Tight junctions of human keratinocytes in primary culture: a freeze-fracture study

Human skin explants obtained from 2- to 5-yr-old patients with harelips were cultured in NCTC 168 medium at 37 degrees C, in a humidified atmosphere containing 5% CO2 in air. After a 2-week incubation period, the newly grown cells were studied with special reference to tight junctions by freeze-fracture electron microscopy. Many completely formed tight junctions were observed between the uppermost living cells of migrating epithelium, and fragmented tight junctions were seen between the lower layer cells. The tight junctions in the uppermost cells developed so well that they formed a belt-like network consisting of two to six rows of strands. This observation may suggest that human keratinocytes have the ability to produce tight junctions perfectly enough to serve as a barrier, although no complete tight junctions were formed in situ.     

Diffusion barriers in the vaginal epithelium during the estrous cycle in guinea pigs

Summary In the present study the permeability barriers of the multilayered vaginal epithelium were examined using tracer perfusion techniques, freeze-fracture and thin sectioning. During diestrus and proestrus the upper layers of mucified epithelial cells exhibit tight-junctional belts, which restrict tracer molecules such as lanthanum and horseradish peroxidase. When the highly mucified cells begin to degenerate toward the end of proestrus the underlying epithelium is already keratinized as typical for estrus. The keratinized epithelial cells have a tight-junctional network that joins the basal plasma membranes with the apical membranes of subjacent cells and blocks paracellular diffusion of the tracer molecules. During conversion of the cornified epithelium to a mucified epithelium in metestrus the intercellular space of the epithelium is stained by tracer molecules even though tight-junctional belts can be observed.These results indicate that during cyclic changes of the vaginal epithelium tight junctions can, in general, be considered for the restriction of paracellular diffusion. In metestrus, however, junctions become functionally leaky although they remain morphologically intact.Intercellular lipids, which are normally common in cornified epithelia, are extremely rare and cannot constitute an effective barrier to diffusion in the vagina of the guinea pig. The significance of a strategy that bases the regulation of the permeability on tight junctions rather than on intercellular lipids is discussed.     

Epithelial water transport in a balanced gradient system.

The relationship between epithelial fluid transport, standing osmotic gradients, and standing hydrostatic pressure gradients has been investigated using a perturbation expansion of the governing equations. The assumptions used in the expansion are: (a) the volume of lateral intercellular space per unit volume of epithelium is small; (b) the membrane osmotic permeability is much larger than the solute permeability. We find that the rate of fluid reabsorption is set by the rate of active solute transport across lateral membranes. The fluid that crosses the lateral membranes and enters the intercellular cleft is driven longitudinally by small gradients in hydrostatic pressure. The small hydrostatic pressure in the intercellular space is capable of causing significant transmembrane fluid movement, however, the transmembrane effect is countered by the presence of a small standing osmotic gradient. Longitudinal hydrostatic and osmotic gradients balance such that their combined effect on transmembrane fluid flow is zero, whereas longitudinal flow is driven by the hydrostatic gradient. Because of this balance, standing gradients within intercellular clefts are effectively uncoupled from the rate of fluid reabsorption, which is driven by small, localized osmotic gradients within the cells. Water enters the cells across apical membranes and leaves across the lateral intercellular membranes. Fluid that enters the intercellular clefts can, in principle, exit either the basal end or be secreted from the apical end through tight junctions. Fluid flow through tight junctions is shown to depend on a dimensionless parameter, which scales the resistance to solute flow of the entire cleft relative to that of the junction. Estimates of the value of this parameter suggest that an electrically leaky epithelium may be effectively a tight epithelium in regard to fluid flow.     

Expression and function of tight junctions in the crypt epithelium of human palatine tonsils.

The human palatine tonsils have surface and crypt stratified epithelium and may be initiated via the epithelium to mount immune responses to various presenting antigens. Here we investigated the expression and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 mRNAs were detected in tonsillar hypertrophy. Occludin and claudin-14 were expressed in the uppermost layer of the tonsil surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium. In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers. In freeze-fracture replicas, short fragments of continuous tight junction strands were observed but never formed networks. In the crypt epithelium of recurrent tonsillitis, the tracer was leaked from the surface regions where occludin and claudin-4 disappeared. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, and -14, but not claudin-7, mRNAs were decreased in recurrent tonsillitis compared with those of tonsillar hypertrophy. These studies suggest unique expression of tight junctions in human palatine tonsillar epithelium, and the crypt epithelium may possess an epithelial barrier different from that of the surface epithelium.     

