O-epitopes when standard
resolution (volume element (voxel): 0.07 µm/0.07 µm/0.35 µm, x/y/z;
or 0.07 µm/0.07 µm/0.2 µm, x/y/z) was used
(Fig. 8A,B). However, when the
cells were viewed at the highest resolution (voxel: 0.04 µm/0.04 µm/0.2
µm, x/y/z), no colocalization of the two states were seen within the
distinct volume elements (Fig.
8B). Thus, at the apical surface there was no molecular state in
which the N- and the O-epitopes were exposed at the same time, and therefore
CEACAM1-L seemed to appear in two mutually different conformational or
supramolecular states in which either the N-epitope or the O-epitopes were
exposed.
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Using the double-staining procedure with the monoclonal/polyclonal anti-CEACAM1 antibodies, we found that hyperosmotic sucrose treatment led to masking of the O-epitopes and exposure of the N-epitopes of CEACAM1-L to a high extent in the lateral domain below the tight junction, but to a much lower extent in the apical domain above the tight junction (Fig. 8C). Thus, hyperosmotic sucrose treatment caused an almost complete shift of the supramolecular/ conformational state of laterally localized CEACAM1-L from the O-epitope state to the N-epitope state.
Confluent MDCK cell layers are a mosaic of cells with different
supramolecular/conformational states of apically localized CEACAM1
The observation that Mab 5.4 did not stain confluent, polarized
CEACAM1-S-expressing cells under any conditions indicated that the N-epitope
of apically localized CEACAM1-S was blocked in a similar way to those in
laterally localized CEACAM1-L, whereas the O-epitopes were exposed. Apically
localized CEACAM1-L, however, appeared in two states with either the N-epitope
or the O-epitopes exposed, as described above. Interestingly, the cells were
heterogeneous with respect to the balance of these two
supramolecular/conformational CEACAM1-L states
(Fig. 8A). The majority of the
cells in an unperturbed monolayer exhibited essentially only the open
O-epitope state, a significant proportion exhibited both
supramolecular/conformational states, whereas the open N-epitope state
dominated in a minority of the cells. This mosaic pattern was seen in all
filter cultures that were analyzed. Since these transfected cell clones were
monoclonal, this observation suggests that the cells in a monolayer appear in
different states with respect to which CEACAM1-L organizational state
dominates.
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Discussion |
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The signal for lateral localization must reside in the cytoplasmic domain
of CEACAM1-L, as CEACAM1-L and CEACAM1-S are identical both in their
extracellular and transmembrane domains. However, the intracellular signal(s)
seems to be necessary but not sufficient for lateral localization, because
there was no localization of CEACAM1-L to lateral borders between
CEACAM1-L-expressing and non-expressing cells. This strongly suggests that
homophilic, antiparallel binding is a prerequisite for maintaining CEACAM1-L
at this location and furthermore emphasizes that laterally localized CEACAM1-L
indeed was engaged in homophilic, antiparallel binding between adjacent cells.
A similar observation was made by Sadekova et al.
(Sadekova et al., 2000) in a
completely different cell system. They expressed CEACAM1-L by microinjection
into fibroblastic 3T3 cells and found that self-association of CEACAM1-L was
necessary for its maintenance at sites of cell-cell contact.
One factor that might regulate the cell surface localization of the CEACAM1
isoforms is the interactions with the submembraneous cytoskeleton. Several
investigators have found that CEACAM1 can bind to actin filaments
(Hunter et al., 1994;
DaSilva-Azevedo and Reutter,
1999
; Schumann et al.,
2001
) and Sadekova et al. found that Rho family GTPases induce a
relocalization of CEACAM1-L from intracellular sites to the cell surface in
3T3 cells (Sadekova et al.,
2000
). Our finding that CEACAM1-L was more firmly associated with
the membrane cortex than CEACAM1-S are compatible with a role for the actin
filament system in the differential surface localization of the two isoforms.
Work is now in progress to identify the sites and amino-acid sequences in the
cytoplasmic domain of CEACAM1-L that are important for its localization to the
lateral domains, as well as for its interactions with the cell cortex.
