Physiol Rev
84: 1229 –1262, 2004; 10.1152/physrev.00001.2004.
Cell Adhesion, Polarity, and Epithelia
in the Dawn of Metazoans
M. CEREIJIDO, R. G. CONTRERAS, AND L. SHOSHANI
Center for Research and Advanced Studies (CINVESTAV), México, Mexico
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I. Introduction
A. For almost a century transepithelial transport was a hard-to-believe hypothesis
B. The puzzle of the first metazoan
II. Model Systems of Transporting Epithelia
III. The Na⫹-K⫹-ATPase
A. The ␣-subunit
B. The ␤-subunit
C. ␣/␤-Subunit interactions
D. The ␥-subunit
E. Assembly and delivery of the Na⫹-K⫹-ATPase
F. The triggering effect of Ca2⫹
G. The polarized distribution of Na⫹-K⫹-ATPase
IV. Tight Junctions
A. Structure
B. Transmembrane proteins
C. Membrane-associated proteins
D. Lipids
E. Diversity arising from processing TJ proteins as well as by the expression of several isoforms
F. Proteins that can localize to the nucleus and adhesion complexes
G. Phosphorylation in TJ assembly and function
H. Biogenesis
V. Septate Junctions
VI. Adherens Junctions
A. Structure and function
B. Relationships between adherens and occluding junctions
VII. Polarity
A. The transporting epithelial phenotype
B. Polarity and the TJ
C. Na⫹-K⫹-ATPase, cell attachment, and the position of the TJ
VIII. Evolution of the Epithelial Vectorially Transporting Phenotype
A. Evolution of the Na⫹-K⫹-ATPase
B. Evolution of junction proteins
IX. The Dawn of Metazoans and Transporting Epithelia
A. The “thrifty sponge”
B. Very flat organisms
C. The “mare nostrum” metazoans
X. Na⫹-K⫹-ATPase and Cell Adhesion
A. Role of the ␤-subunit in cell attachment
B. (P3 A), a pump (P) adhesion (A) mechanism
Cereijido, M., R. G. Contreras, and L. Shoshani. Cell Adhesion, Polarity, and Epithelia in the Dawn of Metazoans.
Physiol Rev 84: 1229 –1262, 2004; 10.1152/physrev.00001.2004.—Transporting epithelia posed formidable conundrums right from the moment that Du Bois Raymond discovered their asymmetric behavior, a century and a half ago.
It took a century and a half to start unraveling the mechanisms of occluding junctions and polarity, but we now face
another puzzle: lest its cells died in minutes, the first high metazoa (i.e., higher than a sponge) needed a transporting
epithelium, but a transporting epithelium is an incredibly improbable combination of occluding junctions and cell
polarity. How could these coincide in the same individual organism and within minutes? We review occluding
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0031-9333/04 $15.00 Copyright © 2004 the American Physiological Society
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CEREIJIDO, CONTRERAS, AND SHOSHANI
junctions (tight and septate) as well as the polarized distribution of Na⫹-K⫹-ATPase both at the molecular and the
cell level. Junctions and polarity depend on hosts of molecular species and cellular processes, which are briefly
reviewed whenever they are suspected to have played a role in the dawn of epithelia and metazoan. We come to the
conclusion that most of the molecules needed were already present in early protozoan and discuss a few plausible
alternatives to solve the riddle described above.
I. INTRODUCTION
A. For Almost a Century Transepithelial Transport
Was a Hard-To-Believe Hypothesis
In the second half of the 19th century Emile Du Bois
Raymond demonstrated that a frog skin separating two
saline solutions exhibits a spontaneous electrical potential difference across it. G. Galeotti (166, 167) proposed
that this potential could be explained by assuming that
the epithelium has a higher Na⫹ permeability in the inward than in the outward direction. This explanation was
not accepted because it would be in violation of the first
and second laws of thermodynamics (66, 72). A second
argument, seeking a source of energy, proposed that it
would be provided by the metabolism of the cells, but was
also refuted because, according to Curie’s principle, phenomena of different tensorial order cannot be coupled,
i.e., Na⫹ transport is a vectorial phenomenon (it occurs in
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B. The Puzzle of the First Metazoan
Life depends on an intense and highly selective exchange of substances between cells and their environment. In the case of single cells living in the sea, this
environment behaves as an infinite reservoir. When cells
belong instead to a higher metazoan organism, they exchange with a very thin layer of interstitial milieu that
would be quickly spoiled were it not for a circulatory
apparatus that constantly restores nutrients and takes
away waste products by carrying them to and from large
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The study of epithelia always posed formidable conundrums. The first one, briefly described below, arose a
century and a half ago when it was discovered that these
tissues have a spontaneous electrical potential between
the outer and the inner side. Today, even when biological
asymmetries (referred to as “polarity”) are still subject to
intense research, we are no longer stuck in a puzzling
situation nor foresee conflicting aspects. The second riddle is posed by the origin of epithelia as part of the
emergence of metazoans. Since its cells perish for lack of
nutrients or excess of metabolic wastes, a metazoan
higher than a sponge exchanges nutrients and wastes with
an internal milieu, which in turn exchanges them through
transporting epithelia, but a transporting epithelium is in
itself a highly elaborated combination of occluding junctions and polarized mechanisms. Therefore, the origin of
the first metazoan would have depended on the incredibly
improbable coincidence, in the same individual and
within minutes, of an elaborated epithelium with occluding junctions and polarity. We propose that the somewhat
baffling episode consisted of a combination of different
species of attaching molecules and even fundamental polarizing processes that were already available in unicellulars. This article is therefore devoted to update some
aspects of cell attachment and polarity that might have
participated in the dawn of transporting epithelia and
metazoans.
the outside to inside direction) and cannot be driven by a
scalar phenomenon (in those days chemical reactions
were assumed to be scalar and therefore could not be
expected to proceed in a given direction of the space).
Half a century later, Hans Ussing devised electric and
tracer methods to unambiguously demonstrate that the
frog skin can actually transport a net amount of Na⫹ in
the inward direction and in the absence of an external
electrochemical potential gradient (456). Of course, this
demonstration, confirmed thereafter in hundreds of laboratories, prompted a closer look at the arguments that had
stood in the way of accepting that metabolism can drive
Na⫹ transport. To start with, a protein like Na⫹-K⫹ATPase cannot interact with Mg2⫹, Na⫹, K⫹, ATP, and
ouabain just anywhere on its surface, but at very specific
sites located either on the outer or on the inner side of the
cell membrane, but not both, i.e., this enzyme is vectorial
at the microscopic level. Yet, when studied in a solution
where millions of molecules point in all directions, vectoriality is lost. But in the orderly molecular orientation of
a cell membrane, Na⫹-K⫹-ATPase is anisotropic both at
the microscopic and at the macroscopic level. In turn, De
Donder and van Rysselberghe (117) proved that chemical
affinity can constitute a driving force, Onsager (340, 341)
demonstrated that all fluxes and all forces present in a
system can be, in principle, coupled, and Kedem (245)
demonstrated that the flux of a given ion can be coupled
to metabolism, so the split of ATP into ADP plus Pi can, in
fact, drive the net flux of Na⫹. In keeping with the idea
that gradients and fluxes of different substances can be
coupled, it was later found that the Na⫹ gradient originated by the Na⫹-K⫹-ATPase can also propel the unidirectional fluxes of sugars, amino acids, Cl⫺, Ca2⫹, H⫹,
etc., via co- and countertransporters.
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
II. MODEL SYSTEMS OF
TRANSPORTING EPITHELIA
Most studies to evaluate unidirectional fluxes across
the apical and the basolateral membrane of epithelial cells
(Fig. 1) followed black box approaches (Fig. 2) (60, 64,
113, 211), but in spite of affording valuable information on
overall epithelial processes (46, 65, 67, 68, 71, 73, 233, 318,
367, 369, 386), these approaches lacked the necessary
resolution to study their cellular and molecular basis and,
more specifically, junctions and polarity. Furthermore,
black box methods could only be applied to mature epithelia where junctions and asymmetry are already established. Therefore, entirely different methods were required.
Oxender and Christensen (343) attempted to assemble an artificial epithelium by sandwiching single Ehrlich
ascites cells between Millipore filters separating two
chambers. However, these cells lacked an intrinsic asymmetry, remained oriented at random, did not attach, and
no asymmetry of the whole preparation was detected.
Further attempts used epithelial cells dislodged from epithelia through treatment with Ca2⫹ chelants and hydrolyzing enzymes. The first step was to separate the epithelium of a frog skin (387) and show that it preserves the
overall properties of dissected epithelia. Therefore, the
second step was to mince the epithelium, dislodge the
cells, and investigate whether these maintain a satisfac-
FIG. 1. Position and structure of diverse
cell junctions. Two neighboring epithelial
cells have their apical domain towards the
outside, with the basal domain contacting
the inside through an extracellular matrix
and the lateral domains contacting each
other. Location of the main junctions mentioned in this review are indicated by
squares and numbered. Cartoons at the bottom left depict the transmembrane molecules of claudin and E-cadherin, which are
the most representative of tight (occludens)
and adherens junctions among many others.
Claudin has 4 transmembrane domains, and
E-cadherin just 1. Both termini of claudin are
intracellular, whereas only the COO⫺ of Ecadherin is intracellular. The extracellular
domain of E-cadherin has 5 repeats and is
glycosylated (small green circles). No molecular details of the macula adherens (desmosome, Ref. 170) nor the gap junction are
represented, as these are not discussed in
the text. The conexons of the gap junctions
(pink) communicate the intracellular compartment of the two neighboring cells.
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areas of epithelia (intestinal, renal, gills, etc.) where they
are finally exchanged with the external environment.
Even when a primeval metazoan might have been a small
organism contained within a single type of epithelium, it
required some sort of sealing between its cells, and these
cells should have had vectorial transport. Therefore, the
emergence of first metazoans higher than a sponge poses
a paradox, because somatic cells gathered in a restricted
space required a transporting epithelium to exchange substances with the external environment, but the first transporting epithelium depended in turn on the highly improbable coincidence, within a few minutes and in the same
organism, of three novelties: 1) assembly of the multicellular organism itself, 2) the establishment of occluding
junctions between epithelial cells, and 3) a polarized distribution of transport mechanisms in the membrane of
these cells.
In this review we explore the possibility that metazoans, occluding junctions, and vectorial transport appeared as manifestations of specific types of binding between certain molecular species, as well as between epithelial cells. The argument requires a brief description of
the preparations developed to investigate the development of the so-called “epithelial transporting phenotype, ”
as well as a review of Na⫹-K⫹-ATPase and cell junctions,
concentrating on those properties that involve specific
bindings between molecules as well as between cells.
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tory ionic steady state and responded to ouabain, amiloride, changes in temperature, or have other evidences that
cells retained the basic properties of natural epithelia
(500, 501). Finally, the third step was to seed the cells on
glass or Millipore filters to form a sort of artificial epithelium. Unfortunately, cells performed poorly in culture,
detached easily, died soon, and prompted us to try established cell lines. We chose the MDCK line derived by
Madin and Darby (288) from canine kidney, which was
generally used to grow viruses, because it retained sufficient differentiation as to secrete fluid (276). We cultured
these cells on translucent supports (a nylon cloth coated
with collagen) on which cells can easily be observed (69,
70, 72, 75), or nitrocellulose filters. A similar preparation
was developed by Misfeldt et al. (315). This approach gave
us an opportunity to study the development of the transporting epithelia phenotype. Thus we used it to study the
synthesis, assembly, and sealing of tight junctions (TJs)
(69, 74, 75, 78, 145, 183, 297), as well as the polarity of
Na⫹-K⫹-ATPase (61, 62, 96, 102); Enrique Rodriguez-Boulan introduced the use of viruses that bud either apically
(e.g., Flu) or basolateraly (e.g., stomatitis) to track the
fate of specific proteins during apical or basolateral polarity (283, 284, 379 –382), and Carlos A. Rabito analyzed
the onset of polarized cotransporters (368, 370, 371). M.