Factors affecting the differentiation of epithelial transport and responsiveness to hormones

Under standard culture conditions, epithelial cells grow with their basal surface attached to the culture dish and their apical surface facing the medium. Morphological and functional markers are located in the appropriate plasma membrane, and transepithelial transport occurs in a variety of cultured epithelia. As a result of the polarity of the cells and the presence of tight junctions between cells, on standard tissue culture dishes there is restricted access of growth medium to the basolateral surface of the epithelium, which is the surface at which nutrient exchange normally occurs. Greater differentiation of epithelial cultures can be achieved by growing primary cultures or continuous cell lines on permeable surfaces such as porous bottom cultures dishes in which the porous bottom is formed by a filter or membrane of collagen, or on floating collagen gels. In many cultures, differentiation varies with the time after the culture was seeded. Certain chemicals that accelerate differentiation in nonepithelial cells also accelerate the differentiation of epithelial cultures. Ultimately, defined media and specific substrates for cell attachment should lead to further differentiation of epithelia in culture.     

Structure and function of intercellular junctions in human cervical and vaginal mucosal epithelia

The mucosal epithelium is a major portal for microbial invasion. Mucosal barrier integrity is maintained by the physical interactions of intercellular junctional molecules on opposing epithelial cells. The epithelial mucosa in the female reproductive tract provides the first line of defense against sexually transmitted pathogenic bacteria and viruses, but little is known concerning the structure and molecular composition of epithelial junctions at this site. In the present study, the distribution of tight, adherens, and desmosomal junctions were imaged in the human endocervix (columnar epithelium) and ectocervix (stratified squamous epithelium) by electron microscopy, and permeability was assessed by tracking the penetration of fluorescent immunoglobulin G (IgG). To further define the molecular structure of the intercellular junctions, select junctional molecules were localized in the endocervical, ectocervical, and vaginal epithelium by fluorescent immunohistology. The columnar epithelial cells of the endocervix were joined by tight junctions that excluded apically applied fluorescent IgG. In contrast, the most apical layers of the ectocervical stratified squamous epithelium did not contain classical cell-cell adhesions and were permeable to IgG. The suprabasal and basal epithelial layers in ectocervical and vaginal tissue contained the most robust adhesions; molecules characteristic of exclusionary junctions were detected three to four cellular layers below the luminal surface and extended to the basement membrane. These data indicate that the uppermost epithelial layers of the ectocervix and vagina constitute a unique microenvironment; their lack of tight junctions and permeability to large-molecular-weight immunological mediators suggest that this region is an important battlefront in host defense against microbial pathogens.     

The fine structure of the parabronchi and the gas exchange area of the Adelie penguin lung

The parabronchi of the Adelie penguin are endowed with wide atria forming pockets between a loose meshwork of bundles of smooth muscle cells lining the parabronchial lumen. The atrial epithelium is of variable thickness and bears numerous microvilli, which are overlain by/or embedded in sheets or whorls of lamellar material ("trilaminar substance", diameter of one lamella 8 ..10 nm) forming layers of very variable thickness. The cells contain either stacks or whorls of this material or roundish lamellated bodies, and are interconnected by desmosomal contacts as well as what presumably represent tight junctions. Underneath the epithelium and within the bundles of muscle cells regularly nerve fibres have been found. The diameter of the morphological air/blood barrier is about 165...210 nm in thin areas, excluding a 12...20 nm thick layer covering the luminal plasma membrane of the air capillary epithelium. The blood capillary endothelium ordinarily is markedly thicker (40...250 nm) than the air capillary epithelium (17...25 nm). The basal lamina between endo- and epithelium is a uniform structure measuring about 95...105 nm. The endothelial cells are interconnected by desmosomal and probably tight junctions.     

Relationship between the saccus endolymphaticus and the fourth ventricle of the brain of the domestic fowl (Gallus gallus f. domestica)

