(Investigative Ophthalmology and Visual Science. 2000;41:1945-1952.)
© 2000
by The Association for Research in Vision and Ophthalmology, Inc.
High Levels of E-/P-Cadherin: Correlation with Decreased Apical Polarity of Na/K ATPase in Bovine RPE Cells In Situ
Janice M. Burke1,2,
Feng Cao1 and
Pamela E. Irving1
From the Departments of
1 Ophthalmology and Cell Biology,
2 Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin.
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Abstract
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PURPOSE. The adherens junction protein E-cadherin induces a basolateral polarity
of Na/K ATPase in most epithelial cells that express it, whereas in
retinal pigment epithelium (RPE) cells, Na/K ATPase is largely apical.
The purpose of this study was to determine whether the distribution of
Na/K ATPase differs in RPE cells in situ, that differ in levels of
junctional E-cadherin.
METHODS. Bovine RPE cells in situ were immunostained with an E-cadherin antibody
(which has some cross-reactivity with the closely related epithelial
cadherin P-cadherin), and RPE cells with different levels of junctional
stain were identified. RPE cells with low and high E-/P-cadherin were
costained in various combinations with Na/K ATPase and interacting
proteins of the membrane cytoskeleton (ankyrin, fodrin, and actin) and
analyzed by confocal imaging.
RESULTS. Individual RPE cells within the same monolayer differed in amount of
Na/K ATPase, with a lower frequency of high expressing cells in the
area centralis. High expressing Na/K ATPase cells were found among
cells with both low and high E-/P-cadherin levels. In cells with low
E/P-cadherin, Na/K ATPase localized to apical microvilli, whereas in
high E-/P-cadherin cells, Na/K ATPase was on basolateral surfaces in
addition to microvilli. Actin staining showed that microvillar domains
were smaller and that lateral membrane domains were taller in
high E-/P-cadherin cells. In high but not low E-/P-cadherin cells,
ankyrin and fodrin levels varied among cells, with a subset of cells
showing distinctly higher expression. Both ankyrin and fodrin had
complex subcellular distribution patterns, although they tended to be
enriched basal to rather than apical to the adherens junction. Cells
with high Na/K ATPase did not necessarily have commensurately higher
levels of ankyrin or fodrin. Where both Na/K ATPase and ankyrin were
high, they codistributed weakly in apical microvilli but more
prominently on the basal cell surface.
CONCLUSIONS. Within the same RPE monolayer, the polarity of Na/K ATPase differs
among cells, with a more basal polarity found in cells with high levels
of junctional E-/P-cadherin. The increased basal Na/K ATPase was due to
a combination of a smaller microvillar domain, a taller lateral domain,
and more basolateral staining for Na/K ATPase, perhaps because of an
enrichment of a basal ankyrin–fodrin membrane cytoskeleton with which
Na/K ATPase is known to associate.
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Introduction
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The calcium-dependent adhesion protein E-cadherin forms
cell–cell attachments at the zonula adherens junction of most
monolayer epithelial cells. Retinal pigment epithelial (RPE) cells were
believed to be an exception and to express N-cadherin rather than
E-cadherin.1
2
3
4
5
6
We recently observed, however, that
E-cadherin is found in adult human RPE cells in situ and in
postconfluent human RPE cell cultures.7
P-cadherin, an
epithelial cadherin that is coexpressed with E-cadherin in many cells,
is also expressed in human RPE,7
and the avian homologue
of P-cadherin (B-cadherin)8
has been found in chick RPE
cells.9
The presence of E-cadherin in RPE cells is of
particular interest because E-cadherin is a morphoregulatory protein,
capable of inducing a specific polarity of cell surface molecules in
the cells that express it. An example is the epithelial cell line MDCK
in which Na/K ATPase and its associated ankyrin–fodrin–actin membrane
cytoskeleton are enriched on lateral membranes, basal to the adherens
junction. This distribution appears to be induced by the formation of
E-cadherin adhesions.10
11
In contrast, in RPE cells in situ, Na/K ATPase is polarized
predominantly to apical microvilli,12
13
14
15
16
17
18
a distribution
that appears inconsistent with the expression of E-cadherin in the
tissue. Furthermore, when a cultured rat RPE cell line was transfected
to express E-cadherin, Na/K ATPase developed a basal
polarity,4
indicating that E-cadherin is capable of
inducing a basal distribution even in a cell type in which the pump is
normally apical. The apparent inconsistency of the observations that
E-cadherin is expressed in RPE cells in situ, where the pump is
reportedly apical, led us to reevaluate Na/K ATPase distribution in RPE
cells within the eye. In the RPE monolayer, cadherin expression levels
vary among fields of cells, both in situ and in vitro.7
We
took advantage of this naturally occurring variation to compare protein
distributions in RPE cells with high and low E-cadherin levels,
focusing on Na/K ATPase and proteins with which it is linked: ankyrin,
fodrin, and actin. Bovine RPE cells were used because cells from the
tapetal region of this species have relatively few melanin and
lipofuscin granules, which obscure detailed visualization of
cytoplasmic and cell surface proteins.
