Comparison of the function of the tight junctions of endothelial cells and epithelial cells in regulating the movement of electrolytes and macromolecules across the cell monolayer

In cell culture, both endothelial and epithelial cell monolayers have been found to generate structurally similar tight junctional complexes, as assessed by thin complexes of the two cell types are, at least in part, responsible for the very different permeability characteristics of native endothelial and epithelial cell monolayers. The purpose of this work was to compare cultured endothelial and epithelial cells with respect to the function of their tight junctional complexes in regulating the movement of macromolecules and ions across the cell monolayers, and define functional parameters to characterize the tight junctional complexes. Bovine aorta endothelial cells and T84 colonic carcinoma epithelial cells were cultured on a microporous membrane support. The permeability coefficients of inulin, albumin, and insulin were determined with the cell monolayers and compared with the permeability coefficients obtained with 3T3-C2 fibroblasts, a cell line that does not generate tight junctions. Electrical resistance measurements across the monolayer-filter systems were also compared. The permeability coefficient of albumin across the endothelial cell monolayer compared favorably with other reported values. Likewise, the electrical resistance across the T84 cell monolayer was in good agreement with published values. Utilizing permeability coefficients for macromolecules as an index of tight junction function, we found that a distinction between a lack of tight junctions (fibroblasts), the presence of endothelial tight junctions, and the presence of epithelial tight junctions was readily made. However, when utilizing electrical resistance as an index of tight junction function, identical measurements were obtained with fibroblasts and endothelial cells. This indicates that more than one index of tight junction function is necessary to characterize the junctional complexes. Although structurally similar, epithelial cell and endothelial cell tight junctions perform very different functions, and, from our data, we conclude that the demonstration of tight junctional structures by electron microscopy is not relevant to the functional nature of the junction: structure does not imply function. A minimal assessment of tight junction function should rely on both the determination of the electrical resistance across the cell monolayer, and the determination of the permeability coefficients of selected macromolecules.     

Actin localization at the tight junctions of invertebrate ciliated epithelia

Indirect immunofluorescencc, rhodamine-phalloidin staining and immunoelectron microscopy performed with the on-grid postembedding immunostaining of Lowicryl K4M sections, were used to identify actin in the branchial epithelium of the lower chordate ascidians. The ciliated cells of these invertebrates present two distinct junctional patterns. One consists only of an extended tight junction whereas in the other the tight junction is accompanied by a prominent zonula adhaerens. Evidence is given of the localization of actin at the tight junction. The absence of reaction in the zonula adhaerens suggests that the definition of this junction in the model here presented must be reconsidered.     

The intercellular junctions of guinea-pig placental capillaries: a possible structural basis for endothelial solute permeability

The endothelial cell junction in guinea-pig placental capillaries consists of a continuous ribbon desmosome (zonula adherens) within which lies a particulate tight junction consisting of between one and five anastomosing strands. The intercellular space at these tight junctions is narrowed and is subdivided by junctional bars which are probably continuous with the intramembrane particle rows seen in freeze-fracture replicas of the junctions. Perfusion with lanthanum salts shows the gaps between the junctional bars to be lanthanum-filled and the entire junction to be lanthanum permeable. The estimated size of the spaces between the junctional bars is consistent with the junctional pore size indicated by previous ultrastructural tracer studies. The wider lateral intercellular space of the ribbon desmosome is spanned by more widely spaced "linkers" which may act as a coarser three-dimensional filter in series with size-limiting pores between the tight junctional bars.     