The fine structure of the wall of the SE was determined exactly and its relationship to the cisternae (the evaginations of the roof of the fourth ventricle extending to the SE) was defined. The way in which the cisterna is formed was defined and the development of its fine structure was described by comparing serial sections from 19-day embryos and adult fowls. Like the SE, the cisternae are lodged in the angle between the cerebellum and the medulla oblongata, in the subarachnoid space. The terminal segment of the cisterna lies in the immediate vicinity of the mesenchymal epithelium bordering the basal labyrinth of the SE cells. Collagen trabeculae keep the SE and the cisternae suspended in the subarachnoid space. The cisternae and trabeculae are wrapped in mesenchymal epithelium. The cisterna is avascular and does not communicate with the SE. The cisterna is lined internally with simple squamous epithelium (modified neural epithelium of the roof of the fourth ventricle). The bodies of the cells bulge into the lumen of the cisterna in the region of localization of their nucleus. The epithelium is seated on a pronounced basal lamina. The surface turned towards the subarachnoid space is lined continuously with mesenchymal epithelium without a basal lamina. The cells of the cisternal epithelium are connected by tight junctions of the type of zonulae occludentes and desmosomes. The basal lamina is continuous and distinct. The mesenchymal epithelium of the subarachnoid space has no basal lamina, as on the subarachnoid surface of the SE, the cisternae, the trabeculae, the pia mater and the arachnoidea.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Modulation of cell junctions during differentiation of the chicken otocyst sensory epithelium.

The differentiation of sensory and support cells within the embryonic chick otocyst is accompanied by alterations in the distribution of preexisting intercellular junctions. Prior to innervation of this epithelium, tight, gap and adhering junctions exist between all cells. Upon differentiation of the epithelium, apical bands of tight and adhering junctions are maintained throughout, while gap junctions and desmosomes are found only between support cells. Thus, some of the gap junctions that join homogeneous epithelial cells prior to innervation are removed as sensory cells differentiate, and a separate population of very large gap junctions is formed between differentiating support cells. Morphological evidence suggests two possible mechanisms which may be responsible for the observed changes in gap junctional distribution: removal of gap junctions by internalization, and formation of gap junctions by aggregation of precursor particles. The temporal correlation between junctional modulation, cytological differentiation of sensory and support cells, and ingrowth of nerve fibers makes the latter event a likely developmental cue for differentiation of this epithelium.     

A freeze-fracture and lanthanum tracer study of the complex junction between Sertoli cells of the canine testis

What appear to be true septate junctions by all techniques currently available for the cytological identification of intercellular junctions are part of a complex junction that interconnects the Sertoli cells of the canine testis. In the seminiferous epithelium, septate junctions are located basal to belts of tight junctions. In thin sections, septate junctions appear as double, parallel, transverse connections or septa spanning an approximately 90-A intercellular space between adjacent Sertoli cells. In en face sections of lanthanum-aldehyde-perfused specimens, the septa themselves exclude lanthanum and appear as electron-lucent lines arranged in a series of double, parallel rows on a background of electron-dense lanthanum. In freeze-fracture replicas this vertebrate septate junction appears as double, parallel rows of individual or fused particles which conform to the distribution of the intercellular septa. Septate junctions can be clearly distinguished from tight junctions as tight junctions prevent the movement of lanthanum tracer toward the lumen, appear as single rows of individual or fused particles in interlacing patterns within freeze-fracture replicas, and are seen as areas of close membrane apposition in thin sections. Both the septate junction and the tight junction are associated with specializations of the Sertoli cell cytoplasm. This is the first demonstration in a vertebrate tissue of a true septate junction.     

Adjudin-mediated Sertoli–germ cell junction disassembly affects Sertoli cell barrier function in vitro and in vivo

Adjudin, an analogue of lonidamine, affects adhesion between Sertoli and most germ cells, resulting in reversible infertility in rats, rabbits and dogs. Previous studies have described the apical ectoplasmic specialization, a hybrid-type of Sertoli cell–elongating/elongated spermatid adhesive junction, as a key target of adjudin. In this study, we ask if the function of the blood–testis barrier which is constituted by co-existing tight junctions, desmosome-gap junctions and basal ectoplasmic specializations can be maintained when the seminiferous epithelium is under assault by adjudin. We report herein that administration of a single oral dose of adjudin to adult rats increased the levels of several tight junction and basal ectoplasmic specialization proteins during germ cell loss from the seminiferous epithelium. These findings were corroborated by a functional in vitro experiment when Sertoli cells were cultured on Matrigel?-coated bicameral units in the presence of adjudin and transepithelial electrical resistance was quantified across the epithelium. Indeed, the Sertoli cell permeability barrier was shown to become tighter after adjudin treatment as evidenced by an increase in transepithelial electrical resistance. Equally important, the blood–testis barrier in adjudin-treated rats was shown to be intact 2 weeks post-treatment when its integrity was monitored following vascular administration of inulin-fluorescein isothiocyanate which failed to permeate past the barrier and enter into the adluminal compartment. These results illustrate that a unique mechanism exists to maintain blood–testis barrier integrity at all costs, irrespective of the presence of germ cells in the seminiferous epithelium of the testis.     