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Methods
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Tissue Preparation
Eyes from adult cattle were obtained approximately 3 to 5 hours
postmortem; approximately 400 eyes were used for this study. The retina
was removed and the eyecup was filled with fixative (phosphate-buffered
3% paraformaldehyde, 4°C). After fixation (1–3 hours), pieces of
RPE with underlying choroid (approximately 5 x 10 mm rectangles)
were dissected from the tapetal region of the superior retina. In early
studies, tissue was taken from throughout the tapetum, immunostained
for various antigens and combinations of antigens (indicated in the
next section), and examined and photographed by epifluorescence
microscopy. After consistent morphologic features were identified, 85
eyes were prepared for confocal imaging, focusing on a constant site
just superior to the optic disc (Fig. 1)
. All proteins were examined in a minimum of 10 different eyes, and the
images that are shown are representative of multiple microscope fields.
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Figure 1. Diagram of the posterior pole of the bovine eye (A),
accompanied by low-power immunofluorescence micrographs of the RPE
monolayer from two animals, immunostained for Na/K ATPase
(B, C). The tissue pieces used for immunostaining
were taken from a region just superior to the optic disc (indicated by
the rectangle and arrow in A). The
small white dots in (B) and (C) are
individual RPE cells with high levels of Na/K ATPase. The frequency of
cells with high Na/K ATPase is higher in eye (B) than in eye
(C). In both eyes, there are fewer high Na/K ATPase cells in
the posterior region (*) than in the more peripheral region
of the tissue piece. Scale bar, 400 µm.
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For some eyes, the retina was not separated from the RPE. Rather, the
choroid and RPE with the retina attached were frozen, and 10-µm-thick
cross sections were cut.
Fluorescence Microscopy and Confocal Imaging
Tissue pieces or frozen sections containing the RPE monolayer were
permeabilized by incubation in 0.5% Triton and immunostained with one
or more of the following primary antibodies: monoclonal
anti–E-cadherin (clone 36; Transduction Laboratories, Lexington, KY),
rabbit polyclonal anti-ankyrin (directed against chicken erythrocytes;
Calbiochem, La Jolla, CA), rabbit polyclonal anti-fodrin/spectrin
(Sigma, St. Louis, MO), and chicken polyclonal anti–Na/K ATPase
(
1ß1; Cortex Biochem, San Leandro, CA). Antibody dilutions and
times of incubation were empirically determined. Appropriate FITC
(fluorescein)-, TRITC (rhodamine)-, or LRSC (lissamine
rhodamine)-conjugated secondary antibodies were from Jackson
ImmunoResearch Laboratories (West Grove, PA). To visualize F-actin,
tissues were stained with fluorescein- or rhodamine-conjugated
phalloidin (Sigma). Frozen sections were counterstained with propidium
iodide to stain nuclei. The clone 36 E-cadherin antibody gives a strong
immunostaining signal in bovine RPE and was therefore used for the
experiments reported here. Weaker junctional staining (not shown) was
also obtained with SHE78-7 and HECD-1 monoclonal E-cadherin
antibodies (Zymed Laboratories, San Francisco, CA). The clone 36
E-cadherin antibody exhibits some cross-reactivity with P-cadherin,
which is coexpressed with E-cadherin in many epithelial cells,
including the RPE7
; we have confirmed by immunoblot
analysis that P-cadherin is found in bovine RPE (not shown). Because of
the cross-reactivity of the clone 36 E-cadherin antibody, cells stained
here are designated as E-/P-cadherin positive.