Relationship of sertoli-sertoli tight junctions to ectoplasmic specialization in conventional and en face views

Ectoplasmic specializations are actin filament-endoplasmic reticulum complexes that occur in Sertoli cells at sites of intercellular attachment. At sites between inter-Sertoli cell attachments, near the base of the cells, the sites are also related to tight junctions. We studied the characteristics of ectoplasmic specializations from six species using conventional views in which thin sections were perpendicular to the plane of the membranes, we used rare views in which the sections were in the plane of the membrane (en face views), and we also used the freeze-fracture technique. Tissues postfixed by osmium ferrocyanide showed junctional strands (fusion points between membranes) and actin bundles, actin sheets, or both, which could be visualized simultaneously. En face views demonstrated that the majority of tight junctional strands ran parallel to actin filament bundles. Usually, two tight junctional strands were associated with each actin filament bundle. Parallel tight junctions were occasionally extremely close together ( approximately 12 nm apart). Tight junctional strands were sometimes present without an apparent association with organized actin bundles or they were tangential to actin bundles. En face views showed that gap junctions were commonly observed intercalated with tight junction strands. The results taken together suggest a relationship of organized actin with tight junction complexes. However, the occasional examples of tight junction complexes being not perfectly aligned with actin filament bundles suggest that a precise and rigidly organized actin-tight junction relationship described above is not absolutely mandatory for the presence or maintenance of tight junctions. Species variations in tight junction organization are also presented.     

Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia

A tight junction-enriched membrane fraction has been used as immunogen to generate a monoclonal antiserum specific for this intercellular junction. Hybridomas were screened for their ability to both react on an immunoblot and localize to the junctional complex region on frozen sections of unfixed mouse liver. A stable hybridoma line has been isolated that secretes an antibody (R26.4C) that localizes in thin section images of isolated mouse liver plasma membranes to the points of membrane contact at the tight junction. This antibody recognizes a polypeptide of approximately 225,000 D, detectable in whole liver homogenates as well as in the tight junction-enriched membrane fraction. R26.4C localizes to the junctional complex region of a number of other epithelia, including colon, kidney, and testis, and to arterial endothelium, as assayed by immunofluorescent staining of cryostat sections of whole tissue. This antibody also stains the junctional complex region in confluent monolayers of the Madin-Darby canine kidney epithelial cell line. Immunoblot analysis of Madin-Darby canine kidney cells demonstrates the presence of a polypeptide similar in molecular weight to that detected in liver, suggesting that this protein is potentially a ubiquitous component of all mammalian tight junctions. The 225-kD tight junction-associated polypeptide is termed "ZO-1."     

Tight junction formation is closely linked to the polar redistribution of intramembranous particles in aggregating MDCK epithelia

We have used freeze-fracture electron microscopy to investigate the relationship between the formation of the tight junction in the establishment of a differential distribution of intramembranous particles (IMPs) between the luminal and basolateral membranes of a canine kidney cell line (MDCK). This involves a characterization of the IMP distribution in these membranes in confluent monolayers of MDCK cells, in EGTA-dissociated cells, and in cells at various stages of reassociation. While normal confluent MDCK monolayer cultures exhibit tight junctions and an IMP differential distribution between the luminal and basolateral membranes, cultures dissociated with EGTA lose both formed tight junctional elements and the differential IMP distribution. We have also found that as tight junctions reform between reaggregating MDCK cells, intramembranous particles appear to rapidly redistribute with respect to them. An asymmetric distribution of these particles in the luminal and basolateral membranes is eventually achieved. Tight junction formation appears so closely linked to the genesis of IMP polarity that at early time points when only a string of tight junctional components spans the junctional zone, differential IMP distributions are seen. Thus, our dissociation studies suggest a close relationship between the integrity of the tight junction and the maintenance of IMP polarity between the luminal and basolateral membranes, while cell reassociation studies suggest that the tight junction may be functionally linked to the genesis of IMP polarity.     

Carbon tetrachloride induced proliferation of tight junctions in the rat liver as revealed by freeze-fracturing

