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 junction development between cultured hepatoma cells: Possible stages in assembly and enhancement with dexamethasone

Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9–11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10^?6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.     

Vertebrate-like tight junctions in the insect eye

Unequivocal vertebrate-like anastomosing tight junctions have been observed for the first time in insect tissues. In freeze-fractured replicas of dipteran compound eyes, the intercellular junctions between certain glial cells in regions distal to the optic neuropile display an extensive network of continuous intramembranous P face (PF) ridges. The intramembranous E face (EF) possesses a reticulum of grooves which occur in the depths of troughs and thereby produce a ‘quilted’ appearance. At PF/EF membrane face transitions, there is an obliteration of the intercellular space at points of membrane fusion; here the PF ridges and EF grooves appear in register and are therefore complementary. Although the septate junctions found here are patent, these tight junctions are occluding to lanthanum and appear to represent the blood-retinal barrier previously demonstrated electrophysiologically in insects. The existence and vertebrate-like structural complexity of these junctions in arthropods supports the concept of the universality of the membrane specializations that mediate cell-to-cell interactions.     

Junctions between sinusoidal endothelial cells in fetal rat liver.

During a freeze-fracture study of tight junctions in fetal rat liver (Montesano et al., '75) unusual patterns of intramembranous particles were observed in regions of contact between sinusoidal endothelial cells. These patterns were mainly represented by arrays of particles, often associated with linear elevations or crests in the membrane A-face; they may represent abortive forms of tight junctions.     

CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

Effects of Ga3 and Ca2+ on barley aleurone protoplasts: a freeze-fracture study

Summary Freeze-fracture electron microscopy was used to study changes in the endomembrane system of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts. Protoplasts were used for this study because their response to calcium and the plant hormone gibberellic acid (Ga₃) can be monitored prior to rapid freezing of cells for electron microscopy. Protoplasts incubated in Ga₃ plus Ca²⁺ secrete elevated levels of a-amylase relative to cells incubated in Ga₃ or Ca²⁺ alone. The endoplasmic reticulum (ER) and Golgi apparatus of protoplasts incubated in Ga₃ plus Ca²⁺ undergo changes that are well correlated with the synthesis and secretion of a-amylase. The ER, which appears as short, single sheets of membrane in Ca²⁺-and Ga₃-treated protoplasts, exists as a series of long fenestrated stacks of membranes following incubation in Ga₃ plus Ca²⁺. The Golgi apparatus is also more highly developed in protoplasts treated with Ga₃ plus Ca²⁺. This organelle is larger and has more vesicles associated with its periphery in protoplasts that actively secrete a-amylase. Evidence that the Golgi apparatus participates in a-amylase secretion is also provided by experiments with the ionophore monensin, which causes pronounced swelling of Golgi cisternae and inhibits the secretion of a-amylase. We interpret these observations as showing that the ER and Golgi apparatus of barley aleurone participate in the intracellular transport and secretion of a-amylase. The plasmalemma (PF face) of barley aleurone protoplasts shows a high density of intramembranous particles (IMPs) which, in general, are evenly distributed. Occasionally, ordered arrays of IMPs are observed, possibly resulting fro m osmotic stress. after 48 hours the plasmalemma of some Ga₃-treated protoplasts show particle-free areas considered to be indications of senescence.abbreviations ER endoplasmic reticulum - Ga₃ gibberellic acid - IEF isoelectric focusing - IMP intramembranous particle - PF protoplasmic fracture - PL plasmalemma     

Tracer and freeze fracture observations on developing tight junctions in fetal rat thyroid

The development of tight junctions in fetal rat thyroid from the sixteenth to the twentieth days of gestation was examined with conventional ultrastructural methods and freeze-fracture preparations. These results were compared with those obtained using lanthanum hydroxide and horseradish peroxidase (HRP) tracers. Tight junctions appear to arise on the plasma membranes of fetal thyroid cells by the aggregation and fusion of linear particle chains which appear at several discrete sites on the plasma membrane of developing follicular cells. Tracer studies show that they are effective barriers to the passage of HRP from the outset, are freely penetrated by La³⁺ at the sixteenth and seventeenth days of gestation, but progressively lose permeability to La³⁺ from the seventeenth to twentieth days of gestation. However, freeze-fracture observations suggest that La³⁺ must penetrate into the follicular lumen through the tight junction elements, for the follicular lumen, when it appears, is always completely surrounded by a continuous though sometimes rudimentary meshwork of tight junction elements. The results suggest that the tight junction forms an effective barrier to the passage of large macromolecules, e.g. thyroglobulin, from very early stages in its development. The La³⁺ results suggest that decreased resistance of the intercellular pathway, possibly related to the development of transepithelial potentials, may occur during this period in development.     