Immunostained preparations were examined and photographed with an
epifluorescence microscope or a Bio–Rad MRC-600 confocal
microscope (Bio-Rad Laboratories, Hercules, CA). For the latter, serial
0.2- or 0.5-µm-thick sections were collected through the RPE
monolayer to generate cross-sectional Z-scans and to produce scans from
which composite en face images could be reconstructed. because the
tissue pieces are not completely flat and because RPE cell height
varies (from approximately 8–15 µm), 10 to 30 scans were collected
above and below the monolayer to assure that the full thicknesses of
all cells in the monolayer were imaged. For some purposes a subset of
scans is shown, and the scan numbers are reported in the figure
legends. The most apical scan was designated as scan 1; it is the scan
in which a microvillar marker (e.g., Na/K ATPase) first appears in the
tallest cell in the field. The scan number of the basal cell surface is
also reported, using markers that are indicated in the legends. Because
the RPE has basal infoldings and because tissue pieces are not flat,
basal antigens occur in several scans (10 or more). The estimate for
the scan number of the basal cell surface is therefore approximate and
not identical for all cells in the field. Comparisons of protein
distributions in low and high E-/P-cadherin fields were made on the
same tissue pieces or on tissue samples that were stained at the same
time and imaged using identical settings. Quantitative comparisons of
the size of different subcellular domains were made only on cells
within the same tissue pieces. Bovine RPE cells contain two classes of
autofluorescent inclusions: small irregular granules in the apical
cytoplasm and base of microvilli and larger lipofuscin/melanolipofuscin
granules in the plane of the adherens junction. These are more
prominent when TRITC is used for the detection fluor. For imaging of
immunostained samples, fields of cells were selected that were
relatively deficient in these granules. For some purposes, the position
of the granules within the cells was used as an endogenous marker of
the apical cytoplasm.
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Results
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Gross Distribution of Na/K ATPase in the RPE Cells of Bovine Eyes
The amount of Na/K ATPase varied markedly among individual RPE
cells, with adjacent single cells in the monolayer showing prominent
immunostaining differences. A subset of cells with distinctly higher
levels of Na/K ATPase can be detected on low-magnification images of
the monolayer (Figs. 1B
1C)
. The frequency of these cells differed
among eyes, but in all eyes they showed a consistent topographical
pattern, being less numerous in a horizontal band superior to the optic
disc, which corresponds to the area centralis. The tissue used for
morphologic analysis was taken from within this region, immediately
superior to the disc (Fig. 1A)
.
To confirm that the cell–cell differences in Na/K ATPase
immunostaining were not due to damage to the RPE during retinal
dissection, frozen sections were prepared from specimens in which the
retina was not removed. Adjacent RPE cells with high and low levels of
Na/K ATPase immunostaining were also seen in these cross sections (Fig. 2A
). Note that the isoform of Na/K ATPase recognized by the antibody
(1ß1) was not found in the outer sensory retina but was found in
the endothelium of the choriocapillaris (Fig. 2A)
.
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Figure 2. (A) Frozen section of the outer retina, RPE, and choroid
immunostained for Na/K ATPase (fluorescein) showing prominent staining
in some RPE cells (e.g., thick arrow) adjacent to cells with
low staining (thin arrows). Nuclei are counterstained with
propidium iodide. (B) Confocal images of the RPE monolayer
dually stained for Na/K ATPase and actin. The upper images
(a, b) show scans in the plane of the apical
microvilli where actin stains the microvillar core (scans 1–10,
0.5-µm scans). The cell base appears in scan 30. The lower images
(c, d) are composites of all scans, through the
entire thickness of the monolayer. For actin, the circumferential
microfilament bundles are the most prominent staining pattern in the
composite image. Arrows and arrowheads indicate
the same positions in all images. Scale bars, 20 µm.
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Additional confirmation of the integrity of RPE cells with different
levels of sodium pump was obtained by confocal imaging of cells
costained for Na/K ATPase and actin (Fig. 2B)
. Because actin forms the
core of apical microvilli and a prominent circumferential bundle in the
apical cytoplasm of all cells, it serves as a marker to show that the
apices of RPE cells with low levels of Na/K ATPase are intact and
indistinguishable from cells with high Na/K ATPase staining (Fig. 2B
,
items a and b).
Comparison of RPE Cells with Different Levels of E-/P-Cadherin:
Na/K ATPase, Ankyrin, and Fodrin
The amount of junctional E-/P-cadherin detected by immunostaining
varied among bovine eyes, and among fields of RPE cells within the same
monolayer. Using identical confocal settings, E-/P-cadherin staining
varied from undetectable (or nearly so) in some fields of cells to a
prominent junctional band in other fields (Figs. 3A , 3B
).