Application of carbon tetrachloride produced a progressive proliferation of tight junctions in the rat liver. This system proved to be rapid and highly reproducable and affords the opportunity for tracing the fate of tight junctions in freeze-fracture replicas, facilitating investigations on their formation and function. Beginning on day one carbon tetrachloride treatments resulted in the progressive loosening and fragmentation of the junctional meshwork. After three to four days the membrane outside the zonulae occludentes was extensively filled with proliferated discrete junctional elements often forming complex configurations. From the fifth day on the zonulae occludentes were restricted again predominantly around the bile canaliculus margins. But the junctional meshwork of the zonulae occludentes remained loosened in comparison to those in the control rats. It could be shown that tight junction proliferation on the lateral surface of the plasmalemma occurred both through de novo formation from discrete centers of growth by addition of intramembranous particles and through reorganization of preexistent junctional strands of the fragmented zonulae occludentes bodies. Whereas the large gap junctions close associated with the zonulae occludentes remained more or less unaffected during the experiments, small gap junctions increased in number after five days and were located at the margin or in the tight junction domain. It is assumed that the degeneration of the tight junctions served as a pool for intramembranous particles which form the gap junctions. The results of these observations are discussed in relation to those obtained in other systems.     

Tight junctions in the choroid plexus epithelium. A freeze-fracture study including complementary replicas

The tight junctions of the choroid plexus epithelium of rats were studied by freeze-fracture. In glutaraldehyde-fixed material, the junctions exhibited rows of aligned particles and short bars on P-faces, the E-faces showing grooves bearing relatively many particles. A particulate nature of the junctional strands could be established by using unfixed material. The mean values of junctional strands from the lateral, third, and fourth ventricles of Lewis rats were 7.5 +/- 2.6, 7.4 +/- 2.2, and 7.5 +/- 2.4; and of Sprague-Dawley rats 7.7 +/- 3.4, 7.4 +/- 2.3, and 7.3 +/- 1.6. Examination of complementary replicas (of fixed tissue) showed that discomtinuities are present in the junctional strands: 42.2 +/- 4.6% of the length of measured P-face ridges were discontinuities, and the total amount of complementary particles in E-face grooves constituted 17.8 +/- 4.4% of the total length of the grooves, thus approximately 25% of the junctional strands can be considered to be discontinuous. The average width of the discontinuities, when corrected for complementary particles in E-face grooves, was 7.7 +/- 4.5 nm. In control experiments with a "tighter" tight junction (small intestine), complementary replicas revealed that the junctional fibrils are rather continuous and that the very few particles in E-face grooves mostly filled out discontinuities in the P-face ridges. Approximately 5% of the strands were found to be discontinuous. These data support the notion that the presence of pores in the junctional strands of the choroid plexus epithelium may explain the high transepithelial conductance in a "leaky" epithelium having a high number of junctional strands. However, loss of junctional material during fracturing is also considered as an alternative explanation of the present results.     

Interaction of filipin with junctional membrane at different stages of the junction's life history

The relationship of filipin-sterol complexes to tight and gap junctions during their formation, maturation, internalization, and degradation was studied in separate cell lines. Filipin-sterol complexes tended to be excluded from mature junctions in tight junction forming COLO 316 cells and gap junction forming SW-13 cells. Once internalized, unlabeled junctional membrane appeared to fuse with heavily labeled vesicles, presumably lysosomes. Although the absence of filipin-sterol complexes from junctional membrane does not necessarily reflect the absolute sterol content of this membrane, the fact that filipin-sterol complexes are largely excluded from these areas indicates that this membrane is different from surrounding membrane. The absence of filipin-sterol complexes also permits the visualization of 'mixing' of this specialized unlabeled membrane domain with other filipin labeled membrane systems.     

Freeze-fracture of capillary endothelium in rat brain

Summary The rat brain capillary was studied with freeze-fracture technique. The attached plasmalemmal vesicles were quite few in number on the luminal front and sometimes numerous on the contraluminal side. The fracture appearance of some tight junctions showed interconnecting ridges on face A and complementary furrows devoid of particles on face B, comparable to the common tight junction in the normal epithelia. Other tight junctions revealed a preferential disposition of quasicontinuous rows of particles on shallow furrows of face B, resembling the tight junctional strands of capillary endothelium in non-cerebral tissues. Either behavior is probably due to the difference in the fracture plane around the single fibril. In addition, the tight junctional strand could surround the perimeter of the endothelial cell completely although the exposed strand of tight junction was limited in length.     

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the "blood-testis" barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated protein ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days, ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions, however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.     