Taub perfected the approach by developing totally defined culture media (440 – 442), and complementary procedures were refined to open and reseal the TJs by removing and restoring Ca2⫹ (69, 97, 101, 184, 186), follow
the cascades of phosphorylation involved (20, 21, 85), the
role of the cytoskeleton (311, 312), the participation of
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protein synthesis and sorting (196, 376, 379, 381), the
polarized distribution of ion channels (317, 359, 360, 361,
421, 439), the involvement of E-cadherin (199 –201), the
effect of agents that induce differentiation (280), and the
vectorial movement of receptors (302). A distinct advantage of cultured monolayers is derived from the
possibility of labeling beforehand a given cell type, and
then mixing them with other types from different epithelia and even different animal species (98, 103, 185),
and other characteristics illustrated below and reviewed
in Cereijido (56) and Cereijido and Anderson (58).1
In addition to the preparation just described, the
information discussed in this review was obtained with
suspended clumps and cysts of epithelial cells (465),
yeasts, Caenorhabditis elegans and Drosophila (479), fertilized eggs, and the first stages of development from egg
to embryo (151).
III. THE Naⴙ-Kⴙ-ATPase
The Na⫹-K⫹-ATPase is not the only pump known
today, yet for many years it was the almost exclusive
focus of attention, and today is recognized to act as the
star in the net transfer of a wide variety of substances
across epithelia. The Na⫹-K⫹-ATPase is a member of the
family of P-type ATPases. ATP hydrolysis by these
ATPases includes a step of transfer of the terminal phos1
For a vignette on the development of this model system, see
Current Contents 32: 46, 1989 and Physiologist 46: 114 –115, 2003.
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FIG. 2. Early black box methods to
study unidirectional fluxes through the
apical or basal domain of epithelial cells.
A frog skin is mounted as a diaphragm
between two halves of a Lucite chamber
with a rectangular exposed area. Ringer
solution is injected in 48 s; the outer one
(apical) has 24Na, so when it reaches the
top the lowest part of the epithelium has
been exposed 48 s, and the rest a fraction
of this time. The skin is quickly frozen
(liquid nitrogen) and washed, and the exposed area is sliced. The 24Na in each
piece is counted. The slope of the curve
is used to calculate J12. Figure at the
bottom right represents an epithelial cell
with the 4 unidirectional fluxes involved
in transepithelial Na flux. Further procedures (not detailed) can be used to calculate the other Jij. [Redrawn from
Cereijido and Rotunno (72).]
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
phoryl group of ATP onto the carboxyl group of an aspartic acid residue that is located in the active site of the
enzyme (42). Among the ions transported by these type of
pumps are protons, calcium, sodium, potassium, and
heavy metals such as manganese, iron, copper, and zinc
(418). The formation of a phosphorylated intermediate
during the catalytic cycle is a characteristic of P-type
ATPases that distinguishes them from V-ATPases and
F-ATPases (347, 348). The Na⫹-K⫹-ATPase is a heteromultimeric membrane enzyme constituted by ␣-, ␤-, and
␥-subunits.
A. The ␣-Subunit
1. ␣-Subunit isoforms
Four isoforms of the ␣-subunit occur in mammals: ␣1,
␣2, ␣3, and ␣4, that exhibit somewhat different properties
in terms of cation binding (231) and are expressed in a
tissue-specific and developmentally regulated manner.
While the ␣1-isoform is expressed in every tissue, ␣2 is
limited largely to skeletal muscle, heart, and brain. The
␣3-isoform is expressed in brain and ovary, and ␣4 is
exclusive of sperm and its precursor cells (41, 406, 481).
The ␣2- and ␣3-isoforms appear early in brain development, and around birth in heart and skeletal muscle.
Na⫹-K⫹-ATPase isoform expression can also be differentially regulated by hormones (214, 342), and ␣-isoform
genes contain 59 specific sequences that are potential
trans-acting factors and hormone binding sites (385). Animals lacking the ␣1-isoform gene do not develop past the
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blastocyst stage (282). Yet, ␣2-isoform-deficient animals
develop to term, as expected, since it is not expressed in
early embryonic development. Interestingly, these animals were either born dead or died within the first few
minutes after birth due to failure in the neuronal activity
of the breathing center in the neonate (321).
B. The ␤-Subunit
This subunit is part of the Na⫹-K⫹- and H⫹-K⫹ATPases but is not included in other P-type ATPases; for
example, Ca2⫹-ATPase of both plasma membrane and
sarcoplasmic reticulum do not have it. It is composed of
⬃370 amino acids, with the first 30 exposed to the cytosol,
and 300-fold to form the extracellular portion that has 3
disulfide bonds. There are 3 N-glycosylation consensus
sequences (NXS or NXT) in the extracellular domain (249,
400). The polypeptide chain of the ␤-subunit weighs 32–35
kDa, and when fully glycosylated can reach an overall
apparent molecular mass of 55– 60 kDa. Analysis of ␤-subunits in which the disulfide bonds were removed through
substitution in baculovirus-infected insect cells, reveals
that the S-S bridges are not important for assembling the
heterodimer, yet they are required for membrane targeting (171, 272). Recent studies using heterologous expression suggest that substitution of the essential asparagine
residues prevents glycosylation, but this has little if any
effect on catalytic activity (30).
The ␤-subunit has been identified as a factor responsible for cell adhesion in nervous tissue (180). The activity
of Na⫹-K⫹-ATPase can be influenced by lectins, in particular, concanavalin A (428), and galectins (various lectins
of animal origin), some of which affect cell adhesion (335)
and provide in this way signal transmission to the catalytic subunit.
1. ␤-Subunit isoforms
Three ␤-isoforms can be attributed to Na⫹-K⫹ATPase in mammals (␤1, ␤2, and ␤3), and a fourth to
gastric H⫹-K⫹-ATPase (␤HK). Significantly, for the nongastric H⫹-K⫹-ATPase, no specific ␤-subunit has been
identified. X-K-ATPase ␤-isoforms exhibit only ⬃20 –30%
overall sequence identity but share several structural features. ␤-Isoforms exhibit a tissue-specific distribution
with ␤1 of Na⫹-K⫹-ATPase being expressed ubiquitously,
␤2 mainly in the heart, skeletal muscles, and glial cells,
and ␤3 in many tissues (for review, see Ref. 42).
Recently, a novel member of the ␤-subunit family has
been identified (␤m) which shares common structural
features and signature motifs with X-K-ATPase ␤-isoforms (355). Despite the similarities with X-K-ATPase
␤-isoforms, ␤m has also some atypical characteristics.
First, it contains two long glutamate-rich regions in the
cytoplasmic NH2 terminus that are not present in X-K-
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The 112-kDa ␣-subunit, often called the catalytic subunit, bears the site for ATP hydrolysis; has the binding
sites for Na⫹, K⫹, and cardiac glycosides; and is homologous to the Ca2⫹-ATPase of the sarcoplasmic reticulum
(SERCA) whose 2.6-Å resolution structure was recently
reported by Toyoshima et al. (449).
Since the first cloning and sequencing of a Na⫹-K⫹ATPase ␣1-subunit (413), hydropathy analyses have deduced 10 transmembrane crossings (241). A combination
of heterologous expression and site-directed chemical
labeling of cysteine mutants found five exposed extracellular loops and showed that both termini are intracellular
(217). The high-resolution structure of SERCA was compared in detail to a Na⫹-K⫹-ATPase projection (429) and
supported the idea that the Na⫹-K⫹-ATPase structure is
very similar to the structure of SERCA (277). There are
three main intracellular structures: a large central loop
between M4 and M5 (210) composed of ⬃430 amino acid
residues, a long NH2-terminal tail of ⬃90 amino acids, and
an intracellular loop of ⬃120 residues between M2 and
M3. These form the N or nucleotide-binding domain, the P
or phosphorylation domain, and the A or actuator domain
(449).
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C. ␣/␤-Subunit Interactions
The subunits of Na⫹-K⫹-ATPase are synthesized independently in the endoplasmic reticulum and assembled
in this organelle. Detergent solubilized ␣␤-heterodimers
of Na⫹-K⫹-ATPase are able to carry out normal Na⫹-K⫹ATPase activity (136, 137). However, without the ␤-subunit the ␣-subunit does not exhibit a detectable activity
(438) and is rapidly degraded (2, 174). The ␤-subunit may
be involved in stabilizing the correct transmembrane folding of the ␣-subunit (2, 172, 173, 399). Amino acid residues
located in the proximity to the COOH-terminal fragment
of the ␤-subunit appear to participate in the association
between the ␣-subunit and the ␤-subunit (29, 95), as does
its transmembrane fragment, which interacts with transmembrane fragments M9-M10 of the ␣-subunit (396).
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In the extracellular M7M8 loop of the ␣-subunit, a
sequence of 26 amino acid residues between N894 and
A919 interacts to produce the associated ␣/␤-complexes
(277), yet in insect cells, which lack endogenous subunits,
infection with baculovirus particles with only ␤-subunits
results in the expression of ␤, which traffics readily to the
plasma membrane (171).
The expression of ␣- and ␤-subunit isoforms follows
a tissue-specific pattern (291, 296, 402). While genes encoding ␣-subunits have a similarity above 90% (412, 413),
the ones encoding ␤-isoforms only share 39 – 48% sequence identity (291, 400). The ␣3- and ␤1-isoforms are
exclusive for the axon, and ␣2-and ␤2-isoforms are exclusive for the Schwann cell, although axonal contacts regulate ␣2- and ␤2-isoform expressions (244). However, multiple ␣- and ␤-isoforms can be expressed simultaneously
in the same cell type (52, 306, 471), suggesting that they
have distinct functions. Conversely, in heterologous expression systems, all Na⫹-K⫹-ATPase ␤-isoforms can associate with all Na⫹-K⫹-ATPase ␣-isoforms and produce
functionally active ␣/␤-complexes with slightly different
transport and pharmacological properties (109, 229).
Moreover, Na⫹-K⫹-ATPase ␣-subunits can produce stable
complexes with H⫹-K⫹-ATPase ␤-subunit which are, however, partially inactive (131, 209, 213). Then again, the
H⫹-K⫹-ATPase ␣-subunit does not associate with the
Na⫹-K⫹-ATPase ␤1-isoform (189, 466). So far, little is
known about the determinants that govern the specificity
of X-K-ATPase ␣/␤-interactions in cells expressing multiple ␣- and ␤-isoforms.
D. The ␥-Subunit
This subunit is a small membrane protein that associates with Na⫹-K⫹-ATPase in the kidney and potentially
in the placenta and the mammary gland (11, 31, 430). It
belongs to the FXYD family, a group of small (7.5–19 kDa)
single-span membrane proteins, characterized by the
presence of the FXYD motif in the extracellular domain,
and that includes phospholemman, channel-inducing factor (CHIF), and other peptides (430). The ␥-subunit is not
required for catalytic activity (207) but modulates Na⫹
pump function by changing the affinity for ATP, Na⫹,
and/or K⫹ (10, 366, 446, 447). It is synthesized from a gene
with two different promoter regions that provide differential expression of the two splice variants known (264,
430). The ␥-subunit or other proteins of the FXYD family,
namely, phospholemman, CHIF, PLMS (a dogfish shark
FXYD family member), and FXYD7, interact with Na⫹-K⫹ATPase and/or modulate its activity, modifying the transport capacity of epithelia in kidney, shark rectal gland,
and choroid plexus, as well as the function of other
tissues in the central nervous system (31, 32, 108, 146, 289,
445). The ␥-subunit and homologous proteins may potentially increase the variety of the Na⫹ pumps.
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ATPase ␤-subunits (357). Second, based on results of cell
fractionation (356) and on its state of glycosylation (357),
␤m appears to be concentrated in the sarcoplasmic reticulum; in contrast, X-K-ATPase ␤-subunits are predominantly found at the plasma membrane. Recent studies
reveal that ␤m is a resident of the endoplasmic reticulum,
which does not act as a chaperone for the maturation of
any of the known X-K-ATPase ␤-subunits, thus representing a protein functionally distinct from other members of
the ␤-subunit family (108).
The ␤2-isoform is strongly expressed in the brain and
moderately in the spleen (181). It was identified as an
adhesion molecule on glia (AMOG) mediating adhesion
between neurons and astrocytes (8, 9). Sequence analysis
of AMOG identified it as a homolog of the ␤1-subunit
(180). The dual function of the ␤2-subunit in cell recognition and ion transport has been hypothesized to couple
cell recognition with regulation of the ionic milieu (180).
