Effects of ovarian hormones on cell membranes in the rat uterus

Freeze-fracture techniques have been used to study tight junctions on the lateral plasma membrane of cells of the luminal epithelium of the rat uterus under various hormonal regimes. Tight junctions from ovariectomized control rats extended some 0.5 μm down the lateral membrane and the junctional strands often formed a network of closely packed, circular compartments. Following treatment of rats with estrogen for 3 days the tight junctional regional still extended 0.5 μm down the lateral membrane, but the strands ran more parallel to the apical surface. They did not enclose circular compartments. After treatment with progesterone, either alone or with estrogen in such a way as to condition the ovariectomized uterus for implantation, a third pattern of junctional organization emerged. In these animals the junctional region extended 1.1 μm down the lateral membrane and the strands frequently crosslinked, enclosing compartments of varying and irregular size and shape. Our observations suggest that ovarian hormones could regulate the contents of the uterine lumen by altering the structure extent of the tight junctions which connect the epithelial cells enclosing the lumen.     

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.     

Tight junction of sinus endothelial cells of the rat spleen

The fine structure of the tight junctions between sinus endothelial cells of the rat spleen and the permeability of such sinus endothelial cells were examined by transmission electron microscopy, using freeze-fracture, triton extraction, and lanthanum-tracer techniques. In freeze-fracture replicas, the segmented strands and grooves of the tight junctions were frequently observed on the basolateral surfaces of the sinus endothelial cells irrespective of the location of the ring fiber. There were one or two wavy-strands or grooves which were, for the most part, oriented parallel to the long cell axis thus forming networks at places. In addition, some strands or grooves were discontinuous while some networks of the junctional strands were not closed. These strands also occasionally lacked intramembranous particles in the tight junctions. The junctional strands run apicobasically at certain sites. In the vertical sections of the sinus endothelial cells treated with lanthanum nitrate, although no tight junctions were observed wherever the endothelial cells were apposed, most of them were situated on the basal part of the lateral surfaces of the adjacent endothelial cells. Several fusions of the junctional membranes were observed in a vertical section of the lateral surfaces of the adjacent endothelial cells. The intercellular spaces of the adjacent endothelial cells except for the fusion of the junctional membranes, were electron dense and the infiltration of lanthanum nitrate was found not to be interrupted by these tight junctions. Based on these observations, the molecular 'fence' and paracellular 'gate' functions of the tight junctions in the sinus endothelial cells are discussed.     

Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical- basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein

Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.     

Adherens and tight junctions: Structure, function and connections to the actin cytoskeleton

Adherens junctions and Tight junctions comprise two modes of cell-cell adhesion that provide different functions. Both junctional complexes are proposed to associate with the actin cytoskeleton, and formation and maturation of cell-cell contacts involves reorganization of the actin cytoskeleton. Adherens junctions initiate cell-cell contacts, and mediate the maturation and maintenance of the contact. Adherens junctions consist of the transmembrane protein E-cadherin, and intracellular components, p120-catenin, β-catenin and α-catenin. Tight junctions regulate the paracellular pathway for the movement of ions and solutes in-between cells. Tight junctions consist of the transmembrane proteins occludin and claudin, and the cytoplasmic scaffolding proteins ZO-1, -2, and -3. This review discusses the binding interactions of the most studied proteins that occur within each of these two junctional complexes and possible modes of regulation of these interactions, and the different mechanisms that connect and regulate interactions with the actin cytoskeleton.     

Adherens and tight junctions: structure, function and connections to the actin cytoskeleton

Adherens junctions and Tight junctions comprise two modes of cell-cell adhesion that provide different functions. Both junctional complexes are proposed to associate with the actin cytoskeleton, and formation and maturation of cell-cell contacts involves reorganization of the actin cytoskeleton. Adherens junctions initiate cell-cell contacts, and mediate the maturation and maintenance of the contact. Adherens junctions consist of the transmembrane protein E-cadherin, and intracellular components, p120-catenin, beta-catenin and alpha-catenin. Tight junctions regulate the paracellular pathway for the movement of ions and solutes in-between cells. Tight junctions consist of the transmembrane proteins occludin and claudin, and the cytoplasmic scaffolding proteins ZO-1, -2, and -3. This review discusses the binding interactions of the most studied proteins that occur within each of these two junctional complexes and possible modes of regulation of these interactions, and the different mechanisms that connect and regulate interactions with the actin cytoskeleton.     