Molecular physiology and pathophysiology of tight junctions V. assault of the tight junction by enteric pathogens

Studies of the impact of enteric pathogens and their virulence factors on the proteins comprising the tight junction and zonula adherens offer a novel approach to dissection of tight junctional complex regulation. Most studies to date provide only tantalizing clues that select pathogens may indeed assault the tight junctional complex. Information on critical human pathogens such as Campylobacter jejuni and Shigella and Salmonella subspecies is lacking. Mechanistic studies are currently sparse, but available results on pathogenic Escherichia coli and specific virulence factors such as the Rho-modifying and protease bacterial toxins indicate four major mechanisms by which these pathogens may act: 1) direct cleavage of tight junctional structural proteins; 2) modification of the actin cytoskeleton; 3) activation of cellular signal transduction; and 4) triggering transmigration of polymorphonuclear cells across the epithelial cell barrier. New therapeutics may evolve from detailed studies of these pathogens and the cellular processes and proteins they disrupt.     

Changes in tight junctions of thyroid epithelium with changes in thyroid activity

The morphology of the tight junction of rat thyroid epithelium was examined in freeze-fractured material fixed in glutaraldehyde and briefly glycerinated. In normal thyroids the overall appearance of this junctional specialization resembled that of other cell types in many respects. Short-term changes in thyroid activity and hypophysectomy for 3 wk did not obviously affect the appearance of tight junctions. Feeding of the goitrogen, thiouracil, which stimulates secretion of thyroid-stimulating hormone, resulted in the appearance of some very narrow and some very wide, tight junctions or sometimes junctions with both wide and narrow regions within the same cell.     

Freeze-fracture studies on the subcommissural organ tight junction in gerbils and mice

Freeze-fracture studies on the tight junction of ependymal cells in the gerbil and mouse subcommissural organ (SCO) show an obvious species-specific variation. The tight junctional structure of the mouse SCO is composed of several strands (7.03 +/- 2.09 strands/cell) and occupies a total depth of 0.88 +/- 0.16 micron with a linear density of 7.12 +/- 2.11 strands/micron. The tight junction of the gerbil SCO is composed of three regions: (1) an apical region: made of 4 to 6 strands, oriented parallel to the free surface, with a high linear density (21.78 +/- 3.98 strands/micron) and small depth (0.049 +/- 0.009 micron); (2) a rather smooth and/or empty intermediate region, and (3) a basal region similar in morphology and morphometry to the junctional area of mouse SCO. These data indicate that the main difference in the SCO tight junction between the gerbil and the mouse is the presence of an apical region of high strand density in the former. We speculate that this apical region may play a role in maintaining the homeostasis of this CNS region in gerbils and possibly other desert animals, and may be part of a mechanism for survival in an extremely dry environment.     

Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219- 232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).     

The ultrastructural basis of the permeability of arterial endothelium to horseradish peroxidase

The thoracic aorta and basilar artery, in which the incidence of atherosclerosis is known to be different, were examined to elucidate the correlation between the structure of the intercellular cleft junction between adjacent endothelial cells and its permeability to HRP. Tannic acid or HRP in the vessel lumen passed through the intercellular clefts of the thoracic aorta into the subendothelial space, whereas in the basilar artery they were unable to penetrate beyond the tight junction of the intercellular clefts. Freeze-fracture replicas revealed that the tight junctions of the thoracic aorta consisted of one to two junctional strands in most areas of the cleaved planes, with discontinuities in some places, whereas those of the basilar artery consisted of a continuous belt-like meshwork of six anastomosing junctional strands on average. These observations confirm that the structure of endothelial junctions in arteries has a close correlation with the permeability of the intercellular clefts to HRP.     