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Figure 3. Confocal images of low and high E-/P-cadherin fields, each triple
stained for E-/P-cadherin (A, B), ankyrin
(C, D), and Na/K ATPase (E,
F). For E-cadherin and ankyrin, selected 0.2 µm scans are
shown for the high E-/P-cadherin region taken from a plane encompassing
the adherens junction and peripheral ankyrin ring (B and
D, scans 23–29). Scan numbering is based on Na/K ATPase
staining, where scan 1 is the apex of the tallest cell and scan 54 is
the cell base. Selected scans at a comparable position within the cells
are also shown for the low E-/P-cadherin region (A and
C, scans 32–38). Scan numbering for low E-/P-cadherin cells
is based on Na/K ATPase for the cell apex (scan 1) and ankyrin for the
cell base (scan 55). The cytoplasmic fluorescence in the cadherin
images is due to autofluorescent inclusions and lipofuscin granules;
lipofuscin/melanolipofuscin granules are indicated by an
asterisk in (A). For Na/K ATPase, cross-sectional
Z-scans are shown; the positions of the Z-scans (E', F') are indicated
by the dotted lines on the composite images (E,
F, respectively). The arrow at the
left of the Z-scans indicates the plane of the cell base.
Scale bar, 20 µm.
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Costaining showed no correlation between amounts of Na/K ATPase and
amounts of junctional E-/P-cadherin; the scattered cells with high
levels of Na/K ATPase were found in fields of cells regardless of level
of cadherin staining (Figs. 3
4
5)
. Although amounts of Na/K ATPase did not differ, the subcellular
distribution of Na/K ATPase did differ in cells with different levels
of E-/P-cadherin. In low E-/P-cadherin cells, Na/K ATPase was
predominantly on apical microvilli (Figs. 3E
3E'
; 4C1
4C2
4C3
; 5A
,
A'
), which were frequently very long (Fig. 3E
3E'
). In high
E-/P-cadherin cells, Na/K ATPase was on lateral and basal cell surfaces
(Figs. 3F 3F'
; 4C1
4C2
4C3
; 5B
, B'
) in addition to apical
microvilli, which were shorter than in low E-/P-cadherin cells (compare
Figs. 3E' and 3F'
, or Figs. 5A'
and 5B'
).
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Figure 4. Composite confocal images of adjacent low and high E-/P-cadherin
(E-/P-cad) regions in the same microscope field, stained for
E-/P-cadherin (A), ankyrin (B), and Na/K ATPase
(C). For Na/K ATPase, selected individual 0.2 µm scans are
also shown in the apical (C1, scan 13), middle
(C2, scan 28), and basal (C3, scan 38) planes of
the cells. For scan numbering, scan 1 is the apex of the tallest cell,
and scan 46 is the cell base. Apical microvillar staining is greater
for low E-/P-cadherin cells (C1), and basal staining is
greater for high E-/P-cadherin cells (C3). In high
E-/P-cadherin cells, perinuclear structures prominently stained for
Na/K ATPase are seen (C2). The nucleus appears as an
unstained hole in the central cytoplasm. Scale bar, 20
µm.
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Figure 5. Confocal images of low and high E-/P-cadherin regions (E-/P-cad;
cadherin staining not shown), each costained for Na/K ATPase and
fodrin. Composite images and cross-sectional Z-scans are shown; the
position of the Z-scans (A' through D'),
which pass through high Na/K ATPase cells, are indicated by the
dotted lines on their respective composite images. Fodrin
immunostaining was more variable among cells in high E-/P-cadherin
regions, where a thin pericellular fodrin band is sometimes seen
(D, inset). The inset shows a single
0.2 µm (scan 27) in the plane of the pericellular fodrin band for the
cell indicated by the asterisk in (D). (Scan
numbering is based on Na/K ATPase staining where scan 1 is the apex of
the tallest cell and scan 66 is the cell base.) Na/K ATPase is largely
microvillar in low E-/P-cadherin regions (A') and on all
cell surfaces in high E-/P-cadherin regions (B'). Fodrin
stained microvilli, but microvillar staining is not prominent and not
greater in microvilli with high Na/K ATPase (arrows in
A' [Na/K ATPase] and C' [fodrin]). The cells
with high Na/K ATPase in the high E-/P-cadherin field are also
indicated by arrows (B' [Na/K ATPase] and
D' [fodrin]). Scale bar, 20 µm.
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Differences in Na/K ATPase distribution could be seen in high Na/K
ATPase cells that were found in adjacent low and high E-/P-cadherin
regions within the same microscope field (Fig. 4)
. Examination of
images at different planes in the apicobasal axis showed distinct Na/K
ATPase–stained puncta in the perinuclear cytoplasm of high but not low
E-/P-cadherin RPE cells (Fig. 4C2
). Examination of cells within the
same field also showed apparent differences in the height of the
lateral membrane of the cell body in cells with different cadherin
levels. A confocal scan taken through a middle plane of the monolayer
(Fig. 4C2 ) passes through the base of the microvilli in low
E-/P-cadherin cells, which have long microvilli and short lateral
membranes. The scan at the same plane through a nearby high
E-/P-cadherin cell passes through the nuclear region because the cell
has a taller lateral membrane and short microvilli. Height differences
between microvillar and lateral membrane domains of cells with
different cadherin levels were examined further by comparing cells
stained for actin (see Fig. 6
).