Polar redistribution of Na+K+ATPase in aggregating MDCK cells

The distribution of Na+K+ATPase was examined immunoelectronmicroscopically along the plasma membranes of aggregating MDCK cells grown in tissue culture. Na+K+ATPase was localized with the use of affinity-purified antibodies employing the double-antibody immunoperoxidase technique. Na+K+ATPase-like immunoreactivity was uniformly distributed along the plasmalemma of cells grown in suspension. Once attached to the substratum, individual MDCK cells exhibited Na+K+ATPase immunoreactivity limited to the microvilli-laden mucosal surface. This exclusive mucosal distribution of Na+K+ATPase disappeared and a basolateral immunoreactivity became apparent once intercellular contacts and the formation of tight junctions occurred between neighboring cells. Thus, both cellular attachment to the substratum, and tight junction formation between aggregating MDCK cells are essential to the genesis of Na+K+ATPase polar distribution. These two events, however, appear to induce opposing distribution patterns for Na+K+ATPase.     

Inverted gap and other cell junctions in cockroach hemocyte capsules: a thin section and freeze-fracture study.

Freeze-fracture and thin-section studies were done on cockroach hemocytes that had encapsulated implanted pieces of Araldite. Desmosome-like junctions and 'B' type gap junction were described. Freeze-fractured gap junctions displayed fused and clustered, but not hexagonally arrayed intramembranous practicles (approximately 130 A) on the B face and pitted areas on the A face of the plasmalemma. Gap junctions were quite numerous and counts of gap and non-gap particles indicated at least a five-fold particle density increase (4000/mu2) compared with B face particle densities (approximately 800/mu2) from free circulating blood cells where gap junctions had not been formed.     

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.     

STUDIES ON DISPERSED PANCREATIC EXOCRINE CELLS : I. Dissociation Technique and Morphologic Characteristics of Separated Cells

A procedure for dissociation of the guinea pig pancreas into individual cells is described which employs enzymatic digestion with pure collagenase, chymotrypsin, and hyaluronidase, utilizes an interposed chelation of divalent cations by EDTA, and is terminated by gentle shearing. Yields of cells are 50–60%, based on DNA recovered. The population comprises ~95% exocrine cells, the remainder consisting of endocrine, duct, and vascular endothelial cells. The exocrine cells, though spherical, retain the structural attributes of their in situ counterparts, including differentiation of the plasmalemma into zones corresponding to the former apical and basal plasmalemma, polarized distribution of organelles indicated by fields of zymogen granules in the cytoplasm underlying the former apex, central location of the Golgi complex, and placement of the rough endoplasmic reticulum and nucleus in the former basal pole of the cell. Electron microscope study of the effects of individual treatments used during dissociation indicates that digestion of basement membrane and collagen is solely due to collagenase activity and that separation of desmosomes (and possibly of zonulae adherentes) results only from exposure to low [Ca⁺⁺] and EDTA and is not effected by the enzymes used. Gap junctions are resistant to enzymes and EDTA; tight junctions resist enzyme treatment but undergo rearrangement upon exposure to EDTA. Both junctions require mechanical shear for complete cell separation. Neither chymotrypsin nor hyaluronidase produces visible alterations in stromal or junctional elements. Dissociation requires the concerted action of enzymes, chelation of divalent cations, and mechanical shear, since the individual treatments are alone ineffective.     