In a recent work Okamura et al. (339) analyzed a
complete list of the P-type ATPase genes in Caenorhabditis elegans and Drosophila melanogaster. The branching points and the inferred evolutionary rates of the invertebrate ␤-subunits suggest that D. melanogaster possesses two distinct types of ␤-subunits, one type (␤1, ␤2,
and ␤3) closer to the vertebrate ␤-subunits and the other
type (␤4, ␤5, and ␤6) sharing more homology with C.
elegans ␤-subunits. The tissue-specific expression patterns of two of the Drosophila melanogaster genes for the
␤-subunit have been studied (Nervana 1 and 2; Nrv).
Nrv1 produces a single ␤-subunit isoform expressed primarily in muscle tissue, whereas Nrv2 codes for two
different isoforms (2.1 and 2.2) expressed in the nervous
system. The tissue-specific expression of each Nrv gene is
independently regulated by the cis-elements present in
the 5⬘-flanking region. The Nrv2 5⬘-flanking DNA directs
expression exclusively to the nervous system, whereas
Nrv1 5⬘-flanking DNA directs expression primarily in
muscle tissue (490).
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
E. Assembly and Delivery of the Naⴙ-Kⴙ-ATPase
F. The Triggering Effect of Ca2ⴙ
1. Extracellular events
Ca2⫹ acts primarily on the extracellular side (97, 100,
101, 186) as indicated by 1) an extracellular Ca2⫹ concentration of 0.1 mM, which suffices to trigger junction formation and polarization, does not increase the level of
cytosolic Ca2⫹ (14 ⫾ 8 nM) that was reduced as a consequence of the preincubation without this ion (20 ⫾ 8 nM);
2) La3⫹ blocks Ca2⫹ penetration, but because of its low
affinity for the extracellular repeats of E-cadherin (see
below), does not prevent this ion from triggering from the
outside, either junction formation or polarization; 3) Cd2⫹
instead blocks both Ca2⫹ influx and the development of
the transporting phenotype (100). The observation that
extracellular Ca2⫹ is sufficient to trigger polarization and
junction formation does not exclude that the subsequent
penetration of the ion into the cytoplasm would produce
a series of additional phenomena (327). Likewise, E-cadherin appears to have a large number of cellular roles
besides that of promoting and maintaining TJs (495).
Physiol Rev • VOL
2. Intracellular events
Once Ca2⫹ binds to the extracellular repeats of Ecadherin, a signal is transduced via two different G proteins, a protein kinase C (PKC), a phospholipase C (PLC),
calmodulin, and mitogen-activated protein kinase (MAPK),
triggering the development of the epithelial transporting
phenotype (20, 21, 79, 184). The assembly and sealing of
TJs occurs so quickly that a fraction of the Na⫹-K⫹ATPase is trapped on the apical side but is thereafter
removed from this location (96). The polarized insertion
of Na⫹-K⫹-ATPase triggered by Ca2⫹ can be blocked with
inhibitors of protein synthesis, yet the proteins whose
synthesis is required are neither the ␣- nor the ␤-subunits
of Na⫹-K⫹-ATPase (96).
G. The Polarized Distribution of Naⴙ-Kⴙ-ATPase
The Na⫹-K⫹-ATPase of epithelial cells serves two
different but integrated roles. The first is the translocation
of ions across the plasma membrane as in other cell types
(417, 418). The second stems from its expression in only
one pole of the cells, in such a way as to carry the
translocation of Na⫹ across the whole epithelium as proposed by Koeffoed-Johnson and Ussing (252). In turn, a
combination between the polarized distribution of Na⫹K⫹-ATPase, specific ionic permeabilities, and the polarized expression of co- and countertransporters drives the
net transport of other solutes across the whole epithelium
(111, 112, 405). In keeping with these roles, Na⫹-K⫹ATPase is found to reside on the basolateral surface in
most epithelial cells (134, 135, 139, 242). In a few other
epithelial cells such as those of the choroid plexus (484),
retinal pigment epithelium (204, 422), and cockroach salivary gland (236), this enzyme is expressed on the opposite side of the cells, but always in a polarized manner.
Koefoed-Johnsen and Ussing (252) conceived their
model on the basis of the macroscopic asymmetries of the
frog skin (electrical potential, currents, and net ion transport) and represented epithelial cells as simple rectangles
where the pump was assumed to be on the inner facing
barrier, that would correspond to the basal side of a more
realistic picture of the cell (Fig. 1). Yet in model systems
of MDCK, either as monolayer (61, 62, 96, 98, 102) or as
cysts (465), Na⫹-K⫹-ATPase is not expressed on the basal
side, but restricted to the lateral plasma membrane facing
the intercellular spaces. For the macroscopic parameters
mentioned above, it is irrelevant whether the pump is
located at the basal or at the lateral side, but as we shall
discuss below, the expression on the lateral side may be
crucial for the polarized distribution of the pump, which
is the basis of the whole asymmetric behavior of epithelial
cells.
Newly synthesized Na⫹-K⫹-ATPase is directly addressed to the basolateral membrane domain in MDCK
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Occluding junctions and apical/basolateral polarity
are lost upon harvesting with trypsin and EDTA, but
regained in a few hours of reseeding at confluence and in
the presence of Ca2⫹. If cells are instead deprived of this
ion and maintained in suspension with a stirrer, the transporting phenotype does not develop. The addition of soluble collagen prompts cells to expose more specific binding sites, but cells still remain unpolarized and fail to form
TJs (395). Ca2⫹ promotes the formation of clumps, in
which suspended cells take each other as substrate, form
TJs and polarize, orienting the apical domain towards the
outer side of the clump (465). Collagen added under this
condition binds to the apical membrane and causes an
inversion of polarity and a relocation of TJs that, by
the way, show the dynamic nature of these structures
(25, 303).
When cells are plated in the presence of Ca2⫹ but this
ion is removed after allowing 20 – 40 min for attachment
to the substrate, the relatively small fraction of Na⫹-K⫹ATPase and ion channels expressed at the plasma membrane cells is not polarized, and these molecules, as well
as TJ-associated molecules, remain trapped in intracellular membrane vesicles (96, 286, 361, 395). Ca2⫹ added at
this time triggers polarization and junction formation in a
few hours, a maneuver called “Ca2⫹ switch” (184). However, the presence of this ion cannot trigger by itself the
development of the transporting epithelial phenotype, as
cell-cell contacts are also required (317, 411, 439).
1235
1236
CEREIJIDO, CONTRERAS, AND SHOSHANI
1. Role of the cytoskeleton
The activity and polarized distribution of renal Na⫹K -ATPase appears to depend on a connection of ankyrin
to the spectrin-based membrane cytoskeleton, as well as
on association with actin filaments. Na⫹-K⫹-ATPase not
only copurifies with ankyrin, spectrin, and actin but also
with three further peripheral membrane proteins: pasin 1
and pasin 2 (258) and moesin (259). A specific binding site
for ankyrin has been localized on the ␣-subunit (120).
Interestingly, the Drosophila Na⫹-K⫹-ATPase ␣-subunit
conserves an ankyrin binding site (498) and codistributes
with ankyrin and spectrin in polarized fly cells (26, 27,
128, 129). However, its polarized distribution was not
altered in spectrin-null mutants (274, 275). Furthermore,
Dubreuil et al. (129) using mutations in the Drosophila
⫹
Physiol Rev • VOL
␤-spectrin gene provided the first direct evidence that
␤-spectrin determines the subcellular distribution of the
Na⫹-K⫹-ATPase and that this role is independent of
␣-spectrin.
Unlike most epithelia, the retinal pigment epithelium
(RPE) distributes the Na⫹-K⫹-ATPase to the apical membrane (44, 51, 204, 422). Early studies on rat RPE monolayers (204) suggested that an entire membrane-cytoskeleton complex is assembled with opposite polarity. Recent
studies have shown that other polarized membrane proteins such as viral envelope membrane proteins do preserve their polarity in RPE cells (43, 44). Furthermore,
Rizzolo and Zhou (377) used the RPE of chicken embryos
and observed that the Na⫹-K⫹-ATPase and ␣-spectrin segregate into different regions of the cell. Despite its segregation from ␣-spectrin, the Na⫹-K⫹-ATPase appears to
associate with a macromolecular complex in microvilli,
suggesting that these properties prevent the Na⫹-K⫹ATPase from complexing with the ␣-spectrin-based cytoskeleton by sequestering the enzyme into the compartment where its activity is required. Discussion of the
polarized distribution of Na⫹-K⫹-ATPase will be pursued
below.
IV. TIGHT JUNCTIONS
Epithelia separate biological compartments with different composition, a fundamental role that depends on
the establishment of occluding junctions. It is generally
assumed that while TJs play this role in vertebrates, septate junctions (SJs) play it in invertebrates. Yet, as discussed below, SJs are transiently present in certain organs of vertebrates during development and permanently
expressed in their nervous system.
TJs were so belittled that more than a century after
they were first described, they were not even represented
in the model of Koefoed-Johnsen and Ussing (252). The
fact that preparations of “tight epithelia”, with a transepithelial electrical resistance (TER) in the order of thousands of ohms per square centimeter, gradually decrease
this parameter after several hours of being mounted between two chambers, led one to assume that epithelia like
the intestinal mucosa or the gall bladder, whose TER only
amounts to ⬍100 ⍀ · cm2 from the outset, did not withstand either dissection or harsh in vitro conditions, yet
the observation that despite their low TER, these epithelia
did transport substances vigorously (see, for instance,
Refs. 47, 121, 122, 123, 373, 374) and many physiologically
important substances flow mainly through a paracellular
route limited by the TJ, led to a reassessment of the
biological role of the thereafter called “leaky epithelia.”
But even then, given its poor ability to discriminate between diverse permeating species, compared with the
plasma membrane, it was expected that the TJ would
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cells (54, 188, 189). This targeting seems to be determined
by the impossibility of this enzyme to board the glycosphingolipid (GSL)-rich rafts that assemble in the Golgi
complex and form vesicles that carry proteins towards
the apical domain. This exclusion may be overcome by
endowing the Na⫹-K⫹-ATPase with a sequence signal
from the fourth transmembrane segment of the ␣-subunit
of H⫹-K⫹-ATPase (TM4 signal) that suffices to readdress
the Na⫹-K⫹-ATPase towards the apical domain. However,
there are nongastric H⫹-K⫹-ATPases that, in spite of lacking the TM4 signal, are nevertheless addressed towards
the apical domain. On the basis of their work with chimeras, Dunbar and Caplan (130) convincingly suggest
that these differences in the polarized expression of
ATPases can be explained as follows: 1) gastric H⫹-K⫹ATPase has the TM4 apical addressing signal already
formed; 2) nongastric H⫹-K⫹-ATPases lack the TM4 signal, but would be able to form it through a particular
arrangement of the molecule that would join two separated segments and thereby complete the signal sequence;
3) also Na⫹-K⫹-ATPase lacks a TM4 but, at variance with
nongastric H⫹-K⫹-ATPases, it is unable to form one by
reconfigurating the ␣-subunit in space; only the insertion
of a TM4 signal by molecular engineering achieves its
apical expression (130, 131).
Based on studies of cultured MDCK cells, three models have been proposed for the development and polarized expression of Na⫹-K⫹-ATPase. One of these models
involves intracellular sorting of newly synthesized proteins at the Golgi apparatus, followed by a vectorial
delivery of the Na⫹-K⫹-ATPase molecules to a distinct
surface domain (54, 378, 499). An alternative model
emphasizes random delivery of newly synthesized Na⫹K⫹-ATPase molecules to the entire plasma membrane,
but selective retention of this protein at specific sites of
the plasma membrane by attachment to the submembrane cytoskeleton (206). A third posibility discussed
below attributes an important role to the ␤-subunit.
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
1237
consist of a couple of rather inconspicuous molecules
placed in neighboring cells and bridged by a mere Ca2⫹
salt link (57).