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Figure 6. Confocal images of a group of high E-/P-cadherin (E-/P-cad) cells
surrounded by low E-/P-cadherin cells, dually stained for the cadherins
and for actin. All images are of the same field but show scans at
different planes. Scans at the plane of the adherens junction (AJ),
where cadherins codistribute with the actin circumferential
microfilament bundle, are shown for both the high E-/P-cadherin
(A and B, scans 52–54) and low E-/P-cadherin
(C and D, scans 61–63) portions of the field.
Scan numbering (0.2 µm scans) is based on actin staining, where scan
1 is the apex of the microvilli of the tallest cell and scan 75 is the
cell base. Junctional staining for E-/P-cadherin is seen at the plane
of the AJ for high (A) but not low (C)
E-/P-cadherin cells. An asterisk indicates lipofuscin
granules at the plane of the AJ (A). Comparative heights of
microvillar and cell body domains in low and high E-/P-cadherin cells
are shown in a cross-sectional Z-scan of actin (B'); the
position of the Z-scan is indicated by dotted lines in en
face actin image (B). In the actin Z-scan (B'),
the heights of the cell body (b) and apical microvillar (m) domains are
indicated for high E-/P-cadherin cells (b*, m*) and flanking low cells
(b, m). The AJ in the high E-/P-cadherin cells is at the plane where
autofluorescent granules are located (base of microvilli) in adjacent
low E-/P-cadherin cells (A). A Z-scan of autofluorescent
granules (A') illustrates the differing heights of the cell
bodies in low and high E-/P-cadherin fields. Scale bars, 20 µm. Scale
bar in (D) is for all composite images; scale bar in
(A') is for the Z-scans.
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For ankyrin, expression levels appeared to correlate with levels of
E-/P-cadherin. In high E-/P-cadherin cell fields (but not low fields) a
subset of cells had markedly higher levels of ankyrin, much of which
was distributed in a distinct peripheral ring (Figs. 3D
4B)
. High
ankyrin cells did not necessarily have high Na/K ATPase (and vice
versa; see also Fig. 8
).
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Figure 8. Confocal images of RPE cells dual-stained for Na/K ATPase (A
through E) and ankyrin (F through J,
respectively). The first pair of images (A and F)
are composites of all scans. Six cells are identified by numbers. These
include cells with high staining for both proteins (cell 2),
high Na/K ATPase and low ankyrin (cells 3 and 6),
low Na/K ATPase and high ankyrin (cells 1 and 5),
or low staining for both (cell 4). The remaining pairs of
images are each composites of three scans (0.6 µm total) at different
planes along the apical-to-basal axis: (B and G;
scans 6–8), apex of the apical microvilli; (C and
H; scans 15–17), base of the microvillar region (terminal
web-like zone); (D and I; scans 20–22), plane of
the prominent peripheral ankyrin ring in cell 2; (E and
J; scans 30 to 32), basal membrane domain. Scan number is
based on Na/K ATPase staining where scan 1 is the apex of the tallest
cell and scan 40 is the cell base. Ankyrin staining was not so
prominent in microvilli as elsewhere in the cell, although
codistribution with Na/K ATPase in microvilli can be detected in cells
with high levels of both proteins (cell 2, arrows
in B and G). In the region of the peripheral
ankyrin ring (cell 2 in I), pericellular Na/K
ATPase is diminished (cell 2 in D). (All cells in
the field are not of identical height so the plane of this pair of
scans was selected to show the zonular ring of ankyrin in the cell
[cell 2] which is highly stained for both ankyrin and Na/K
ATPase.) At the basal membrane, Na/K ATPase and ankyrin codistribute
(E and J). In this domain, cells that showed low
Na/K ATPase in other subcellular domains showed distinct basal staining
only in cells with high ankyrin (see cells 1 and
5). At the basal surface, the pattern of cells with distinct
staining for both ankyrin and Na/K ATPase appears to be the same. Scale
bar, 20 µm.