The presence of G1 and G2 populations in normal epithelium of rat urinary bladder

The cell population kinetics of transitional epithelium of the rat urinary bladder was analysed by (3H) thymidine autoradiography and pararosanilin Feulgen DNA cytofluorometry. By flash and 72 h continuous DNA labelling, the generative cells of the transitional epithelium were found to be well localized in the basal layer, and it was postulated that che cells produced by cell proliferation in the basal layer would migrate towards the surface, maintaining direct attachment to the basement membrane by anchorage of a cellular process. Analyses of normal and wounded transitional epithelium revealed that 58.8% of all basal cells are G0 cells in G1 phase (G1-population), and 59.0% of the remaining basal cells reside in prolonged (75.1-108.0 h) G2 phase, preserving the ability to divide (G2-population). The cell cycle time of the generative basal cells including the long G2 phase was calculated as 129.1-162 h. All the cells existing in upper layers were found to be also G0 cells in G1 phase, with the DNA amounts of 2C class. No polyploid cells could be detected except for 2C-2C binucleated cells in the superficial layer. The existence of a G2-population may serve for the urgent need of cell incrementation to repair cell loss as the cells in G2 phase can divide without the time-delay needed for DNA synthesis. The rat transitional epithelium, which is composed exclusively of proliferating and potentially proliferative cells, will have much greater capability to repair damage than stratified squamous epithelia.     

THE FINE STRUCTURE OF THE TRANSITIONAL EPITHELIUM OF RAT URETER

The fine structure of the transitional epithelium of rat ureter has been studied in thin sections with the electron microscope, including some stained cytochemically to show nucleoside triphosphatase activity. The epithelium is three to four cells deep with cuboidal or columnar basal cells, intermediate cells, and superficial squamous cells. The basal cells are attached by half desmosomes, or attachment plates, on their basal membranes to a basement membrane which separates the epithelium from the lamina propria. Fine extracellular fibres, ca. 100 A in diameter, are to be found in the connective tissue layer immediately below the basement membrane of this epithelium. The plasma membranes of the basal and intermediate cells and the lateral and basal membranes of the squamous cells are deeply interdigitated, and nucleoside triphosphatase activity is associated with them. All the cells have a dense feltwork of tonofilaments which ramify throughout the cytoplasm. The existence of junctional complexes, comprising a zonula occludens, zonula adhaerens, and macula adhaerens or desmosome, between the lateral borders of the squamous cells is reported. It is suggested that this complex is the major obstacle to the free flow of water from the extracellular spaces into the hypertonic urine. The free luminal surface of the squamous cells and many cytoplasmic vesicles in these cells are bounded by an unusually thick plasma membrane. The three leaflets of this unit membrane are asymmetric, with the outer one about twice as thick as the innermost one. The vesicles and the plasma membrane maintain angular conformations which suggest the membrane to be unusually rigid. No nucleoside triphosphatase activity is associated with this membrane. Arguments are presented to support a suggestion that this thick plasma membrane is the morphological site of a passive permeability barrier to water flow across the cells, and that keratin may be included in the membrane structure. The possible origin of the thick plasma membrane in the Golgi complex is discussed. Bodies with heterogeneous contents, including characteristic hexagonally packed stacks of thick membranes, are described. It is suggested that these are "disposal units" for old or surplus thick membrane. A cell type is described, which forms only 0.1 to 0.5 per cent of the total cell population and contains bundles of tubular fibres or crystallites. Their origin and function are not known.     

Kinetics of cell replication in the uterine cervix. VI. Loci of DNA-synthesizing cells and arrested mitoses in the basal-cell layer

The mode of proliferation in the basal-cell layer of the squamous cervical epithelium was investigated in C57B1 mice with the aid of 3H-thymidine and vincristine. Six hours after vincristine injection and two hours after thymidine injection, 33% of the basal cells were in DNA synthesis and 12% in mitosis. Of these, only 23% of the cells in DNA synthesis and 45% of those in mitosis were found as single cells. The remaining cells proliferated in clusters of two or more cells. As many as 59% of the cells in DNA synthesis and 30% of those in mitosis occurred in colonies of three or more consecutive cells, indicating that multicell clustering is a rather common pattern of basal cell proliferation. Multicell loci of DNA-synthesizing cells occurred contemporaneously with but independently of multicell loci of mitotic cells (the loci were nonconsecutive). Basal-cell replication in the squamous cervical epithelium thus appears to be an organized process of cell renewal.     