The scope has since drastically changed, due to a
series of observations: 1) TER ranges from 10 (e.g., proximal kidney tubule) to ⬎10,000 ⍀ · cm2 (e.g., urinary
bladder), indicating that TJs can adjust the degree of
tightness to physiological needs (91, 92). 2) The TJ has
been found to consist of a cluster of protein species
(ZO-1, ZO-2, ZO-3, cingulin, occludin, claudins, etc.; see
Fig. 3A and Table 1) that stretches from its membrane lips
to the cytoskeleton (425, 451). 3) Some of these proteins
contain nuclear addressing and nuclear export signals
(76, 187, 224). 4) TJ molecules change their degree of
phosphorylation in response to physiological conditions
and pharmacological challenge (21, 86). 5) In a given
moment, TJs may relax their tightness to allow the passage of macrophages toward an infected site, or spermatozoa in their travel from the Sertoli cell to the lumen of
the seminiferous tube (49, 81, 351). 6) There is an increasing number of autoimmune diseases associated with
faulty TJs that allow the passage of peptides from the
intestinal flora. The immune system develops antibodies
Physiol Rev • VOL
that not only attack the microbial antigen, but neurons,
␤-cells from the pancreas, the thyroid gland, and other
targets of the host, whose proteins have sufficient homology with the intruder molecule (58). 7) Some TJ-associated molecules contain consensual segments with tumor
suppressor proteins (182, 483, 488). 8) TJs act as a barrier
that prevents lipids in the apical and the basolateral membrane from mixing (126, 127, 460, 461). 9) Lipids are
themselves part of the structure of the TJ (50, 334).
Physiologists have traditionally described cell functions such as the action potential, exchange diffusion, and
cotransport decades before the specific molecules that
performed these roles could be found. The picture is now
totally reversed, as we know of dozens of proteins (e.g.,
the ones mentioned in the section above) whose function
is mostly unknown. Some mutations in claudin genes
severely impair the TJs (e.g., deletion of claudin-16/paracellin suppresses the ability of kidney nephrons to reabsorb Mg2⫹ and Ca2⫹) (414), and mutations that result in
the absence of claudin 14 are responsible for autosomal
recessive deafness (39, 474). Knockouts of claudins 1, 5,
and 11 produce severe damage of the epidermal, bloodbrain, and myelin barriers, causing dehydration in the
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FIG. 3. Effect of ouabain on nucleus and adhesion complexes (NACos). The diverse types of cell junctions have
specific molecular components that, whenever the degree of attachment with the neighboring cell is weakened below a
certain grip, abandon the scaffold and travel to the nucleus where they switch on or off genes that have to do with
proliferation, differentiation, etc. A: essential components of cell-cell junctions. The membrane-attaching molecule is
represented in pale purple, and highly glycosylated (green). Proteins that shuttle from cell junctions to the nucleus
(NACos) are represented in yellow. Cytoskeletal linkers are in pink and other signaling proteins are in blue. B: monolayer
of MDCK cells under control condition, showing the distribution of ␤-catenin (green) mainly on the lateral borders. C:
in monolayers treated with ouabain, ␤-catenin (green) shuttles to the nucleus (blue). D: same monolayer as in B, showing
the typical distribution of ZO-1 (red). E: ouabain does not provoke shuttling of ZO-1 toward the nucleus. ZO-1 and
␤-catenin in ouabain-treated monolayers are endocytosed, and the chicken fence pattern starts to segment. B and D (as
well as C and E) correspond to the same area and same optical slice taken at the middle of the nucleus. (From C.
Flores-Maldonado and I. Larre, unpublished results.)
1238
TABLE
CEREIJIDO, CONTRERAS, AND SHOSHANI
1.
Functional classification of molecules in the different types of cell junctions
Junction
Transmembrane
Protein
Nectin
E-cadherin
Tight
Claudins
JAMs
Occludin
Desmosomes
Desmoglein
Desmocollin
␣-Integrin
␤-Integrin
Focal
Signaling Proteins
ARVCF
␤-Catenin
␣-Catenin
AJUBA
ZONAB
CDK4
Symplekin
huASH1
ZO-2
SRC family
Plakoglobin
PKC
Paxilin
Calreticulin
FHL-2
Hic-5
PINCH
CDK6
Zyxin
SRC family
ILK
FAK
PKA
Heterotrimeric G proteins
PKC
Rho-family GTPases
Cytoplasmic Plaque Proteins*
AF6/AFADIN
Vinculin
␤-Catenin
␣-Catenin
Cingulin (ZO-2, ZO-3, AF-6, and JAM)
PAR-3 (JAMs, PAR-6, atypical PKC)
PAR-6 (PAR-3)
PATJ (Pals-1, ZO-3, claudin)
PALS-1 (CRB1, PATJ)
ZO-1, 2, 3 (occludin, claudin)
Desmoplakin
␣-Actinin
Vinculin
Talin
Filamin
Transmembrane proteins are those that interact with homotypic or heterotypic proteins of adjacent cell or extracellular matrix. NACos are
proteins that can localize to the nucleus and adhesion complexes. * Some of the specific proteins that interact with the plaque molecules are listed
in parentheses. ARVCF, armadillo repeat gene deleted in VCFS (Velo-cardio-facial syndrome) thought to contribute to the morphoregulatory
function of the cadherin-catenin complex (294); NECTIN, a Ca2⫹-independent immunoglobulin-like cell-cell adhesion molecule at AJs associated
with E-cadherin through their respective peripheral membrane proteins, afadin, and catenins (432); AF7/AFADIN, a peripheral membrane protein
that connects nectin to the actin cytoskeleton (432); AJUBA, a cytosolic protein found at sites of cell adhesion and a member of the zyxin family
of LIM proteins (293); CRB1, a human ortholog of Drosophila Crumbs, a transmembrane protein that plays an important role in epithelial cell
polarity (384); zyxin and paxillin, the prototypes of two related subfamilies of LIM domain proteins that are localized primarily at focal adhesion
plaques (468); CDK4 and CDK6, cyclin-dependent kinases that function as nuclear regulators of the cell cycle (19); PINCH, a five LIM domain and
an integrin-link kinase (ILK) binding protein involved in the regulation of integrin-mediated cell adhesion (463); Hic-5, a paxillin-related focal contact
protein that upregulates transcription of c-fos gene and is a coactivator of steroid receptor-activated transcription (494); calreticulin, ␣-integrininteracting protein with nuclear function, originally identified as a chaperone in the endoplasmic reticulum (116); FHL-2, an integrin-interacting
LIM-domain protein which modulates the transcriptional activity of nuclear ␤-catenin (473); FAK, focal adhesion kinase triggers signaling cascades
important for the regulation of cell proliferation and survival (216); filamin and talin, actin binding proteins which form mechanical links to the
cytoskeleton and are involved in the organization of actin networks (493); plakoglobin, known also as ␥-catenin is a major component of the
submembrane plaque of desmosomes where it binds to desmoglein; like its close homolog ␤-catenin, it can bind also to E-cadherin and can interact
with LEF/TCF transcription factors (278); desmoplakin, a member of the plakin family at desmosomes that connects cytoskeletal elements to each
other and to the junctional complex, plays a crucial role in orchestrating cellular development and maintaining tissue integrity (278).
mouse (161), selective alteration of transport of molecules of 800 Da or less in endothelia (329), and slow nerve
conduction (191, 319), respectively.
It can be argued that, although there is ample evidence that TJs restrict diffusion through the paracellular
permeation route, they might act as our lips, which enable
our mouth to hold water, yet are not sewn together. Data
supporting its role as an anchoring structure are somewhat scarce. The possibility exists that homologous interactions between transmembrane proteins located in
neighboring cells (e.g., claudin/claudin) would stabilize
them in this position but might not constitute a strength
element holding cells together.
A. Structure
Proteins of the TJ have been divided according to
several criteria, depending on whether they cross the
membrane and pop up on the extracellular side, associate
specifically with the cytoplasmic side of the TJ, have PDZ
Physiol Rev • VOL
segments that bind them to other protein species forming
stable scaffolds, anchor this structure to the cytoskeleton,
belong to the TJ only transiently, for instance, during
junction assembly and sealing, or because they are proteins like the NACos (see sect. IVF) that shuttle between
the TJ and the nucleus where they act as transcription
factors (20, 63, 76, 182, 301, 451).
B. Transmembrane Proteins
Occludin and claudins belong to the tetraspanin superfamily (256), which has four membrane-spanning domains, two extracellular loops, and the NH2 and COOH
termini in the cytoplasm (159, 162). The extracellular
loops of occludin are characteristically rich in glycine and
tyrosine residues (300). The COOH-terminal domain binds
a number of TJ plaque proteins including ZO-1 (142, 163,
226), ZO-2 (225, 477), ZO-3 (208), and cingulin (105). The
NH2-terminal and the first extracellular domain modulate
transepithelial migration of neutrophils (110, 219). Occlu-
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Adherens
NACos
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
C. Membrane-Associated Proteins
The cytoplasmic domain of TJ membrane proteins
binds to a complex cluster of intracellular proteins like ZO-1,
ZO-2, ZO-3, and Pals, which belong to the membrane-associated guanylate kinase homolog family (MAGUK) (141, 187,
239, 384, 483). Stevenson et al. (425) identified the first
known TJ protein, ZO-1 (zonula occludens) (see also Ref. 5),
that is a prominent associated peptide, judging from the
number of associations and changes in phosphorylation it
goes through. ZO proteins contain a series of domains, such
as PDZ, which enable these peptides to interact with other
ZO, occludins, claudins, and with the actin cytoskeleton
(141–143, 226, 477, 478). MAGI (MAGUK inverted protein)
colocalizes with ZO-1 (124). MAGI-1b localizes to the basolateral membrane and forms complexes with ␤-catenin and
E-cadherin during junction formation (222) and in pallidoluysian atrophy (191). Notice that although ␤-catenin and
E-cadherin are the key components of adherens junctions,
they influence the TJ (see below). Tumorigenic proteins of
adenovirus and papilomavirus target for degradation, or sequester MAGI-1 (179). MAGI-1 appears to be a tumor suppressor (179) that interacts at the TJ with the signaling
molecule GEF, a guanine nucleotide exchange factor participating in the Rho signaling pathway (313). MAGI-2 and -3
interact with PTEN, an inositol trisphosphate phosphatase
Physiol Rev • VOL
(488, 489), and MAGI-2 binds megalin (multi-protein receptor mediating endocytosis in kidney proximal tubules; Ref.
345). PARs are partitioning-defective proteins involved in
embryonic polarity. PAR-3 localizes at the TJ through association to the COOH terminus of JAM (227) and forms a
complex with PAR-6, a single PDZ-possessing molecule with
a CRIB domain (232), and atypical PKCs ␭ and ␨ (PAR-3/
ASIP, atypical PKC isotype-specific interacting protein)
(228). Par-6 inhibits TJ reassembly after junctional disruption induced by Ca2⫹ depletion (169). MUPP1 (multi-PDZ
domain protein 1) functions as a cross-linker between claudin-based TJ strands and JAM oligomers in TJs (205) and
retains MAGUK protein Pals1 through its MAGUK recruitment domain (MRE) (384).
AF-6 is transiently expressed at tight (491) and adherens junctions (292), suggesting a role in the formation of
junctions (182). It is also an ALL-1 fusion partner at chromosome 6 associated with acute leukemia (363). PATJ is
another junction-associated protein, which contains 10 PDZ
domains that bind it to ZO-3 and that can also be found at the
apical plasma membrane (383, 384). Cingulin is a molecule
with globular domains and a central ␣-helical rod region
necessary for dimerization (88). Through several domains
spread along the molecule, specially the ZIM (ZO-1 interacting motif), cingulin cross-links with ZO-2, ZO-3, AF-6, and
JAM to the F-actin and myosin cytoskeleton (28, 105, 106,
114, 115). ZONAB is a transcription factor that regulates
ErbB-2 and paracellular permeability (19, 22). Other TJassociated proteins are symplekin, which is related to the
polyadenylation of mRNA (431), the transcription factors
HuASH1 and AP-1 (40, 325), MAGI-3, an atypical PKC (13,
179, 228, 427), JEAP, which appears to anchor other TJassociated protein species (328), 7H6 (247), as well as Pilt
(243). Another group of TJ-associated proteins is involved in
vesicle traffic: Rab13 (298, 409, 497), Rab3b (426), and Sec6/8
complex (198). Finally, TJs also contain proteins associated
with G proteins like Gi-0␣, Gi-2␣, G12␣, Gs␣ (119, 125, 389,
390).
D. Lipids
TJs appear on freeze-fracture replicas as continuous
anastomosing strands. These strands are thought to be composed of either protein (420) or lipids (237, 358). The “protein model” depicts the strands as polymers of protein molecules in the plane of the plasma membrane that join,
through their external domains, to the corresponding polymer of the neighboring cell. The preponderant participation
of proteins is strongly supported by their content of transmembrane proteins such as occludin (162) and claudins
(159). Transfected claudins even promote formation of
strands resembling those of epithelial TJ in fibroblasts (164,
262). The “lipid model” in turn depicts the strands as cylindrical micelles with the polar groups of the lipids directed
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din contributes to the structure and sealing of the TJ (158,
265, 304, 309, 458, 480). Although the roles of occludins in
TJ assembly and functions are clearly demonstrated, null
mutant mice expressed well-developed TJs (391–393).