Formation of tight and gap junctions in the inner enamel epithelium and preameloblasts in human fetal tooth germs

Human fetal primary tooth germs in the cap stage were fixed with a glutaraldehyde-formaldehyde mixture, and formative processes of tight and gap junctions of the inner enamel epithelium and preameloblasts were examined by means of freeze-fracture replication. Chains of small clusters of particles on the plasma membrane P-face of the inner enamel epithelium and preameloblasts were the initial sign of tight junction formation. After arranging themselves in discontinuous, linear arrays in association with preexisting or forming gap junctions, these particles later began revealing smooth, continuous tight junctional strands on the plasma membrane P-face and corresponding shallow grooves of a similar pattern on the E-face. Although they exhibited evident meshwork structures of various extents at both the proximal and distal ends of cell bodies, they formed no zonulae occludentes. Small assemblies of particles resembling gap junctions were noted at points of cross linkage of tight junctional strands; but large, mature gap junctions no longer continued into the tight junction meshwork structure. Gap junctions first appeared as very small particle clusters on the plasma membrane P-face of the inner enamel epithelium. Later two types of gap junctions were recognized: one consisted of quite densely aggregated particles with occasional particle-free areas, and the other consisted of relatively loosely aggregated particles with particle-free areas and aisles. Gap junction maturation seemed to consist in an increase of particle numbers. Fusion of gap junctions in the forming stage too was recognized. The results of this investigation suggest that, from an early stage in their development, human fetal ameloblasts possess highly differentiated cell-to-cell interrelations.     

Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo

The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.     

Osmotic reversal induces assembly of tight junction strands at the basal pole of toad bladder epithelial cells but does not reverse cell polarity

Summary This paper reports the effect of reversing the osmotic environment between luminal and serosal compartments of a toad urinary bladder on the polarity of assembly of tight junction strands. Toad bladders were filled with Ringer's solution (220 mOsm) and were immersed in distilled water at room temperature or at 37°C. Within two minutes, new tight junction strands are assembled. The new tight junctional strands unite the basal pole of epithelial cells with the apical side of basal cells. Physiological studies show that oxytocin, a synthetic analog of antidiuretic hormone, is still capable of inducing increases in water transport in epithelia which were osmotically reversed. This capacity decreases significantly for longer periods of osmotic reversal. Osmotic reversal does not alter the original polarity of epithelial cells: 1) the apical tight junction belt, at the apical pole, is not displaced; 2) the freeze-fracture morphology typical of apical plasma membrane (particle-rich E faces; particle-poor P faces) is not altered; 3) oxytocin and cyclic AMP induce aggregates which are observed only at the apical plasma membrane. Massive assembly of junctional elements occurs even in epithelia preincubated in the presence of cycloheximide (an inhibitor of protein synthesis) or of cytoskeleton perturbers. Our experiments show that the polarity of assembly of tight junction strands depends on the vectorial orientation of the osmotic environment of the epithelium.     

Regulation of tight junctions and loss of barrier function in pathophysiology

The mechanism by which epithelial and endothelial cells interact to form polarized tissue is of fundamental importance to multicellular organisms. Dysregulation of these barriers occurs in a variety of diseases, destroying the normal cellular environments and leading to organ failure. Increased levels of growth factors are a common characteristic of diseases exhibiting tissue permeability, suggesting that growth factors play a direct role in elevating permeability. Of particular concern for this laboratory, increased expression of vascular endothelial growth factor may enhance vascular permeability in diabetic retinopathy, leading to vision impairment and blindness. However, the mechanism by which growth factors increase permeability is unclear. Polarized cells form strong barriers through the development of tight junctions, which are specialized regions of the junctional complex. Tight junctions are composed of three types of transmembrane proteins, a number of peripheral membrane structural proteins, and are associated with a variety of regulatory proteins. Recent data suggest that growth factor-stimulated alterations in tight junctions contribute to permeability in a variety of disease states. The goal of this review was to elucidate potential mechanisms by which elevated growth factors elicit deregulated paracellular permeability via altered regulation of tight junctions, with particular emphasis on the tight junction proteins occludin and ZO-1, protein kinase C signaling, and endocytosis of junctional proteins. Understanding the molecular mechanisms underlying growth factor-mediated regulation of tight junctions will facilitate the development of novel treatments for diseases such as brain tumors, diabetic retinopathy and other diseases with compromised tight junction barriers.