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Fodrin showed highly complex distributions among and within RPE
cells in situ (Fig. 5)
. In cells with low E-/P-cadherin, levels of
fodrin were relatively homogeneous among cells, and the protein was
distributed throughout the cytoplasm, with somewhat higher staining
between the nucleus and lateral cell borders, basal to the adherens
junction (Fig. 5C
5C'
). Microvillar staining for fodrin was detectable
but not prominent and was not higher in cells with high levels of
microvillar Na/K ATPase (Fig. 5C'
). In fields of cells with higher
E-/P-cadherin levels, fodrin staining intensity was more variable among
cells (Fig. 5D)
. There were groups of cells with distinctly higher
levels of fodrin, and in some of these cells fodrin distributed in a
discrete, thin apicolateral ring (Fig. 5D
, inset), in addition to an
enrichment of the more diffuse basolateral cytoplasmic staining.
Comparison of RPE Cells with Different Levels of E-/P-Cadherin:
Size of Microvillar and Lateral Membrane Domains
Microvillar size in RPE cells with different levels of
E-/P-cadherin was examined using specimens in which microvilli were
visualized by staining their actin core (Fig. 6)
. Actin staining was
also used to estimate the height of the lateral membrane domain of the
cell body using the circumferential actin bundle as a marker. The actin
bundle is associated with the adherens junction, which demarcates
apical and basolateral membranes. The distance from the circumferential
bundle to the cell base, therefore, provides a measure of the height of
the lateral border of the cell body. Using actin staining, differences
in height of both the microvillar and the lateral domains were seen in
adjacent fields of RPE cells with different levels of E-/P-cadherin
(Fig. 6)
. Confocal Z-scans (Fig. 6B'
) showed that cells with low levels
of E-/P-cadherin have relatively long microvilli and short cell bodies
compared with cells with higher levels of E-/P-cadherin. Sequential en
face confocal scans through adjacent low and high E-/P-cadherin cells
from the plane of their adherens junctions to the cell base could be
used to estimate the differences in height of lateral membranes of the
cell body. The plane of the adherens junctions was approximately 2 µm
higher in cells with higher amounts of E-/P-cadherin, reflecting their
taller cell bodies. The different heights of the cell bodies could also
be shown using endogenous autofluorescent granules rather than the
actin bundle as a marker of the cell apex (Fig. 6A
6A'
). The granules
are distributed in the apical cytoplasm and in the base of microvilli;
in cells with high E-/P-cadherin the distance from the granules to the
cell base was visibly greater than in low E-/P-cadherin cells (Fig. 6A'
).
As shown in Figures 3D
and 4B
4a
subset of high E-/P-cadherin cells
also has high levels of ankyrin, with ankyrin forming a prominent
peripheral ring. Among high E-/P-cadherin cells, cells with high levels
of ankyrin have an even taller lateral cell domain than adjacent cells
(Fig. 7)
. As shown, the distance from the ankyrin ring to the cell base is
greater at the borders between adjacent high ankyrin cells than at the
borders between adjacent high and low ankyrin cells (Fig. 7A'
). Using
the actin bundle-to-base as a measure of basal cell height (as in Fig. 5 ) in cells costained with actin and ankyrin, high ankyrin cells had a
basal domain that is approximately 2 µm taller than adjacent low
ankyrin cells. Actin and ankyrin–costained specimens also showed that
the prominent ankyrin ring is basal to the circumferential actin bundle
(not shown).
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Figure 7. Composite confocal image (A) and two Z-scans
(A', A'') of a high E-/P-cadherin region
immunostained for ankyrin showing a cluster of high ankyrin cells.
(Cadherin staining is not shown.) The positions of Z-scans A' and A''
are indicated by dotted lines A' and A'', respectively, on the composite
image. In Z-scan (A'), a single shorter low ankyrin cell
(arrow in A and A') is flanked by
taller high ankyrin cells. Z-scan (A'') illustrates the
height of the cell body domain, from the peripheral ankyrin ring to the
cell base. Cell height is greater at the borders of two high ankyrin
cells (brackets h-h) than at the borders of adjacent high
and low ankyrin cells (brackets h-l). Scale bar, 20 µm.
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RPE Cells with High Ankyrin: Codistribution with Na/K ATPase
Within fields of RPE cells with high E-/P-cadherin, individual
cells may have high or low ankyrin (Figs. 3D
4B)
, and high or low Na/K
ATPase (Figs. 3F
4C)
, but ankyrin and Na/K ATPase are not necessarily
high in the same cells. Fields were selected that contained cells with
all combinations of staining patterns to examine the subcellular
codistribution of the two proteins (Fig. 8)
. Ankyrin staining was seen in apical microvilli, although the staining
was not prominent and was not greater in cells with high levels of Na/K
ATPase (Figs. 8B
8G)
. Ankyrin was enriched in a plane at the base of
microvilli, which has been called a terminal web-like
region,19
where it codistributed with Na/K ATPase (Figs. 8C
8H)
. Na/K ATPase did not codistribute with the prominent peripheral
rings of ankyrin (Figs. 8D
8I
, cell 2). However, Na/K ATPase and
ankyrin distinctly colocalized in the cell bases; even cells with low
overall levels of Na/K ATPase but high ankyrin showed a basal
codistribution of the sodium pump with ankyrin (Figs. 8E
8J)
.