Formation of tight junctions and desmosomes protects MDCK cells against hyperthermic killing

Cell density is known to modify the survival of mammalian cells exposed to elevated temperatures. We have examined the role that cell–cell contact plays in this phenomenon. The formation of cell–cell contact is carried out by cells' junctional complex, i.e., tight junctions, desmosomes, and gap junctions. Lack of formation of tight junctions and desmosomes, or their opening, could interfere with the functions and structures of cell membrane. Membrane damage is at least partially responsible for cell death at elevated temperatures. MDCK cells with high density plated in low calcium medium form confluent monolayers devoid of the formation of tight junctions and desmosomes but quickly assemble them after Ca²⁺ restoration. We used MDCK cells and the calcium switch technique to investigate effects of cell–cell contact and, independently, of cell density on hyperthermic cell killing. We found that MDCK cells that formed tight junctions and desmosomes were more resistant to hyperthermic treatment than those that did not. Blocking the formation pathway of tight junctions made cells sensitive to heat. Cells growing at lowdensity showed almost the same survival as did cells at high density in the absence of the formation of tight junctions and desmosomes. The results suggest that the formation of tight junctions and desmosomes play a more important role in determining hyperthermic response than does density per se. The formation of tight junctions and desmosomes appears to protect cells modestly against hyperthermic killing. © 1994 Wiley-Liss, Inc.     

Junctional diversity in two regions of the epidermis of Oikopleura dioica (Tunicata,Larvacea)

Summary A simple continuous epithelium surrounds the body of the pelagic larvacean. It consists of two zones of cells: oikoplast cells and flattened cells. The oikoplast cells are columnar and produce a thick extracellular house that ensheathes the body of the organism. These cells are joined laterally by wide tight junctions (zonulae occludentes). The tail of the animal is surrounded by exceedingly thin cells which are joined by narrow tight junctions under which lie intermediate junctions (zonulae adhaerentes) and gap junctions. A web of fibrous material inserts into the intermediate junctions. The transitional cells between the two epithelial zones have one lateral border with a wide tight junction, and the other lateral border with a narrow tight junction and a wide intermediate junction. In freeze-fracture replicas, the wide tight junction has a number of anastomosing ridges, in comparison with the narrow tight junction, which usually consists of only a single row of intramembranous particles. In replicas, the thin epithelial cells show unusual parallel arrays of particles in clusters on their apical plasma membranes. This simple epithelium, therefore, exhibits striking differences between the two cellular zones, in the structural characteristics of both the lateral borders and the apical membrane.     

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.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions

In the central nervous system (CNS) of pupal Calliphora, dramatic alterations occur in the perineurial and glial gap junctions. Having formed macular plaques by late larval stages, in early pupae cell migration causes the EF intramembranous junctional particles to disaggregate and move apart into linear and then disorganised arrays as shown by freeze-fracture. After nerve and glial cell reorganisation into the adult pattern, the gap junctions begin to reform in the late pupae, again seemingly by particle migration into linear arrays and clusters. Ultimately the particles form numerous macular plaques between both perineurial and glial cells. Statistical analyses support the contention that these are performed EF particles which undergo translateral movement from macular larval junctions into the disaggregated particles of early pupae and that the same particles appear to undergo realignment and reclustering in late pupae to form the mature gap junctions of adults. This is the first report to indicate breakdown and reformation of gap junctions in vivo involving reutilisation of the same intramembranous particles. Perineurial “tight” junctions are not to be found in early pupal stages and their absence can be correlated with the free entry of ionic lanthanum into the CNS observed during that period. In late pupae, when the tight junctional moniliform ridges have apparently reformed, the entry of the tracer lanthanum becomes restricted to the level of the perineurium, penetrating no deeper. This is also the case in the adult, where the blood-brain barrier is maintained. PF particles in the form of short linear ridges and clustered particle arrays in nerve cell membranes are present throughout pupal and adult stages; their continued presence throughout the whole of development suggests some role in neuronal function, as yet unclear.     

