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Cell Adhesion, Polarity, and Epithelia in the Dawn of Metazoans

Center for Research and Advanced Studies (CINVESTAV), México, Mexico

ABSTRACT

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 {alpha}-Subunit

1. {alpha}-Subunit isoforms

B. The {beta}-Subunit

1. {beta}-Subunit isoforms

C. {alpha}/{beta}-Subunit Interactions

D. The {gamma}-Subunit

E. Assembly and Delivery of the Na+-K+-ATPase

F. The Triggering Effect of Ca2+

1. Extracellular events

2. Intracellular events

G. The Polarized Distribution of Na+-K+-ATPase

1. Role of the cytoskeleton

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 {beta}-Subunit in Cell Attachment

B. (PA), a Pump (P) Adhesion (A) Mechanism

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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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.

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 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 P_i 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.

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 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.

	II. MODEL SYSTEMS OF TRANSPORTING EPITHELIA

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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.

View larger version (44K): [in this window] [in a new window]   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 E-cadherin 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.

View larger version (25K): [in this window] [in a new window]   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).]

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 Na+-K+-ATPase

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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 phosphoryl 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

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- 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).

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 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-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).

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).

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.

E. Assembly and Delivery of the Na⁺-K⁺-ATPase

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).

F. The Triggering Effect of Ca²⁺

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).

2. Intracellular events

Once Ca²⁺ binds to the extracellular repeats of E-cadherin, 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 Na⁺-K⁺-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 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.

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 -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

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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 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 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).

View larger version (73K): [in this window] [in a new window]   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.)

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 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). Occludin 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).

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 (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 TJ-associated 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 TJ-associated 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 G_i-0, G_i-2, G₁₂, G_s (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 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).

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-⁺ 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-^– 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 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). 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 cAMP-dependent 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).

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^– 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 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,<