The Sertoli cell junctional complex: structure and permeability to filipin in the neonatal and adult guinea pig

The development and maintenance of the Sertoli cell junctional complex were investigated in prepubertal and adult guinea pigs. To correlate the structure of the blood-testis barrier with its permeability, the polyene antibiotic filipin (a cholesterol-binding agent of low molecular weight: 570.70) was added to the fixative as a tracer visible in freeze-fracture replicas. Discontinuous zonules, intermediate junctions (i.e., adhering fasciae) and gap junctions all proved permeable to filipin in the two age groups. Only the continuous occluding zonules characteristic of the adult guinea pig's testis were impermeable to the tracer. In pubertal animals, the establishment of the blood-testis barrier coincided with the completion of the junctional strands in occluding zonules. The formation of occluding zonules was similar in the newborn and the adult. In the adult, the Sertoli cell junctional complexes contained three types of cell junctions: occluding, adhering, and gap junctions. The sequence of occluding and adhering junctions from the base to the apex of the epithelium was the reverse of that demonstrated in most epithelia. The impermeable continuous occluding zonules at the base showed parallel patterns of uninterrupted junctional strands, whereas the permeable discontinuous zonules found higher in the epithelium showed a meandering pattern of broken strands. Our observations indicate that (1) Sertoli cell junctional complexes form near the young germinal cells at the base of the seminiferous epithelium and break down near the older germinal cells toward the apex; (2) the various patterns and orientations of the junctional strands reflect, respectively, the different stages of disintegration of the occluding zonules and the conformation of the mature Sertoli cell to the irregular contours of the germinal cells; (3) there is no relationship between permeability and junctional strand orientation; and (4) the cellular contacts between Sertoli cells and germinal cells situated below the blood-testis barrier may represent the early stages of formation of junctional elements which ultimately become incorporated into the Sertoli cell junctional complex.     

Structure and permeability of goblet cell tight junctions in rat small intestine

Summary Two major cell types, goblet and absorptive cells, dominate the epithelial lining of small intestinal villi. We used freezefracture replicas of rat ileal mucosa to examine the possibility that tight junction structure, known to relate to transepithelial resistance, might vary with cell type. Tight junctions between absorptive cells were uniform in structure while those associated with villus goblet cells displayed structural variability. In 23% of villus goblet cell tight junctions the strand count was less than 4 and in 30% the depth was less than 200 nm. In contrast, only 4% of absorptive cell tight junctions had less than 4 strands and only 9% had depth measurements less than 200 nm. Other structural features commonly associated with villus goblet cell tight junctions but less commonly with absorptive cell tight junctions were: deficient strand cross-linking, free-ending abluminal strands, and highly fragmented strands. Bothin vivo ileal segments and everted loops were exposed to ionic lanthanum. Dense lanthanum precipitates in tight junctions and paracellular spaces were restricted to a subpopulation of villus goblet cells and were not found between villus absorptive cells. After exposure of prefixed ileal loops to lanthanum for 1 hour, faint precipitates of lanthanum were found in 14% of tight junctions and paracellular spaces between absorptive cells compared to 42% of tight junctions and paracellular spaces adjacent to villus goblet cells. When tested in Ussing chambers, the methods used for lanthanum exposure did not lower transepithelial resistance. Everted loops exposed to ionic barium and examined by light microscopy showed dense barium precipitates in the junctional zone and region of the paracellular space of villus goblet cells but not in these regions between absorptive cells. However, the macromolecular tracers, microperoxidase, cytochromec and horseradish peroxidase, were excluded from both villus goblet cell and absorptive cell paracellular spaces inin vivo segments. These findings suggest that a subpopulation of villus goblet cells may serve as focal sites of high ionic permeability and contribute to the relatively low resistance to ionic flow which characterizes the small intestinal epithelium.     