Claudins are the major transmembrane proteins of
tight junctions (159, 164). The mammalian claudin family
comprises 25 members (182, 218, 451) that are the main
constituents of the characteristic strands of TJs observed
in freeze-fracture replicas. Claudin by itself is able to form
these strands and recruit endogenous ZO-1 when expressed in fibroblasts (164, 319, 320). The COOH terminus
is intracellular and has a domain that binds the PDZ
proteins ZO-1, ZO-2, ZO-3 (225), PATJ (383), and MUPP-1
(205) (see below). Different claudins copolymerize along
the same TJ strand and bind certain types of claudins of
the neighboring cell (165). The type of claudin expressed
is responsible for some specific functions of the TJ in a
very strict spatial-temporal fashion (93, 94, 255, 457). Thus
epithelial cells of the proximal tubule and MDCK have a
characteristic low TER phenotype due to the expression
of claudin-2 (160, 250, 375).
Junction adhesion molecule (JAM) is a protein of
epithelial and endothelial cells with a single transmembrane domain. This protein is important for neutrophil
migration through the endothelial cell layer (295). Its
intracellular domain binds AF-6 (132), ASIP/Par 3 (133,
227), ZO-1 (28, 132), and cingulin (28).
1239
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CEREIJIDO, CONTRERAS, AND SHOSHANI
E. Diversity Arising From Processing TJ
Proteins as Well as by the Expression
of Several Isoforms
ZO-1 has three splicing variables (17, 187). ZO-1-␣ⴙ
has an 80-amino acid domain, called the ␣-motif, inserted
in the COOH-terminal half (17, 476). This variant is associated with the development of TJs and is abundant in
epithelia (17, 263, 455). ZO-␣ⴚ lacks the ␣-motif, is abundant in epithelia that express very dynamic TJs, like Sertoli cells, endothelia (17), and even in cells like podocytes
that do not express TJs (263).
Occludin is produced by a single gene (human chromosome band 5q13.1, Ref. 391) and is modified during
Physiol Rev • VOL
posttranslation. Two alternative splicing isoforms are
known: occludin 1B, a longer variant that has a 56-amino
acids insertion in the NH2 terminus (324), and occludin
T4M(⫺), a short variant that lacks the fourth transmembrane domain and causes the COOH terminal to be exposed to the extracellular space (177).
The ZO-2 gene employs two alternative promoters
that give rise to two ZO-2 isoforms differing at their
amino-terminal portion by 23 amino acids. Although both
isoforms are present in normal tissues, the longer one is
absent in most pancreatic cancers (82– 84).
F. Proteins That Can Localize to the Nucleus
and Adhesion Complexes
Some junction-associated proteins contain nuclear
address and nuclear export signals (86, 187, 224) that
enable them to shuttle back and forth between the TJs as
well as from other types of adhesion complexes, to the
nucleus (Table 1; Fig. 3). Upon arriving at the nucleus,
proteins that localize to the nucleus and adhesion complexes (NACos) activate transcription, modify mRNA processing and trafficking, activate c-jun kinase, alter the
G1/S phase transition of cell cycling, modulate histone
methyltransferase, proliferation, cell density, oxidative
stress, as well as interact with other molecules within the
nucleus and submembrane scaffolds determine cell fate
during embryonic development and are involved in tumorigenesis (23). In other words, the diverse cell junctions have specific molecular components that whenever
the degree of attachment is weakened below a certain
grip, they abandon the scaffold and travel to the nucleus
where they switch on or off genes that have to do with
proliferation.
G. Phosphorylation in TJ Assembly and Function
As mentioned above, both the assembly (20) and
disassembly of the TJ (85) involve important phosphorylation steps. ZO-1 and ZO-2 become phosphorylated on
Ser, Thr, and Tyr residues. Cingulin is phosphorylated on
Ser residues, and occludin has several levels of phosphorylation on Ser and Thr that are required for its incorporation into the TJ (86, 394). Cingulin is phosphorylated in
serine residues by a kinase insensitive to PKC activators
or inhibitors (87). Likewise, the assembly of TJs depends
on vinculin phosphorylation on Ser and Thr (352). ZO-1 in
epithelia with low TER is more Ser-phosphorylated than
in high-TER epithelia (424). Hypoxia in brain microvessels enhances phosphorylation, decreases expression,
and delocalizes ZO-1 (149). However, low phosphorylation of ZO-1 has been detected in cells that lack TJs or
have ZO-1 disassembled through calcium depletion (215).
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toward the axis, and the hydrophobic tails immersed in the
lipid matrix of the plasma membrane of both neighboring
cells (237). It is in keeping with the fact that the lipid-soluble
probe dipicrylamine can be transferred with the aid of an
applied voltage from the cell membrane of a previously
loaded cell to the membrane of a neighboring one (453). It is
also supported by the recovery of large bleached areas with
lipids diffusing through the TJ (192) and by the cytochemical
localization of phospholipids with gold complexed phospholipase A2 (240). However, several studies have failed to
support or discard this model (126, 127, 326, 401, 419, 460,
461). Lipidic messengers, like diacylglycerol, play a role in
TJ formation (20, 21). Lipid phosphatase PTEN is also associated with the TJ and participates in tumor suppression
(488, 489).
The possibility exists that strands would not be made
exclusively of proteins or lipids, or that lipids would
participate indirectly as a source of a second messenger
that may in turn regulate TJs. Recent evidence supports
both possibilities. Thus changing the total composition of
phospholipids, sphingolipids, cholesterol and the content
of fatty acids does not alter either TER or the structure of
the strands, but enrichment with linoleic acid increases
the paracellular flux of dextran without detectable modifications of either TER or TJs (50). Cholesterol depletion
elicited by treating MDCK cells with methyl ␤-cyclodextrin transiently increases TER (156, 287). MDCK I and
Fisher rat thyroid cells spontaneously express high TER
in normal culture conditions, yet when their synthesis of
glycosphingolipid is inhibited with 1-phenyl-2-hexadecanoylamino-3-morphilino-1-propanol-HCL (PPMP), TER
markedly decreases without a noticeable structural
changes in the TJs (279). Another experimental evidence
pointing to lipid involvement in the TJ is that hyperphosphorylated occludin and ZO-1 are found in lipidic rafts.
These rafts are detergent-insoluble fractions, enriched
with glycolipid, cholesterol, and proteins like caveolin-1.
These TJ proteins coprecipitate and partially colocalize
with caveolin-1 and are displaced from the “raftlike” compartment after TJ disassembly by calcium chelation (334).
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
integrity, possibly by anchoring to the cytoskeleton (151,
153). Occludin and ZO-1␣⫹ are assembled in the last stage
(32 cells) (408, 410). In mouse and human, the TJ mRNAs
of claudin-1, JAM, occludin TM4⫹ and TM4, and ZO-1␣⫺
are initially inherited from maternal transcription and
followed by embryonic transcription from the two-cell
stage onward, and remain present throughout preimplantation development (154, 155, 176). Only ZO-1␣⫹, ZO-2,
and desmocolin-2, a component of desmosomes, are transcribed later on from the embryonic genome, during 16- to
32-cell stage in human and mouse (176, 408).
Development of TJs in Xenopus embryos is quite
different from that of mouse, human, or bovine, due to the
pressure imposed on Xenopus to develop rapidly swimming tadpoles, which are able to escape predators. The
Xenopus egg has a very impermeable membrane, due to
removal of transport proteins like the Na⫹-K⫹-ATPase
through endocytosis (147, 151, 398). Xenopus embryos
develop a polarized distribution of cadherins XB/U after
the first cleavage, in the two-cell stage, coincidentally
with the nascent blastocoele (6, 238). Sealed TJs develop
at this stage, although in basal position and without constituting a fence for basolateral proteins (147, 310). TJs
are initially assembled in the membrane at the animal
pole, by the end of zygote cytokinesis. Cingulin is the first
TJ protein to be incorporated, followed by ZO-1, then
occludin. Interestingly, formation of the new apical membrane and the generation of cell polarity in Xenopus precede initiation of TJ formation, and TJ proteins assemble
at the apical-basolateral boundary in the complete absence of intercellular contacts, indicating that the mechanism of TJ biogenesis relies on intrinsic autonomous
cues (147).
V. SEPTATE JUNCTIONS
H. Biogenesis
Biogenesis of the TJs is closely related to development. In compactation, blastocyst formation, gastrulation, and neurulation, epithelia separate compartments of
distinct composition. The formation of primordial epithelia results from maternal (e.g., in Xenopus) or embryonic
(e.g., in mice) expression programs (151, 153). The first
stage of TJs formation starts 1–2 h after compactation,
with the assembly of plaque proteins ZO-1␣ⴚ and rab13 at
the eight-cell stage (150, 409). This process depends on
E-cadherin, protein synthesis, and assembly of microtubules. In a second stage, cingulin plaque protein is incorporated at the 16-cell stage and stabilized in a process
dependent on E-cadherin-mediated adhesion (152, 230).
E-cadherin seems to be necessary not only for spatial
organization of the TJs, but also for maintaining their
Physiol Rev • VOL
The first description of SJs in Hydra was made by
Wood (482). While TJs are characteristic of chordates,
nonchordates have instead SJs (269). SJs can be found in
the blood-brain and blood-testis barriers of adult arthropods and even in the Ranvier nodes of vertebrates. Green
and Bergquist (194) found that sponge junctions are difficult to locate due to their transient nature but have
extremely regular intercellular spacing even in the absence of any septa. In cross-sectional views of the different types of SJ, there is little variation, as virtually all
septa span a 15- to 18-nm intercellular space. Coelenterata
have clear SJs. Mollusca and Arthropoda possess the
well-known mollusk-arthropod pleated septate junctions
(420). Tunicata do not have SJs, but rather a variation of
the vertebrate TJ (175, 285). Some insects have TJs in the
central nervous systems and rectal pads (266 –268, 271).
Furthermore, Toshimori et al. (448) described a type of TJ
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Vascular endothelial growth factor increments Tyr phosphorylation and paracellular permeability (7). Epidermal
growth factor induces Tyr phosphorylation of ZO-1 and
relocalizes this protein towards the apical membrane of
A431 cells (459), and tumor necrosis factor increases TJ
permeability through Ser and Thr phosphorylation of occludin (89, 90). During TJ assembly ZO-1 is phosphorylated in tyrosine residues in a MAPK regulated fashion
(79). Phorbol esters disassemble TJs and downregulate
claudin-2. ZO-1 associates and is a substrate of ZAK, a
Ser/Thr kinase (18) and of PKC (13). ZO-2 is Tyr phosphorylated when epithelial cells are transfected with v-src
(433), phosphorylated in Ser and Thr residues by cAMPdependent protein kinase (PKA) and atypical PKC when
TJs are either absent or disassembled by Ca2⫹ removal
(13). Occludin phosphorylation in Ser and Thr residues is
required to recruit occludin to the TJ (394), whereas
phosphorylation in Tyr is involved in disassembly and
opening of TJs (118).
Although we still have no dynamic model to explain
the interplay of Ca2⫹ levels, small GTP-binding proteins,
phosphorylation, and TJ-associated molecules, there is
ample evidence that this cascade involves the cytoskeleton that is linked to E-cadherin through p130, ␣-, ␤-, and
␥-catenins, vinculin, ␣-actinin, fodrin, and spectrin (496).
In fact, actin also combines with ZO-1, ZO-2, and ZO-3,
occludin, cingulin, and other TJ molecules. The cytoskeleton is likely to constitute a structural framework as well
as an informative network conveying signals from the
adherens junctions to the TJ and back. Drugs interfering
with microfilaments and microtubules block junction formation (311, 312) and polarization during a Ca2⫹ switch,
and reverse these processes in fully polarized cells with
already established TJs (496).
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CEREIJIDO, CONTRERAS, AND SHOSHANI
in the cyst envelope of the silkworm testis that does not
form extensive anastomosing arrays. These structures
might not be true TJs (48, 193, 331). However, Lane and
Chandler (270) clearly demonstrated the existence of
what appear to be true TJs in the central nervous system
of arachnids.