The differences that were observed in cell height and Na/K ATPase
polarity between bovine RPE cells with low and those with high
E-/P-cadherin levels are summarized in the diagram shown in Figure 9
.
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Figure 9. Diagram summarizing the protein distributions in low E-/P-cadherin RPE
cells (A) and in high E-/P-cadherin cells with either low
(B) or high (C) ankyrin levels. In
(A), Na/K ATPase polarity is predominantly apical due to a
large microvillar domain where Na/K ATPase distributed and a short
lateral domain of the cell body (*) with no detectable Na/K
ATPase. As compared with cells in (A), cells in
(B) have a reduced apical polarity of Na/K ATPase due to a
reduced size of the microvillar domain and an increased lateral domain
(*) where Na/K ATPase was detected. Cells in (C)
have an even less apically polarized Na/K ATPase due to a greater
enlargement of the lateral domain (*) and an enrichment of
Na/K ATPase in basolateral membranes.
|
|
|
Discussion
|
|---|
One general observation resulting from examining protein
distributions in individual RPE cells in situ was that protein
expression levels among cells are distinctly heterogeneous. All
proteins examined in the present study, with the exception of F-actin,
showed this heterogeneity. RPE cells appear uniform in phenotype due to
their regular hexagonal organization, but this gross uniformity gives a
misimpression of the molecular heterogeneity of the cells. Differences
among RPE cells in the expression levels of the intermediate filament
protein vimentin were previously shown,20
21
and amounts
of melanin also vary among cells.22
Because large expanses
of the monolayer were examined here, we found that the cell–cell
variations in expression for different proteins occurred in different
patterns. Cells with high expression levels for a given protein could
occur predominantly as single cells (Na/K ATPase), small clusters or
rows of cells (ankyrin), larger groups of cells (fodrin), or more
expansive fields of cells (E-/P-cadherin). The monolayer is, therefore,
a mosaic, with individual cells having the potential to exhibit a
complicated spectrum of protein expression levels. We previously
hypothesized that RPE cells are phenotypically heterogeneous in culture
because of a naturally occurring heterogeneity in expression levels of
proteins that affect cell shape.21
23
One such protein is
E-cadherin, which has a morphoregulatory
function,11
24
25
26
and which was found in the present
study to vary among cells. This observation, coupled with the
observation that E-cadherin is expressed by some but not all phenotypes
of cultured RPE cells,7
lends support to this hypothesis.
The protein with the most striking intercellular variability was Na/K
ATPase, which was found at markedly high levels in a subset of RPE
cells. These cells varied in frequency among eyes but were consistently
less numerous in the posterior pole. This unexpected observation helps
explain our previous observation that the number of sodium pump sites,
quantified by ouabain binding, varies among eyes and is lower in the
bovine area centralis relative to the peripheral retina.27
Similar results were obtained when macular and extramacular regions of
human eyes were compared.27
At the time we assumed that
the entire population of RPE cells in the macula/area centralis had an
overall lower pump site density than more peripheral cells. It appears
now that lower macular pump density is due to a lower frequency of
cells with high Na/K ATPase expression levels. The functional
ramifications of this observation remain obscure. Ion transport for the
entire tissue may be similar whether all cells have the same number of
pumps or individual cells have varying numbers of pumps, provided that
the total number of sodium pump sites is adequate for the tissue
surface area. However, it seems inevitable that large intercellular
differences in sodium pump density will impact ion transport in the
microenvironment of individual RPE cells and the photoreceptors with
which they interact.
RPE cells have been known for many years to have a predominantly apical
distribution of Na/K ATPase within eyes.12
13
14
15
16
17
18
This
polarity was confirmed in the present study in that Na/K ATPase
immunostaining was clearly seen on the apical microvilli of bovine RPE
cells. However, closer examination revealed heterogeneity in the
distribution of the sodium pump among cells. Certain subcellular
distributional patterns of Na/K ATPase could be predicted by levels of
E-/P-cadherin coexpression in the same cells. Compared with RPE cells
with low E-/P-cadherin levels, cells with high levels showed reduced
apical polarization of Na/K ATPase, especially if the cells also
expressed high levels of ankyrin. The reduced apical polarity of Na/K
ATPase in RPE cells with high E-/P-cadherin level resulted from a
combination of a smaller microvillar domain, a taller lateral domain,
and increased immunodetectable Na/K ATPase in basolateral membranes
(see diagram in Fig. 9
). E-cadherin has been shown to induce a basal
polarity of Na/K ATPase in epithelial cells in vitro,10
11
including in an RPE cell line.4
The increased basal
distribution of Na/K ATPase in RPE cells in situ with higher levels of
E-/P-cadherin is consistent with the "basal" inductive
properties of E-cadherin.