Cell junctions in the subcommissural organ of the rabbit as revealed by use of ruthenium red

Summary Tight junctions were found in the apical junctional complex of the adult rabbit subcommissural organ (SCO) in addition to zonulae adhaerentes and gap junctions of typical ependymal cells. Ventricular perfusion of ruthenium red before fixation was found to give excellent results for distinguishing between gap and tight junctions at the ependymal surface. The implication of tight junctions as a mechanical means of sealing off the SCO area from the cerebrospinal fluid and the use of ruthenium red as a tracer substance are discussed.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen.     

Route of Passive Ion Permeation in Epithelia

“Tight junctions” between cells in some epithelia actually provide the main route of passive ion permeation. The degree of junctional tightness may underlie important functional differences between different epithelia.     

Morphological changes in the junctional complex of cells in the arachnoid layer of the rat after cold injury

Summary The junctional complexes of cells in the outer arachnoid layer overlying the cerebral cortex of 2-week-old rats were examined with freeze-fracture electron microscopy up to 60 min after transcranial cold injury to the dorsal surface of the brain. Within 30 min after injury, areas of gap and tight junctions with morphological features characteristic of junction formation and/or junction disruption were found scattered among normal junctional complexes in some arachnoid cells. Within 60 min after injury, tight junctions with features typical of less leaky zonulae occludentes were present in all arachnoid cells examined. These morphological features include increases in the number of tight junctional strands and the number of strand-to-strand anatomoses. Gap junctions were interspersed among the tight junctional strands, and many were completely encircled by the strands. The increase in the number and complexity of the tight junctional strands in response to brain injury may be the morphological basis for the maintenance of the cerebrospinal fluid-blood dural barrier.This study was supported by the National Institute of Neurological and Communicative Disorders and Stroke Grant NS20590. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported herein were conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Pub. No. (NIH) 78-23     

Tight junctions of human uterine epithelial cells change during the menstrual cycle: a morphometric study.

Tight junctions between luminal epithelial cells of the human uterus were studied by freeze-fracture electron microscopy. It was found that junctional complexity decreased during the menstrual cycle, and we explore how this finding may contribute to the role of the uterus in facilitating implantation.     

Transmembrane proteins of tight junctions

Tight junctions contribute to the paracellular barrier, the fence dividing plasma membranes, and signal transduction, acting as a multifunctional complex in vertebrate epithelial and endothelial cells. The identification and characterization of the transmembrane proteins of tight junctions, claudins, junctional adhesion molecules (JAMs), occludin and tricellulin, have led to insights into the molecular nature of tight junctions. We provide an overview of recent progress in studies on these proteins and highlight their roles and regulation, as well as their functional significance in human diseases.     

Transmembrane proteins of tight junctions

Tight junctions contribute to the paracellular barrier, the fence dividing plasma membranes, and signal transduction, acting as a multifunctional complex in vertebrate epithelial and endothelial cells. The identification and characterization of the transmembrane proteins of tight junctions, claudins, junctional adhesion molecules (JAMs), occludin and tricellulin, have led to insights into the molecular nature of tight junctions. We provide an overview of recent progress in studies on these proteins and highlight their roles and regulation, as well as their functional significance in human diseases.     

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.     

PCC4azal teratocarcinoma stem cell differentiation in culture: II. Morphological characterization

The ultrastructural morphology of the PCC4azal embryonal carcinoma cells and their differentiated counterparts, endoderm-like cells and giant cells, was characterized and compared with that of the cells of embryoid bodies. The ultrastructure of the PCC4azal embryonal carcinoma cells is similar to that of the embryonal carcinoma cells of the embryoid body. These cells are small, with a large nucleus and relatively few cytoplasmic organelles. Gap junctions and modified adherens junctions are formed at some areas of intercellular contact between the embryonal carcinoma cells. The differentiated PCC4azal endoderm-like cells have a more developed cytoplasm, containing an extensive endoplasmic reticulum with large Golgi regions. Most striking is the de novo appearance of epithelial-like junctional complexes which join the apical borders between the endoderm-like cells, thus polarizing the cell monolayer. The zonula occludens junctions of the junctional complex are extensive, consisting of six or more strands of tight junctional ridges. Terminal webs are present in the apical regions that are inserted into the zonula adherens region of the junctional complex. Gap junctions continue to join neighboring cells, and some gap junctions are intercalated within tight junctional ridges. The ultrastructure of the differentiated endodermal cells of the embryoid bodies is very similar to that of the PCC4azal endoderm-like cells. The embryoid body endodermal cells form similar junctional complexes which also contain continuous belts of tight junctions that are intercalated with gap junctions. As the PCC4azal endoderm-like cells are transformed to giant cells, a massive cytoskeleton is formed, consisting of a large complex system of 10-nm filaments, microtubules, and 7-nm microfilaments. The junctional complexes that were present during the endodermal stage are partially disassembled as the giant cells migrate apart. Thus, the differentiation process in this system is characterized by significant and distinctive morphological changes.     