Our finding that CEACAM1-L occurred in two different states is of great
interest. The state in which the N-domain epitope (the N-epitope) was blocked
was clearly involved in homophilic, antiparallel binding between adjacent
cells, which is reasonable since it has been demonstrated that homophilic
binding is mediated by a reciprocal binding between the N-domains of CEACAM1
molecules presented by opposing membranes
(Wikström et al., 1996).
In this state the other epitopes (the O-epitopes), recognized by the
polyclonal antibodies, were accessible. In the state where the N-epitope was
exposed it was intriguing to find that the O-epitopes were blocked.
Interestingly, we did not observe any states in which both the N-epitope and
the O-epitopes were exposed simultaneously. Although these two states might be
explained by switching between different conformations, the simplest
explanation is that they reflect monomeric and dimeric/oligomeric forms of
CEACAM1-L. We have previously demonstrated that CEACAM1 can form parallel
dimers in the plane of the membrane
(Hunter et al., 1996
).
Although monomers and dimers are in mass-action-regulated equilibrium with
each other, we showed that the cells could influence the extent of
dimerization (Hunter et al.,
1996
). Furthermore, we found that the monomeric forms of CEACAM1
can mediate cell-cell adhesion by antiparallel, homophilic binding between
N-domains (Hunter et al.,
1996
; Wikström et al.,
1996
). Thus, the state in which the N-epitope was blocked and the
O-epitopes were exposed might represent antiparallel, homophilic binding
between monomers, whereas the state in which the N-epitope was exposed and the
O-epitopes were blocked could represent parallel dimers or clustered oligomers
that did not participate in antiparallel, homophilic binding. The masking of
the O-epitopes could then be explained by blocking owing to dimerization or
oligomerization. According to the present results, monomeric CEACAM1 would
then only exist when antiparallel, homophilic binding occurs between adjacent
membranes. In the absence of antiparallel binding, CEACAM1 would be driven
into the dimer state or clusters of parallel oligomers.
A reasonable explanation for the effect of hyperosmotic treatment on the
organizational state of CEACAM1 is that shrinkage of the cells caused a
mechanical rupture of the antiparallel, homophilic bonds between the CEACAM1-L
molecules in the lateral cell surface domains. This would cause CEACAM1 to
switch from antiparallel-bound monomers to dimers/oligomers not engaged in
cell-cell binding. Such an explanation is supported by the observation that
the hyperosmotic treatment caused a much more pronounced change of the
CEACAM1-L organizational state in the lateral cell-cell contact areas than
above the tight junctions (Fig.
8C). However, the hyperosmotic treatment might also affect the
organization of CEACAM1-L in other ways. Cells have osmotic sensors, and
several signaling pathways, including the classical MAP kinase pathway, are
triggered by increased extracellular osmotic pressure
(Roig et al., 2000;
van der Wijk et al., 2000
;
Weiergräber and Häussinger,
2000
). This might lead to altered phosphorylation of the
cytoplasmic domain of CEACAM1-L, which in turn could change the monomer/dimer
equilibrium and facilitate dimer/oligomer formation and breaking of
homophilic, antiparallel bonds.
The two different organizational states of CEACAM1-L were also observed in
the apical surfaces of the polarized MDCK cells, which suggest that the
homophilic, antiparallel binding state exists, and even dominates, in this
location as well. While, at the first glance this seems illogical, it can
easily be explained as an interaction between adjacent membranes of the apical
microvilli. When MDCK cells polarize, densely clustered microvilli of uniform
size are developed in a brush-border-like configuration on the apical surfaces
(Butor and Davoust, 1992). Our
present confocal microscopical data showed that both isoforms of CEACAM1 were
expressed along the entire length of the apical microvilli of the polarized
MDCK cells. Immunoelectron microscopy has demonstrated that endogenous CEACAM1
is abundantly expressed along the sides of the microvilli as wells as in
intestinal epithelial cells and hepatocytes (B.O., unpublished)
(Kuprina et al., 1990
; Mowery
and Hixson, 1991; Frängsmyr et al.,
1999
), and we have previously suggested that CEACAM1 may mediate
adhesive bonds between adjacent microvilli
(Hansson et al., 1989
). In
intestinal and renal tubular brush borders, microvilli are closely arranged in
a hexagonal pattern, and unidentified surface molecules on the microvillar
membranes form bridges between adjacent microvilli
(Copenhaver et al., 1971
). In
order to test whether this model would be compatible with the present data and
could offer an explanation to the high resolution confocal staining patterns
of CEACAM1 in the apical MDCK cell surfaces, we constructed the model that is
shown in Fig. 8D. In this model
we have drawn microvilli, the intermicrovillar distances and CEACAM1 to scale.