Changes in the blood-brain barrier of the central nervous system in the blowfly during development, with special reference to the formation and disaggregation of gap and tight junctions. I. Larval development

In the central nervous system (CNS) of full-grown larvae of the blowfly Calliphora erythrocephala, the glial-ensheathed nerve cells are completely surrounded by a layer of perineurial cells which form a “blood-brain barrier” between the circulating haemolymph and the CNS. A variety of intercellular junctions, including gap and tight junctions, are found between adjacent perineurial cells and some also between apposing glial cells; these have been characterized by freeze-fracturing as well as by tracer studies and analysis of thin sections. They are found not to be present between such cells in the undifferentiated CNS in the newly hatched larvae, nor are the nerve cells encompassed by glial cells; ionic lanthanum can penetrate to the axonal surfaces at this stage. However, over the 5 days of larval growth and development the glial cells produce attentuated cytoplasmic processes that ensheath the nerve cells, and the perineurium is formed; junctional complexes are assembled and a larval blood-brain barrier is produced which excludes tracers. Freeze-fracture preparations suggest that the inverted gap junctions which develop have done so by migration of individual intramembranous EF particles to form, at first, linear arrays and small clusters and, ultimately, macular aggregations in the perineurium; these lie between the undulating rows of PF particles forming the septate junctions. These septate junctions are formed by the organization of arrays of PF particles into multiple rows. Extensive PF particles fusing into ridges with EF grooves to form perineurial “tight” junctions are also observed, seemingly in the process of development; entry of exogenous lanthanum followed by its exclusion parallels the completion of ridge formation. These ridges are simple linear arrays of particles which may be discontinuous, lying in parallel with one another and the surface. Clustered particle arrays as well as scattered short ridges on the axonal PF, however, appear to be present unchanged throughout larval life; their role may therefore be associated with neural membrane function although there are suggestions that some may form axo-glial junctions. This is the first report on the lateral migration of intramembranous particles as the mode of formation of gap junctions in the nervous system of an invertebrate.     

Connexins Induce and Maintain Tight Junctions in Epithelial Cells

Connexins (Cx) are considered to play a crucial role in the differentiation of epithelial cells and to be associated with adherens and tight junctions. This review describes how connexins contribute to the induction and maintenance of tight junctions in epithelial cells, hepatic cells and airway epithelial cells. Endogenous Cx32 expression and mediated intercellular communication are associated with the expression of tight junction proteins of primary cultured rat hepatocytes. We introduced the human Cx32 gene into immortalized mouse hepatic cells derived from Cx32-deficient mice. Exogenous Cx32 expression and the mediated intercellular communication by transfection could induce the expression and function of tight junctions. Transfection also induced expression of MAGI-1, which localized at adherens and tight junction areas in a gap junctional intercellular communication (GJIC)–independent manner. Furthermore, expression of Cx32 was related to the formation of single epithelial cell polarity of the hepatic cells. On the other hand, Cx26 expression, but not mediated intercellular communication, contributed to the expression and function of tight junctions in human airway epithelial cells. We introduced the human Cx26 gene into the human airway epithelial cell line Calu-3 and used a model of tight junction disruption by the Na⁺/K⁺-ATPase inhibitor ouabain. Transfection with Cx26 prevented disruption of both tight junction functions, the fence and barrier, and the changes of tight junction proteins by treatment with ouabain in a GJIC–independent manner. These results suggest that connexins can induce and maintain tight junctions in both GJIC-dependent and –independent manners in epithelial cells.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

The TRPV4 Channel Contributes to Intercellular Junction Formation in Keratinocytes

Transient receptor potential vanilloid 4 (TRPV4) channel is a physiological sensor for hypo-osmolarity, mechanical deformation, and warm temperature. The channel activation leads to various cellular effects involving Ca²⁺ dynamics. We found that TRPV4 interacts with β-catenin, a crucial component linking adherens junctions and the actin cytoskeleton, thereby enhancing cell-cell junction development and formation of the tight barrier between skin keratinocytes. TRPV4-deficient mice displayed impairment of the intercellular junction-dependent barrier function in the skin. In TRPV4-deficient keratinocytes, extracellular Ca²⁺-induced actin rearrangement and stratification were delayed following significant reduction in cytosolic Ca²⁺ increase and small GTPase Rho activation. TRPV4 protein located where the cell-cell junctions are formed, and the channel deficiency caused abnormal cell-cell junction structures, resulting in higher intercellular permeability in vitro. Our results suggest a novel role for TRPV4 in the development and maturation of cell-cell junctions in epithelia of the skin.