Cell-cell interactions in regulating lung function

Tight junction barrier formation and gap junctional communication are two functions directly attributable to cell-cell contact sites. Epithelial and endothelial tight junctions are critical elements of the permeability barrier required to maintain discrete compartments in the lung. On the other hand, gap junctions enable a tissue to act as a cohesive unit by permitting metabolic coupling and enabling the direct transmission of small cytosolic signaling molecules from one cell to another. These components do not act in isolation since other junctional elements, such as adherens junctions, help regulate barrier function and gap junctional communication. Some fundamental elements related to regulation of pulmonary barrier function and gap junctional communication were presented in a Featured Topic session at the 2004 Experimental Biology Conference in Washington, DC, and are reviewed in this summary.     

THE INTER-SERTOLI CELL TIGHT JUNCTIONS IN GERM CELL-FREE SEMINIFEROUS TUBULES FROM PRENATALLY IRRADIATED RATS: A FREEZE-FRACTURE STUDY

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood—testis barrier in the normal testis.     

Gap and tight junctions in tunicates. Study in conventional and freeze-fracture techniques

Intercellular junctions have been investigated in epidermis and pharyngeal epithelium of larvae and adults of various species of tunicates with conventional and freeze-fracture techniques. Gap and tight junctions were found, similar to those observed in vertebrate tissues. Gap junctions were frequent in glandular epithelia and in larval tissues. They were interpreted as ways of intercellular communication in these developing tissues. They were also particularly numerous in Phallusia pharyngeal cells. Tight junctions were found preferentially in adult pharyngeal and epidermal epithelia, where they were arranged in strands of distinct particles forming a belt-like network at the apical part of cells. These junctions were interpreted as providing a tight barrier between the internal medium and the external environment. In larvae, tight junctions were found only between epidermal cells of the tail. These junctions thus characterized completely differentiated tissues, where they might play, in tunicates and in vertebrates, the same role as septate junctions do in invertebrates.     

Intercellular junctions during development of the definitive kidney in lampreys: freeze fracture and morphometric analyses

The changing morphology of intercellular junctions in renal morphogenesis during lamprey metamorphosis was followed using freeze-fracture replicas and morphometry. Gap junctions and particle aggregates among strands of occluding junctions are conspicuous in the differentiating podocytes of the renal corpuscle and in the early ciliated neck and proximal segments but not in the distal segment. The cells of the segmented nephron arise from alpha nephrogenic cells which have a focal aggregate (macula occludens) of 4.8 junctional strands. Upon the initiation of metamorphosis the number of strands increases to 8 in undifferentiated cells with either maculae or zonulae occludentes. Differentiation of both neck and proximal segments is accompanied by gradual transformation of a 7-strand zonula occludens to a 4-strand junction but it becomes more shallow in the latter segment. Two types of undifferentiated cell are recognized through five of the seven metamorphic stages. Cells of two types of distal segments begin differentiation at the midpoint of transformation and immediately show zonula occludens of different morphology. Distal segment I (pars recta) has 5 strands and a 0.250 mum depth whereas segment II has 8-9 strands with twice the apical-basal depth. The larval archinephric duct undergoes a moderate transformation in junctional morphology with the addition of 2 strands and no increase in apical-basal depth in zonulae occludentes during metamorphosis. Changes and development of types of intercellular junctions along the nephron in lampreys are discussed with reference to known regional functional specialization in this organism and with the morphology of renal tubular intercellular junctions in other vertebrates.