VI. ADHERENS JUNCTIONS
A. Structure and Function
Physiol Rev • VOL
B. Relationships between Adherens
and Occluding Junctions
Both types of junctions maintain a fundamental, but
a highly complex relationship. TJs depend on the E-cadherin/E-cadherin interaction throughout the life of the
epithelium, as TJs in mature monolayers can be opened
by interfering with E-cadherin. E-cadherin does not localize in the TJ but triggers the cascade of chemical events
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The adherens junction is a large multiprotein cluster
that plays crucial roles in tissue organization and patterning. It mediates homotypic cell-cell adhesion and the
transduction of extracellular signals. E-cadherin, a classical Ca2⫹-dependent homophilic cell adhesion molecule
(434), is characterized by five extracellular structural repeats (EC1 to EC5) and a highly conserved cytoplasmic
domain that associates with several cytoplasmic proteins,
most prominently the catenins (344), which associate in
turn to actin cytoskeleton (203, 251, 470, 495). E-cadherin
is a well-known polarity-inducing protein that is able to
polarize Na⫹-K⫹-ATPase in fibroblasts (307). In polarized
MDCK cells, E-cadherin is localized to the lateral membrane (34). Newly synthesized E-cadherin is preferentially
sorted to the lateral membrane (273) and binds strongly to
the ␤-catenin (212), a protein that is necessary for efficient delivery of the complex to the plasma membrane
(80). Basolateral delivery of E-cadherin relays upon a
double leucine motif present in the intracellular domain
(314). Ca2⫹ interacts with the extracellular part of Ecadherin (100, 186), more specifically with a motif of three
amino acids (364). Ca2⫹ binds to the junctions between
extracellular repeats of E-cadherin, changing its conformation (333, 407), and allowing it to aggregate with other
E-cadherin molecules on the same cell, an arrangement
that favors binding to the E-cadherin of neighboring cells
(495). When adherent junctions are blocked with specific
antibodies against the extracellular domain of E-cadherin,
TJs do not form and, if already established, they reopen
(103, 199, 200, 201). When the amino acid triplet His-AlaVal (HAV) from the extracellular domain of E-cadherin is
blocked with specific peptides containing an HAV sequence, the functions of the E-cadherin/catenin complex
are interfered (330). However, HAV cannot act by itself, as
flanking sequences are important in the binding selectivity of HAV peptides to E-cadherin (290, 372). Peptides
from the bulge and groove regions of the EC1 domain of
E-cadherin can inhibit cadherin-cadherin interactions, resulting in the opening of the cellular junctions (416).
The initial contact is restricted to small regions called
“puncta” (3, 462). Vasioukhin et al. (462) demonstrated
that Ca2⫹ stimulates the formation of very dynamic filopodia that penetrate and embed into neighboring cells.
The initial AJ is extended in a process similar to a “zipper”
closure.
This chain of reactions is interrupted by the failure in
␣-catenin (470) and ␤-catenin, a critical component of the
Wnt signaling cascade (305, 349). When wnt extracellular
signals instruct cells to proliferate, dephosphorylated
␤-catenin is targeted to the nucleus and associates with
the transcription factor LEF/TCF (37). In nonproliferating
cells, phosphorylated ␤-catenin is removed from cell contacts and, once in the cytoplasmic pool, is bound to the
axin and adenomatous polyposis coli protein complex
(APC), where it is ubiquitinated and targeted to the proteasome for degradation (1, 190). Hence, the regulation of
adhesion seems to be exerted, at least in part, by the
titration of ␤-catenin from the cell contacts by APC. APC
also binds to microtubules and participates in the formation of parallel tubulin bundles (316) involved in asymmetric cell division (353, 492).
Epithelial to mesenchymal transition (EMT) is the
loss of cell-cell interactions and acquisition of a migrating
phenotype of cells that changes the adhesive epithelial
phenotype to a fibroblastic-like migrating one (35, 36, 38,
423). It requires loosening of the E-cadherin/catenin-dependent strong cell-cell adhesion of epithelial tissues
(202, 220, 435), as well as silencing the expression of
E-cadherin and TJs proteins (53, 223). Cano et al. (53) and
Pérez-Moreno et al. (354) have identified the E-cadherin
transcriptional repressors Snail and the mouse E12/E47
that are members of the zinc finger family and product of
the E2A gene, respectively. Both interact with the Ecadherin palindromic element E-pal, present in E-cadherin promoter, and silence its expression. In addition to
its effect on the E-cadherin, Snail binds directly to the
E-boxes of the promoters of the claudin/occludin genes,
resulting in complete repression of their promoter activity
(223). In epithelial cells EMT can be triggered by the
binding of the hepatocyte growth factor/scattering factor
(HGF/SF) to its receptor, c-met, through the activation of
the ras and MAPK signaling pathway (350). HGF/SF induces the ubiquitination of E-cadherin by Hakai, which is
a member of the ubiquitination machinery, that induces
endocytosis and dynamic recycling or degradation of Ecadherin, and the subsequent perturbation of cell-cell
adhesions (157).
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
VII. POLARITY
The life of a cell depends on thousands of different
chemical reactions controlled by a myriad of enzyme
species. Life would be impossible if these molecules had
access to each other. Whenever a tissue is homogenized,
temperature should be lowered below 4°C, and an abundant batteries of inhibitors should be added beforehand.
Furthermore, each protein species should be targeted
from the site of synthesis to the place in which it works
(the Golgi apparatus, the cytoskeleton, peroxisomes,
etc.), and degraded if found out of place (for a recent
review, see Ref. 59). Therefore, all proteins should be
assigned a specific place in the cell. In a wide sense,
polarity is the consequence of targeting and selectively
retaining different protein species to different sides of the
cell, but the term is often restricted to the asymmetric
expression of cellular features, such as microvilli, flagella,
receptors, ion channels, pumps, and co- and countertransporters on a given pole of the cell, that constitute the
structural basis of the asymmetric behavior of epithelia
referred to in section I.
Until a couple of decades ago, it was taken for
granted that polarity was a peculiar property of epithelial
cells and neurons. Today it is the other way around: all
cells from single ones like bacteria, yeast, and spermatozoa to hepatocytes and osteoblasts have some degree of
polarization. Were it not for membrane targeting and
polarity, the monomers of a receptor synthesized and sent
at random to the plasma membrane would have to ramble
and diffuse on the plane until they meet by chance the
other partners. Polarity may therefore reveal a principle
of economy that speeds up the assembly of membrane
structures. The fact that polarity is so old and widely
distributed is reflected in the constancy of its fundamental
mechanisms and by the fact that molecules and mechanisms underlying polarity, such as E-cadherin and vinculin or genes such as PAR 3 and PAR 6, can be found to
play similar roles from yeasts to Caenorhabditis, Drosophila, and humans (for recent reviews, see Refs. 59,
479). However, it is important to stress that there was a
FIG. 4. The tight junction is a promiscuous structure, yet it depends on adherens junctions which are
not. Epifluorescence image of a monolayer formed by a
mixed population of MDCK and LLC-PK1 cells. LLCPK1 cells were labeled beforehand with 6.3 ␮M
CMTMR.
Physiol Rev • VOL
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resulting in TJ formation from afar (20). Nevertheless,
Troxell et al. (450) found typical junctional strands and
some TJs markers, such as ZO-1 and occludin, in cells
whose expression of endogenous E-cadherin had been
severely reduced. It is likely that E-cadherin would be
also needed to hold the two neighboring cells together, so
TJs can be formed. Heterotypic TJs can be established
between cells from different organs of the same animal,
and even from different animal species (185). This is
illustrated in Figure 4, where a monolayer of cocultured
MDCK (dog, unstained) and LLC-PK1 cells (pig, red) that
exhibits homophilic (MDCK/MDCK and LLC-PK1/LLCPK1) as well as heterophilic TJs (MDCK/LLC-PK1) revealed by the continuous distribution of occludin and
ZO-1 molecules (green), and by the development of a TER
(not shown). E-cadherin instead is only observed at
homologous MDCK/MDCK borders, as indicated by
DECMA-1 antibody, a result that agrees with the observation that the cell-cell contact provided by E-cadherin is a
highly specific one (495). The antibody used has no specificity for E-cadherin in LLC-PK1 cells but binds to Ecadherin of MDCK cells in homotypic borders. Taken
together, these results indicate that E-cadherin triggers
and supports TJs at the junction between different types
of epithelia cells, like the ones commonly occurring in the
nephron and the gastrointestinal tract (103).
The life of higher organisms would be impossible
without epithelial barriers that separate the internal from
the environmental milieu. Therefore, it is conceivable that
the most fundamental and evolutionary conserved role of
the TJs is to act as a diffusion barrier and that finer
regulation of TJ permeability, as well as their participation in the variety of functions mentioned in section I,
would constitute a later development (for a recent review,
see Refs. 23, 301). In this respect, it is interesting that
Furuse et al. (165) deleted the COOH-terminal cytoplasmic domain of claudins and observed that these lose their
ability to associate with ZO-1, ZO-2, and ZO-3. Nevertheless, these TJs still exhibit the characteristic meshwork of
fibrils in freeze-fracture replicas.
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CEREIJIDO, CONTRERAS, AND SHOSHANI
very long evolution before polarity became triggered by
contact clues and, besides of serving the own necessities
of the cell, started to participate in the assembly of multicellular organisms (472).
C. Naⴙ-Kⴙ-ATPase, Cell Attachment, and the
Position of the TJ
A. The Transporting Epithelial Phenotype
B. Polarity and the TJ
Clear-cut demonstrations that TJs are not responsible for the polarized distribution of membrane proteins
stem from the study of receptors for immunoglobins,
ferritin, and cholesterol-binding-protein and from the observations already mentioned above that upon switching
confluent MDCK cells to Ca2⫹, TJs form so rapidly that a
fraction of Na⫹-K⫹-ATPase is trapped in the apical
(wrong) side, but is thereafter removed and the cell attains the normal polarization of this enzyme in the next
couple of hours (96). However, as discussed below, TJs
may be in fact responsible for the polarized transport of
fluid.
A TJ itself is polarized, as some of its molecules are
targeted very precisely to and from the TJ to other places
such as the basolateral membrane (165, 224, 299, 394).
Actually, the experimental dissection between TJs and
polarity is hard to achieve, as interference with evolutionarily conserved signaling cascades starting at membrane
contacts affect both TJs and polarity. Proteins, such as
PAR3, PAR4, and PAR6, identified originally in the nematode Caenorhabditis elegans (246) have homologs in
Drosophila and mammals (281, 337, 427, 443, 444). Furthermore, Baas et al. (16) have recently identified a human homolog of PAR4, called LKB1, whose activation
triggers the development of the whole transporting epithelial phenotype even in single, noncontacting cells. ZO-1
is a member of the same protein family as Drosophila
Discs-large tumor suppressor (452, 476). The situation is
in fact much more complex, because different TJ-associated signal-transduction systems might influence each
other (for a recent review, see Ref. 301).
Nevertheless, the TJ is important to preserve the
polarization of mobile membrane molecules such as lipids
(126). But even this role does not seem to be that of a
The complex mechanisms described above are the
product of a long evolution and might not have been
present in primeval metazoans of pre-Cambrian times,
more than 600 millions years ago. The first metazoans
exchanging through epithelia must have had much simpler mechanisms. One of the simplest occurring in an
epithelium would be a membrane molecule with the capacity to attach to a similar one in the neighboring cell, so
the pair would get stuck at the contacting border (Fig. 5).
The Na⫹-K⫹-ATPase might constitute a case in point.
Axelsen and Palmgren (15) propose that the third subunit
of bacterial K⫹-ATPase (KdpC) may be the ancestor of the
X-K-ATPase ␤-subunit, due to the similar assembly functions they both perform. As discussed above, in higher
species the ␤-subunit is tightly and specifically bound to
the ␣-subunit (42), and the association begins as soon as
they are synthesized in the endoplasmic reticulum (55) so
that both reach the plasma membrane forming already a
hard-to-dissociate dimer, in which ␤ helps ␣ to trap K⫹ in
a pocket during the pumping cycle (464). However, it is
not expected that they be firmly bound in early organisms
Caenorhabditis elegans because sequence analyses show
no association domains (338). Therefore, it is conceivable
that the ␣-subunit became a stable resident of the lateral
space of transporting epithelia by virtue of being bound to
a ␤-subunit, which in turn has the ability to dwell in this
position because of a linkage it makes with another ␤-subunit placed in the plasma membrane of a neighboring cell
(Fig. 5).