Degradation of the radula in the snails Biomphalaria glabrata say and Limnaea stagnalis L. (Gastropoda,Pulmonata)

Summary The radula of snails is formed at the posterior end of the radular gland or pocket, and degraded at the same rate at its anterior end. Degradation is due to different secretory activities of the inferior epithelium of the radular gland. Its secretions seem to degrade enzymatically the matrix of the radular membrane and basal plates of teeth, leaving only chitin containing microfibres and degradation products. The sclerotized parts of the teeth remain unchanged, but as they are now only loosely connected with the radular membrane. they are torn off easily during feeding movements. The rest of the degraded and frayed radular membrane and the subradular membrane are also lost by abrasion during feeding. The cells of the inferior epithelium are connected with each other by septate desmosomes and an elaborate system of deep lateral interdigitation which may provide tensile strength. Extrusion of degraded cells of the inferior epithelium into the subradular membrane takes place, although the thick basal lamina forms a continuous sheath which is closely adjoined to the basal parts of the inferior epithelium. Nerve fibres containing vesicles with electron dense neurosecretory material (deduced from the diameter of 200–250 nm) are attached to this sheath or penetrate into it; they may be involved in the regulation of production and degradation processes during radula replacement. Problems of the forward transport of radula and inferior epithelium are discussed.     

The ability of an epithelium to survive removal of the basal lamina by enzymes: fine structure and content of sodium and potassium of the midgut epithelium of the larva of Tenebrio molitor after withdrawal of the basal lamina by elastase--a short note

A method is described to separate the epithelial cells of the posterior part of the mealworm midgut from their thick basal lamina using elastase. After removal of the basal lamina the naked epithelial cells remain connected with each other, still forming a midgut tube. The ultrastructural changes observed are enlargement of the lateral spaces between the cells and simultaneous destruction of junctional structures other than desmosomes and tight junctions. This enlargement is most probably due to shrinkage of the epithelial cells as a consequence of osmotic stress, which the cells normally seem to be protected against by the basal lamina. The content of sodium and potassium in the epithelium is not influenced by the elastase treatment, indicating that the midgut tube stays alive with intact plasma membranes.     

Caco 2 cell culture as intestinal epithelium model for hexose transport studying

Distribution of SGLT1 and GLUT2 hexose transporters as well as that of fibrillar actin and tight junction proteins in cultured Caco2 cells incubated in medium with different hexose concentrations has been considered. Glucose absorption by the cells from incubation medium has been determined. Fibrillar actin was concentrated in the microvilli and closely to tight junction. The actin distribution was not dependent on the glucose concentration. There was no SGLT1 association with brush border actin and the transporter localization was not dependent on the concentration of hexose. GLUT2 was localized in the basal part of Caco2 cells after low concentration hexose load (2.5 mM). The transporter was colocalized with microvilli actin in the apical part of the cells after high concentration hexose load (25 mM). The tight junction proteins, occludin and claudin 1, 3, 4 were not dependent on glucose concentration. Claudin 2 was not detected in Caco2 cells. Caco2 cell culture can be used as a model for studying of hexose transport in small intestine epithelium.     

Fine structure of the retinal pigment epithelium of the mallard duck (Anas platyrhynchos)

As part of a comparative morphological study, the fine structure of the retinal pigment epithelium (RPE), the choriocapillaris and Bruch's membrane (complexus basalis) has been investigated by light and electron microscopy in the mallard (Anas platyrhynchos). In this species the RPE consists of a single layer of cuboidal cells which display numerous very deep basal (scleral) infoldings and extensive apical (vitreal) processes which enclose photoreceptor outer segments. The RPE cells are joined laterally by prominent basally-located tight junctions. Internally smooth endoplasmic reticulum is the most abundant cell organelle with only small amounts of rough endoplasmic reticulum present. Polysomes are abundant as are basally-located mitochondria which often displayed a ring-shaped profile. The cell nucleus is large and vesicular. Melanosomes are plentiful only within the apical processes of the RPE cells in the light-adapted state. Myeloid bodies are large and numerous and very often have ribosomes on their outer surface. Bruch's membrane (complexus basalis) shows a pentalaminate structure but with only a poorly represented central elastic lamina. Profiles of the choriocapillaris are relatively small and the endothelium of these capillaries while extremely thin facing the retinal epithelium is but minimally fenestrated.