One of the mechanisms for polarizing Na/K ATPase to basolateral
membranes of epithelial cell lines is selective retention via ankyrin
linkage to the basal fodrin–actin membrane
cytoskeleton.28
Ankyrin and fodrin were reported to show a
parallel reversal with Na/K ATPase to the apical surface of rat RPE
cells,18
suggesting that an ankyrin-fodrin–selective
retention mechanism may contribute to polarizing the pump to apical
membranes in the RPE. There is a question, however, about the role
played by at least fodrin in contributing to apical pump polarity.
During later stages of chick embryogenesis as microvilli elongate,
fodrin (spectrin) no longer codistributes with Na/K
ATPase.19
Rather, fodrin is enriched at the base of
microvilli (terminal web-like region) and in the base of
cells.19
In our analysis of bovine RPE cells in situ shown
here, ankyrin and fodrin were detected in apical microvilli, but
neither protein distributed prominently to this region. Of note, cells
with high levels of Na/K ATPase did not necessarily show comparably
elevated levels of ankyrin and fodrin, and distinct codistribution with
apical Na/K ATPase was difficult to find, even in cells with high
levels of both Na/K ATPase and ankyrin or fodrin. The lack of prominent
codistribution between Na/K ATPase and ankyrin–fodrin in apical
microvilli raises the possibility that another linking system
contributes to stabilizing apical Na/K ATPase in bovine RPE cells, as
has been previously suggested for chick RPE.19
The ankyrin–fodrin cytoskeleton may play a greater role in segregating
Na/K ATPase to the basal membranes of RPE in situ, particularly in high
E-/P-cadherin cells. Only in high E-/P-cadherin cells were high levels
of ankyrin and fodrin seen, and both proteins distributed more
prominently basal to the adherens junction where a specific
codistribution of at least ankyrin with Na/K ATPase was seen.
E-cadherin expression may change the expression of fodrin and ankyrin
and induce their basolateral enrichment, thereby contributing to a
basolateral enrichment of Na/K ATPase. Support for this possibility
comes from studies of cultured RPE cells transfected to express
E-cadherin.4
Transfected cells showed increased amounts of
fodrin and a change in ankyrin isoform accompanying the development of
a basal Na/K ATPase. Whether the higher levels of ankyrin seen here in
some high E-/P-cadherin RPE cells reflect a different ankyrin isoform
is currently unknown because the ankyrin antibodies that were used
recognize all isoforms.
We focused in the present study on ankyrin–fodrin–cytoskeletal
linkage as a mechanism for polarizing Na/K ATPase, because regulating
the expression of membrane cytoskeletal proteins and their position
within cells are functions that have been attributed to E-cadherin.
However, there are several mechanisms for polarizing Na/K ATPase that
are differentially used by different epithelial tissues,29
and it is unlikely that mechanisms triggered by E-cadherin are acting
alone in RPE cells. Indeed, RPE cells in situ with high E-/P-cadherin
levels show an increased basal polarity of Na/K ATPase, but the cells
nonetheless retain distinct apical Na/K ATPase. If the increased basal
distribution is induced by E-/P-cadherin, then it appears that
cadherin-mediated mechanisms compete, not entirely successfully, with
other mechanisms in RPE cells that maintain an apical sodium pump. What
the latter mechanisms are, and why E-/P-cadherin is only partially
effective, remain to be determined.
|
Acknowledgements
|
|---|
The authors thank Emmber Foods, Inc. for generously providing cow
eyes.
|
Footnotes
|
|---|
Supported by Grants R01-EY10832 (JMB) and P30-EY01931 from the National Eye Institute; and by unrestricted grants from the Posner Foundation (Milwaukee, Wisconsin) and Research to Prevent Blindness (New York, New York). JMB is the recipient of a Senior Scientific Investigator Award from Research to Prevent Blindness.
Submitted for publication November 15, 1999; revised January 18, 2000; accepted January 31, 2000.
Commercial relationships policy: N.
Corresponding author: Janice M. Burke, The Eye Institute, 925 North 87th Street, Milwaukee, WI 53226-4812. jburke{at}mcw.edu
|
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Top
Abstract
Introduction