However, a Na⫹-K⫹-ATPase located in a primitive
epithelium as depicted in Figure 5 would be inefficient, as
roughly one-half of the pumping fluid would leak back to
the outer environment. Combinations of Na⫹-K⫹-ATPase
at the cell-cell border with TJs would probably select
epithelia in which TJs would be pushed toward the outermost end of the intercellular space, because it would
ensure a maximal yield of fluid transported vectorially
towards the inside (Fig. 5).
VIII. EVOLUTION OF THE EPITHELIAL
VECTORIALLY TRANSPORTING
PHENOTYPE
Evidence that mollusks can absorb ions from dilute
media was already presented by August Krogh (261) (see
Ref. 122). Water, ions, lipids, amino acids, and sugars
were found to be transported by similar mechanisms in
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The model put forward by Koefoed-Johnsen and
Ussing (252, 456) had three basic asymmetries: 1) only the
outward-facing membrane (apical) has a specific permeability to Na⫹; 2) only the inner facing membrane (basolateral) has a K⫹ specific permeability; and 3) Na⫹-K⫹ATPase is asymmetrically situated on the inner facing
membrane of the cell. In the following four decades the
model of Koefoed-Johnsen and Ussing has been amply
confirmed and complemented with important new findings summarized below.
Physiol Rev • VOL
simple fence. Thus transfection of occludin with its
COOH terminus truncated impairs the ability of the TJ to
retain a fluorescent lipid probe in the apical domain (24).
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
1245
the intestine in Ascaris, earthworms, freshwater clams,
insects, as well as in the mammalian intestine (4, 107, 221,
235, 365).
Evolutionists refer to homology when two organs
have the same role and evolutionary origin, but reserve
analogy (an anatomical term) and homoplasy (its molecular counterpart) for organs that, in spite of playing the
same physiological role, result from different phylogenetic processes. Classical examples are the wings of the
butterfly and those of the bat. In this respect, the relationship between different occluding junctions is still debatable. Thus they play the role of occluding junctions (148,
308, 420) and share considerable homology between some
of their molecules, such as claudins, ZO-1, and dlg (12,
485). Nevertheless, these types of junctions do show considerable differences in composition as well as in their
evolution (301, 444).
The next step in evolution is constituted by Cnidaria, whose most conspicuous representative is the hydra, a freshwater coelenterate first described by Abraham
Tembley in 1744 (as cited by Gierer and Meinhardt, Ref.
Physiol Rev • VOL
178). It reproduces through stem cells, and although its
cells are not known to have apical/basolateral polarity,
the organism does show regional differentiation, attributed to gradients of substances secreted by some of its
cells (178). Fei et al. (144) have found that Hydra vulgaris
has a homolog of ZO-1, termed HZO-1, that is a MAGUK
protein, a family with members such as ZO-2, dlg-A, and
TamA, which are involved in a wide variety of cellular
functions such as TJ formation, cell proliferation, differentiation, and synapse formation.
A. Evolution of the Naⴙ-Kⴙ-ATPase
Thanks to the increasing number of organisms whose
whole genome has been sequenced, Okamura et al. (338)
could correlate the molecular evolution of the 11 subfamilies of the P-type ATPases within the establishment of the
kingdoms of living things. Interestingly, heavy metal
transporters (type 1B) and intracellular Ca2⫹-ATPases
(type 2A) are probably the most fundamental for life,
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FIG. 5. Evolution of transepithelial transport. Hypothetical molecular (A) and cellular (B) events that may have
intervened in the generation of a primeval transporting epithelium. Each yellow block represents an epithelial cell, single
in A and paired in B. A: the ␣-subunit of Na⫹-K⫹-ATPase is represented as a membrane protein with several transmembrane domains (yellow), and the ␤-subunit has the characteristics of a cell-attaching molecule: a short cytoplasmic
domain (blue), a single transmembrane domain (light blue), and a long extracellular fragment (purple). Both subunits
associate (top row, middle) and are sorted to the plasma membrane (top row, right). B, left: the complex of ␣- plus
␤-subunits becomes stabilized at the lateral border of the cell, because it is the only position in the plasma membrane
where the ␤-subunits of the two neighboring cell can interact. Middle: Na⫹-K⫹-ATPases pump ions into the intercellular
space, the osmolarity of the ions attract an amount of water, and the fluid pushes intercellular material (gray) towards
the outermost end of the intercellular space (TJ). In principle, the pressure exerted by the fluid may push the attaching
material towards both ends of the intercellular space. However, those that push it towards the outermost end would be
favored by selection, because in this configuration the intercellular space is open towards the inner side of the body and
allows a net absorption of nutrients. Cell at the bottom right shows a hypothetical later step, in which the incorporation
of co- and countertransporters into a given pole of the cell completes the machinery for the vectorial movement of
substances across the epithelium. To avoid crowding, E-cadherin, whose paramount role is discussed in the text, is not
represented in this drawing.
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CEREIJIDO, CONTRERAS, AND SHOSHANI
B. Evolution of Junction Proteins
When Dictyostelium cells run out of nutrients, they
polarize and adhere to form a multicellular structure that
supports the spore head (260). During this process, the
cells at the top of the stalk tube form a constriction with
Physiol Rev • VOL
rings of cells that express AJs with the ␤-catenin analogous aardvark associated to the actin cytoskeleton. Aardvark is also independently required for cell signaling (197)
and shares significant homology with Saccharomyces cereviseae protein Vac8, Saccharomyces pombe SPBC354,
and plant Arabidopsis thaliana AB016888 sequences.
This demonstrates that in spite of lacking stable junctions, protozoa appear to have molecules that coordinate
cytoskeletal dynamics, the position of the mitotic spindle
and cell polarity, and that may have been precursors of
molecules that make AJs and TJs in metazoans (197, 353).
␤-Catenin that, as discussed above, has a dual role in
cell-cell adhesion and cell signaling, is present in the
nonmetazoan amoeba Dictyostelium discoideum, indicating that it evolved before the origins of metazoan
(197). It seems plausible that ␤-catenin may have been a
requisite for all multicellular development (197). In this
sense, Dictyostelium has ␤-catenin that plays signaling
and adhesion roles as in vertebrate. C. elegans instead
diverged, because it has two catenins: HMP-2 for adhesion
and Bar-1 for signaling to the nucleus (257).
Apical junctions in Caenorhabditis elegans have tetraspan VAB-9, which is a claudin-like membrane protein,
that colocalizes with E-cadherin (HMR-1), and this localization depends on HMR-1, ␣-catenin (HMP-1), and
␤-catenin (HMP-2) (415).
Hua et al. (218) analyzed the phylogenetic trees of
connexins, claudins, and occludins and found no sequence or motif similarity between the different families
studied, indicating that, if they did evolve from a common
ancestral gene, they have diverged considerably to fulfill
separate and novel functions.
It has been recently demonstrated that Sinuous, a
homolog of claudin in Drosophila, not only localizes to
the septate junctions, but also confers to this structure its
barrier function (33, 487). Kollmar et al. (256) only find a
single claudin gene in the urochordate Halocynthia roretzi and concluded that claudins emerged when TJs
replaced septate junctions. However, Asano et al. (12)
found that C. elegans has no less than four different forms
of claudins participating in the epithelial barrier.
Cadherins form a superfamily with at least six distinct subfamilies: classical or type-I cadherins, atypical or
type II cadherins, desmocollins, desmogleins, protocadherins, and Flamingo cadherins. In addition, several cadherins clearly occupy isolated positions in the cadherin
superfamily (454). Nollet et al. (332) suggest a different
evolutionary origin of the protocadherin and Flamingo
cadherin genes versus the genes encoding desmogleins,
desmocollins, classical cadherins, and atypical cadherins.
In contrast to classic cadherins, which bind catenins by
the cytoplasmic domains and have genes with as much as
12 introns, nonclassic cadherins do not interact with
catenins and genes have many fewer introns (485). Phylogenetic analyses suggest that there are four paralogous
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since these two types are found in every kingdom. They
appeared very early in evolution and played critical roles
in ion homeostasis. On the other hand, the ancestors of
animals probably found the Na⫹-K⫹-ATPase and H⫹-K⫹ATPase (type 2C) and got rid of the proton pump (type
3A) occurring in the lineage of plants and fungi. While the
divergence of substrate specificity (ion selectivity) occurred early in the evolution of P-type ATPases and has
been conserved ever since (15), the divergence of the
P-type ATPase isoforms occurred after vertebrates and
invertebrates separated (436). Recently, the study of evolution of P-type ATPases has been amplified with new
information from the database for the nematode Caenorhabditis elegans and the fly Drosophila melanogaster.
Focusing on key domains of both ␣- and ␤-subunits, such
as the ouabain binding site and the ␣/␤-assembly site, a
set of novel isoforms that retain an ancestral characteristic of the Na⫹-K⫹- and H⫹-K⫹-ATPase have been identified (338, 339, 437). In the phylogenetic analysis, the
␤-subunits of C. elegans and D. melanogaster form a
unique single cluster. Interestingly, D. melanogaster possesses two distinct types of ␤-subunits, one type closer to
the vertebrate ␤-subunits and the other sharing more
homology with the C. elegans ␤-subunits. As mentioned
before, the functional expression of the Na⫹-K⫹-ATPase
requires the assembly of the ␣- and ␤-subunits. In this
regard, the newly identified invertebrate subunit isoforms
(Ce2C3 and Ce2C4 and Dm␤4 – 6) are of particular interest because they lack the characteristic domains that have
been demonstrated to be critical for ␣/␤-assembly (140,
277, 467). Therefore, Takeyasu et al. (437) and Okamura
et al. (338, 339) suggested that these nonassembling subunits may exist as lonely subunits and may play not-yetidentified function(s) other than in ATP-driven ion transport. Taken together, their studies suggest that the P-type
ancestor first lacked the inhibitor binding site and the
assembly domain, and therefore existed as a single subunit in lower invertebrate organisms. In its evolution, this
ancestral form acquired the abilities to bind ouabain and
to assemble with ␤-subunit, becoming the immediate
ancestor of the currently known Na⫹-K⫹- and H⫹-K⫹ATPase family. Although the information on the molecular
evolution of the ␤-subunit is comparatively scarce, it has
been suggested that it derives from the third subunit of
bacterial K⫹-ATPase (KdpC). Alternatively, the ␤m protein
of sarcoplasmic reticulum could also represent a primitive form of the ␤-subunit family of the X⫹-K⫹-ATPases.
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
IX. THE DAWN OF METAZOANS
AND TRANSPORTING EPITHELIA
ture (e.g., D. discoideum) and migrate somewhere else.
Likewise, organisms such as Porifera, without real tissues
nor organs, have no internal milieu because the environment can circulate through the body, all cells can exchange directly with the environment, and should not
concern us here. We refer instead to stable metazoans
that survive thanks to an internal milieu whose stable
composition depends on transporting epithelia. Although
the fossil record starts some 560 – 600 million years ago,
there is evidence that they were already existing as early
as 800 –1,000 million years ago (104, 388). Ideas about
metazoan phylogeny are numerous and somewhat conflicting (see Refs. 14, 104, 322, 323, 404, 475). Furthermore, our search for an ancestor of metazoans with true
epithelia may start with a plausible idea of how such an
organism should have been, regardless of whether it is
still surviving or has been long extinct. The elaboration on
a priori possible organisms might at least afford a useful
working hypothesis to solve the conundrum mentioned in
section I on the origins of metazoans and transporting
epithelia.