The average diameter of brush border microvilli is 150 nm, the smallest
intermicrovillar distance is around 30 nm, and the length of the 4 Ig domain
extracellular domain of CEACAM1 is approximately 15 nm (from homology with the
structure of CEA as solved by Bates et al.
(Bates et al., 1992
) and Boehm
et al. (Boehm et al., 1996
).
The hexagonal arrangement of the microvilli allow for antiparallel
CEACAM1-CEACAM1 binding at the narrowest inter-microvillar distances, whereas
at other locations CEACAM1 would not be engaged in antiparallel binding. We
then put a raster on this model with squares corresponding to 40x40 nm
or 70x70 nm. Squares (pixels) that contained only antiparallel, bound
CEACAM1 (corresponding to the state with masked N-epitope and exposed
O-epitopes) were given a red color, squares (pixels) that contained only
CEACAM1 not engaged in antiparallel binding (corresponding to the state with
exposed N-epitope and masked O-epitopes) were given a green color, and squares
(pixels) that contained both states of CEACAM1 were given a yellow color (no
color grades owing to varying relative amounts of the two states within
distinct pixels were introduced in this model). This showed that with a pixel
size of 70x70 nm the majority of the pixels became yellow, whereas
pixels of 40x40 nm became almost exclusively red or green. Given that
the plastic microvilli are not perfectly circular in cross-section and not
organized in an absolute hexagonal pattern, this is exactly the pattern that
is seen in Fig. 8B.
Accordingly, this analysis lends support to a model in which CEACAM1 mediates
adhesive binding between closely packed microvilli on the apical surfaces of
polarized epithelial cells.
Interestingly, CEACAM1-S seemed to be almost exclusively in the adhesive
state in the apical surfaces, whereas CEACAM1-L occurred both in the adhesive
and non-adhesive states, although the adhesive state dominated
(Fig. 8A). This indicates that
CEACAM1-S has a stronger tendency to form adhesive bonds, when in a cellular
context, than CEACAM1-L, a tendency that also has been observed in direct cell
adhesion experiments (Wikström et
al., 1996). The monomer/dimer model can rationally explain this
difference between the two isoforms. Both the extracellular domain of CEACAM1,
which is identical in the two isoforms, and the cytoplasmic L-domain, which is
lacking in CEACAM1-S, contribute to dimer formation
(Hunter et al., 1996
) (I.
Hunter and B.O., unpublished). Therefore, CEACAM1-L has a stronger tendency to
form dimers than CEACAM1-S. The monomer/dimer equilibrium would then be more
shifted towards monomer formation for CEACAM1-S than for CEACAM1-L, which in
turn would favor homophilic adhesion. Obviously, in this model coexpression of
the two isoforms that allow for heterodimer formation would alter the balance
between the adhesive and non-adhesive states
The observation that confluent monolayers of monoclonal, polarized CEACAM1-L-expressing cells constituted a mosaic of cells in which either the adhesive or the non-adhesive state of CEACAM1-L dominated suggests that the cells can regulate the adhesive properties of CEACAM1 in a dynamic manner. Cycling of CEACAM1 between adhesive and non-adhesive states might regulate microvillar motility, which could have an important function for stirring of the liquid layer close to the apical cell surface. This would facilitate absorption processes. The osmotic regulation of the adhesive state of CEACAM1 suggests that osmotic pressure could be a controlling factor for nutrient absorption by the small intestinal epithelial cells, which are constantly subjected to variations in the extracellular osmotic pressure owing to exposure to digested food stuffs.
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