A. The “Thrifty Sponge”
The corelationship between multicellularity and
transporting epithelia is stronger than the considerable
variety of adaptative structures known to exist in many
organisms. But a mere accumulation of cells might not
qualify as metazoan. Thus, when nutrients are exhausted
or wastes accumulate, cells may temporarily shut off their
living processes, or form a transient multicellular struc-
It is conceivable that in a given moment a group of
cells became surrounded by a primitive epithelium, i.e.,
one whose cells did not exhibit vectorial transport, but
that attach and become an impermeable barrier (Fig. 6,
top). Secreted molecules such as nutrients or cAMP may
thereby be momentarily conserved and used as food stuff
FIG. 6. Hypothetical steps towards a metazoan surrounded by a transporting epithelium. Top panels:
“thrifty sponge.” A: in a given moment, cells are surrounded by a primitive epithelial layer of nonpolarized
cells, and secret a valuable substance (a nutrient? a
signal?). The surrounding cells not only lack polarity, but
do not have a selective permeability either. B: although
cells profit from the valuable substance they secrete, the
internal milieu is exhausted of nutrients and polluted by
wastes. C: as a consequence of intoxication, or because
an increase in osmolarity of the internal milieu bursts the
“thrifty sponge,” the organism is flushed by the outer
environment. D: a conglomerate of cells adopts a very
flat structure and is surrounded by a primitive epithelium
without specific permeability, yet the area-to-volume ratio is enough to ensure a satisfactory exchange with the
environment. Peristaltic movements (arrow) might help
stir the internal milieu. E: “mare nostrum.” A polarized
cell divides. Its descendants stay attached to each other,
generate an internal milieu, and because of the orientation of the mitotic spindle, all descendant cells have the
same polarization. F: perpendicular orientation of the
mitotic spindle provokes the development of an internal
body of cells (pink). Alternatively, this development can
be originated through a process resembling epithelialmesenchymal transition.
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subfamilies (E-, N-, P-, and R-cadherins) of vertebrate
classic cadherin proteins (168) and that these genes duplicated early in evolution (362).
Amphioxus cadherin BbC localizes to adherens junctions in the ectodermal epithelia of embryos and confer
homotypic adhesive properties. It has a cytoplasmic domain whose sequence is highly related to the cytoplasmic
sequences of all known classic cadherins, but its extracellular domain lacks the classical five extracellular cadherin repeats and is similar to the extracellular domain of
nonchordate cadherins (336).
Choanoflagellates, a group of unicellular and colonial
flagellates that resemble cells found only in Metazoa,
express sequences of proteins that encode cadherin repeats. Phylogenetic analyses reveal that choanoflagellate
cadherins are most similar to protocadherins and to the
Flamingo class of cadherins, demonstrating that these
proteins evolved before the origin of animals and were
later co-opted for development (248).
1247
1248
CEREIJIDO, CONTRERAS, AND SHOSHANI
or signals, yet the entrapped milieu would gradually spoil
and force a transient breakage of the epithelium, thus
allowing the external medium to gain access to the cells
and flush the organism. One may even conceive a certain
synchrony between periods of secretion and epithelial
tightness. In this respect, Green and Bergquist (193–195)
have found that in sponges, cell junctions are apparently
formed only when required for a specific purpose.
B. Very Flat Organisms
C. The “Mare Nostrum” Metazoans
Up to this point we have been asking how did primeval metazoans develop their first epithelium. We cannot
discard the opposite situation though. Because polarization is already observed in unicellulars, the possibility
exists that an already polarized cell would proliferate
without completing the separation of its descendants, and
somehow profit from preserving an internal milieu (Fig. 6,
bottom). Furthermore, proliferating epithelial cells usually have their mitotic spindle parallel to the surface of the
epithelium so that proliferation expands the area of the
organ (for a review, see Ref. 353). Yet when the spindle is
perpendicular to the epithelium, it provokes the formation of an outgrowth similar in many respects to the body
mass of a higher organism.
X. Naⴙ-Kⴙ-ATPase AND CELL ADHESION
A. Role of the ␤-Subunit in Cell Attachment
B. (P3 A), a Pump (P) Adhesion (A) Mechanism
Na⫹-K⫹-ATPase arrives to the lateral membrane, interacts with ankyrin, and becomes anchored to the cytoskeleton that stabilizes the enzyme in this position
(206), yet we still ignore why this enzyme binds to the
cytoskeleton at the lateral borders but does not bind to
this structure when expressed at other cell borders. Furthermore, Na⫹-K⫹-ATPase in Drosophila epithelia does
not bind to ankyrin but is nevertheless polarized (26). To
develop a more plausible explanation, we took as hints
that 1) when a monolayer of cultured MDCK cells is
Until a few years ago, it was assumed that, while
synapses exchange valuable signals, contacts between
other cell types were neutral and uneventful, and the role
of cell attachment was reduced to secure the firm and
definitive position of cells in a tissue. Then it was realized
that attachments have to be somehow regulated, as revealed in the following circumstances: 1) when cells divide in the crypt of microvilli and migrate to the apex, and
2) cells of different types mixed in a suspension sort
themselves spontaneously and group into islets of specific
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Another possibility would be a very flat organism
with large surface-to-volume ratio, sufficient to allow exchange with the surroundings (Fig. 6, middle). Although
these putative organisms might not have had circulatory
apparatuses, peristaltic movements might have helped to
stir the internal milieu and favor exchange. Actually, nature seems to have resorted to large surface-to-volume
body plans and ease the survival of primitive metazoans.
Thus Cnidaria, just one evolutionary step above sponges,
have wide and flat structures.
treated with EGTA, the chicken fence image of Na⫹-K⫹ATPase (Fig. 7A) splits into two moieties and each neighboring cell retrieves its own (Fig. 7B), indicating that each
cell contributes the pumps and opening the possibility
that these interact across the intercellular space. 2) In
keeping with such conjunction, MDCK cells express Na⫹K⫹-ATPase at the lateral but not at the basal borders, as
if only at the intercellular space the enzyme would find an
attaching partner on an opposite cell. 3) When cocultured
with Ma104 cells (epithelial from monkey kidney), an
MDCK cell only expresses Na⫹-K⫹-ATPase in a given
border provided its neighbor is another MDCK cell (Fig.
7C), suggesting that the putative Na⫹-K⫹-ATPase/Na⫹-K⫹ATPase interaction is a specific one (Fig. 5). 4) As mentioned above, Gloor et al. (180) have shown that the
␤2-subunit of glial cells acts as a cell adhesion molecule
and has the corresponding structure: a short cytoplasmic
tail, a single transmembrane domain, as well as long and
glycosylated extracellular fragment. MDCK cells have instead the ␤1-isoform, but its structure is almost identical
to that of ␤2. This suggests that the ␤-subunit of Na⫹-K⫹ATPase may establish a cell-cell contact as suggested in
Figure 5. 5) Furthermore, this subunit is required for the
formation of the septate junction of Drosophila (486). 6)
As discussed below, the ␤-subunit of Na⫹-K⫹-ATPase
does participate in cell attachment.
On this basis, we have proposed that the polarized
position of Na⫹-K⫹-ATPase at the lateral borders of epithelial cells depends on the binding ability of its ␤-subunit. The expression of this enzyme at the plasma membrane facing the intercellular space would be the only
place where the ␤-subunits of neighboring cells can interact (77, 78, 98). This view has recently received experimental support from the studies illustrated in Figure 7, D
and E. Briefly, it is observed that MDCK cells only express
their ␤-subunits at the point of contact with another
MDCK cell, but not in contacts with CHO fibroblasts.
However, when CHO cells are transfected beforehand
with ␤-subunit from the dog (i.e., same species as MDCK
cells), MDCK cells now do express their ␤-subunits in
heterotypic MDCK/CHO contacts.
ADHESION AND POLARITY IN THE DAWN OF METAZOANS
1249
cell types. 3) The points where cells attach to a neighbor
or to the substrate are like tips of icebergs sunk into the
cytoplasm, whose molecules form scaffolds, associate
with the cytoskeleton, send and receive specific molecules to the nucleus and other organelles, as well as
exhibit the complex patterns of phosphorylation/dephosphorylation in specific amino acids that we mentioned
above. 4) The nucleus changes the pattern of gene expression in response to the arrival of the NACos (Fig. 3, Table
1). 5) After severing the attachment with its neighbors,
cells may undergo proliferation, differentiation, migration, or apoptosis, in which batteries of genes are turned
on or off. 6) The differentiation of a given cell type
depends on the nature of contacts with its neighbors.
Nowhere is this process more astonishing than in the
self-assembly of a brain, where genes, by obeying just
chemical rules, produce the most complex three-dimensional ordered object known, depending on the species of
attaching molecules coded, and the timing of their expression in axons and dendrites of the neurons. 7) There is a
growing amount of evidence of cancers and autoimmune
diseases associated with failures in molecules involved in
cell contacts (76, 77, 182). On this basis we tentatively
attribute the very emergence of metazoans to special
combinations of attachments between specific cells as
well as between specific molecules.
Physiol Rev • VOL
In keeping with the importance of the processes
mentioned in the previous paragraph, cell-cell attachment
seems to be strictly regulated. Yet the information presently available on this regulation is very scant. We found
a mechanisms termed P3 A, which transduces the occupancy of the Na⫹-K⫹-ATPase (P) by ouabain, into a signaling to molecules involved in cell-cell and cell-substrate
attachment (A), that prompts these to release the grip and
results in detachment of the cell (102). Thus MDCK cells
treated with ouabain increase Tyr phosphorylation and
content of active MAPK, redistribute molecules involved
in cell attachment (occludin, ZO-1, desmoplakin, cytokeratin, ␤-actinin, vinculin, and actin), and detach. Genistein
and UO126, inhibitors of protein tyrosine kinase and
MAPK kinase, respectively, block this detachment. The
content of P190Rho-GAP, a GTPase activating protein of
the Rho small G protein subfamily, is increased by
ouabain, suggesting that both the Rho/Rac and MAPK
pathways may be involved.
The P3 A mechanism may play several potential
roles, in views of the following: 1) damaged epithelial
cells endanger the permeability barrier between higher
organisms and the environment. An injured cell would
conceivably produce less ATP, the activity of the Na⫹-K⫹ATPase would decrease, thus triggering the P3 A mechanism that promotes its own detachment and replace-
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⫹
⫹
FIG. 7. Patterns in the expression of Na -K ATPase in epithelial cells. A: in MDCK cells cultured in
monolayers, Na⫹-K⫹-ATPase is expressed at the lateral
borders, forming a chicken fence pattern. B: cell detachment provoked by 2.0 mM EGTA shows that each
cell carries Na⫹-K⫹-ATPase in its borders, implying
that the image in Figure 7A is given by the enzyme
present in the two neighboring cells. C: a monolayer
prepared with a mixed population of MDCK cells and
Ma104 cells, which were previously stained with the
red dye CMTMR. The use of a mouse antibody specific
for the ␤-subunit of MDCK, followed by a goat antimouse fluoresceinated antibody, indicates that this
subunit only occupies the cellular border of an MDCK
cell, provided the neighboring cell is another MDCK
one (green), but not when the border contacts an
Ma104 cell. D: in mixtures of MDCK with CHO cells
(fibroblasts derived from Chinese hamster ovary),
MDCK cells only express Na⫹-K⫹-ATPases in homotypic (MDCK/MDCK) but not in heterotypic MDCK/
CHO borders (open arrows). However, when CHO cells
are transfected beforehand with dog ␤-subunit, MDCK
cells do express their ␤-subunits in heterotypic contacts (E, green arrows). Bar ⫽ 20 ␮m.
1250
CEREIJIDO, CONTRERAS, AND SHOSHANI
Physiol Rev • VOL
tions of ouabain, but there are a variety of cell-cell contacts, as well as subtler degrees of detachment. Figure 3
illustrates the case of a NACo (␤-catenin) that is addressed to the nucleus by ouabain, and another that is not
(ZO-1). Taken together, this information suggests that by
expressing a variety of ␣-subunits with different ouabain
sensitivities (214, 385, 438), the Na⫹-K⫹-ATPase may be
controlled by the hormone ouabain, and activate a P3 A
mechanism that deeply modifies the structure and function of a given cell.
In summary, we cannot provide an answer to the
question raised in section I about the origin of transporting epithelia and metazoans, but after systematizing the
information obtained from widely heterogeneous fields, it
seems that ancestral unicellulars already possessed most
of the precursor molecules to build up transporting epithelia and form a perhaps transient metazoan that evolution might subject to selective pressures. Attachment,
both at the molecular and the cellular level, seems to have
played a paramount role.
We thank Dr. Catalina Flores-Maldonado and Lic. Isabel
Larré for helpful discussion and furnishing their unpublished
results in Figure 3, B–E. We also acknowledge the efficient and
pleasant help of Elizabeth del Oso.
This work was supported by the National Research Council
of Mexico (CONACYT).
Address for reprint requests and other correspondence: M.
Cereijido, Center For Research and Advanced Studies, Dept. of
Physiology, Biophysics, and Neurosciences, Avenida Instituto
Politécnico Nacional 2508, Código Postal 07360, México D.F.,
Mexico (E-mail: [email protected]).
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