VARIATIONS IN TIGHT AND GAP JUNCTIONS IN MAMMALIAN TISSUES

The fine structure and distribution of tight (zonula occludens) and gap junctions in epithelia of the rat pancreas, liver, adrenal cortex, epididymis, and duodenum, and in smooth muscle were examined in paraformaldehyde-glutaraldehyde-fixed, tracer-permeated (K-pyroantimonate and lanthanum), and freeze-fractured tissue preparations. While many pentalaminar and septilaminar foci seen in thin-section and tracer preparations can be recognized as corresponding to well-characterized freeze-fracture images of tight and gap junction membrane modifications, many others cannot be unequivocally categorized—nor can all freeze-etched aggregates of membrane particles. Generally, epithelia of exocrine glands (pancreas and liver) have moderate-sized tight junctions and large gap junctions, with many of their gap junctions basal to the junctional complex. In contrast, the adrenal cortex, a ductless gland, may not have a tight junction but does possess large gap junctions. Mucosal epithelia (epididymis and intestine) have extensive tight junctions, but their gap junctions are not as well developed as those of glandular tissue. Smooth muscle contains numerous small gap junctions The incidence, size, and configuration of the junctions we observed correlate well with the known functions of the junctions and of the tissues where they are found.     

Development and evolution of chordate cartilage

Deuterostomes are a monophyletic group of animals containing vertebrates, lancelets, tunicates, hemichordates, echinoderms, and xenoturbellids. Four out of these six extant groups-vertebrates, lancelets, tunicates, and hemichordates-have pharyngeal gill slits. All groups of deuterostome animals that have pharyngeal gill slits also have a pharyngeal skeleton supporting the pharyngeal openings, except tunicates. We previously found that pharyngeal cartilage in hemichordates and cephalochordates contains a fibrillar collagen protein similar to vertebrate type II collagen, but unlike vertebrate cartilage, the invertebrate deuterostome cartilages are acellular. We found SoxE and fibrillar collagen expression in the pharyngeal endodermal cells adjacent to where the cartilages form. These same endodermal epithelial cells also express Pax1/9, a marker of pharyngeal endoderm in vertebrates, lancelets, tunicates, and hemichordates. In situ experiments with a cephalochordate fibrillar collagen also showed expression in pharyngeal endoderm, as well as the ectoderm and the mesodermal coelomic pouches lining the gill bars. These results indicate that the pharyngeal endodermal cells are responsible for secretion of the cartilage in hemichordates, whereas in lancelets, all the pharyngeal cells surrounding the gill bars, ectodermal, endodermal, and mesodermal may be responsible for cartilage formation. We propose that endoderm secretion was primarily the ancestral mode of making pharyngeal cartilages in deuterostomes. Later the evolutionary origin of neural crest allowed co-option of the gene network for the secretion of pharyngeal cartilage matrix in the new migratory neural crest cell populations found in vertebrates.     

Cell Junctions in Arthropod Ion-transport Systems

The epithelial cells involved in the movement of ions and waterform a major subset of all epithelial cell types. Both the formand the functions of cell junctions present in these cells areessentially the same as those found elsewhere. Gap junctionsare believed to regulate intercellular communication; desmosomesand hemidesmosomes provide mechanical anchorage to other cellsand the extracellular matrix; septate junctions play roles inproviding cell to cell anchorage, and perhaps in sealing thelateral surfaces of adjacent cells together to prevent paracellularfluid and solute movement; tight junctions (of limited distributionin insects) are seals between adjacent cells. They form a barrierto the paracellular movement of solutes and water. Examination of the junctions in salivary glands and midgut provideinsight into the roles of these junctions in the developmentand function of ion transport systems. In Manduca sexta (Johannsen)the cells of the salivary gland are joined by pleated septateand gap junctions. Individual salivary cells have numerous foldsand canaliculi. The walls of the canaliculi consist of extensivelyfolded plasma membrane in intimate association with mitochondria.Gap junctions connect adjacent parts of the same cell acrossmembrane folds, effectively shortening diffusion distances inthe cells. Hemidesmosomes are present in the walls of developingcanaliculi. They are attached to pore filaments that occupythe lumen of the developing canaliculi. The hemidesmosomes andpore filaments may have a morphogenetic role as they disappearafter the canaliculi are formed. In Manduca sexta the midgut cells are joined by gap and septatejunctions. These junctions differ in morphology from their counterpartsin the salivary gland; physiological studies show the gobletcells are not coupled to neighboring tall columnar cells. Wehave shown the gap junctions joining them are typical of non-couplingjunctions. Preliminary studies suggest that the gap junctionschange form when the cells are coupled.     

Regulation of Tight Junction Assembly and Epithelial Polarity by a Resident Protein of Apical Endosomes

The establishment of tight junctions and cell polarity is an essential process in all epithelia. Endotubin is an integral membrane protein found in apical endosomes of developing epithelia when tight junctions and epithelial polarity first arise. We found that the disruption of endotubin function in cells in culture by siRNA or overexpression of the C‐terminal cytoplasmic domain of endotubin causes defects in organization and function of tight junctions. We observe defects in localization of tight junction proteins, reduced transepithelial resistance, increased lanthanum penetration between cells and reduced ability of cells to form cysts in three‐dimensional culture. In addition, in cells overexpressing the C‐terminal domain of endotubin, we observe a delay in re‐establishing the normal distribution of endosomes after calcium switch. These results suggest that endotubin regulates trafficking of polarity proteins and tight junction components out of the endosomal compartment, thereby providing a critical link between a resident protein of apical endosomes and tight junctions.     

Structure and distribution of gap junctions in lens epithelium and fiber cells

Summary We report a comparative study of gap junctions in lens epithelia of frog, rabbit, rat and human, using a double mounting method for freeze-fracture electron microscopy. The gap junctions on the narrow sides of hexagonal cortical fiber cells of various species were also studied with the same technique. Gap junctions were commonly present between epithelial cells of the entire undifferentiated epithelium, between fiber cells on both wide and narrow sides, and between epithelial cells and fiber cells. Structural diversity of gap junctions, based on connexon arrangements, was evident in lens epithelia among the four species studied. Gap junctions with random arrays of connexons were found predominantly in frog lens epithelium, while the crystalline and striated configurations were mainly observed in the epithelia of human and rat, and of rabbit, respectively. On the other hand, there was no structural variation of gap junctions observed on either wide or narrow sides of lens fiber cells from any species studied. Only the random-type gap junction was found. However, the distribution of gap junctions was unique on the narrow sides. There was a single row of junctional plaques along the middle of the narrow sides, whereas the wide sides showed an uneven distribution pattern. The gap junctions between epithelial cells and fiber cells had a random packing of connexons.     

Vaccinia virus entry, exit, and interaction with differentiated human airway epithelia

Variola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors.     

Intercellular junctions and rhombic particle arrays in the developing and adult dorsal ocelli of the honeybee

Intercellular junctions and particle arrays in the developing and mature dorsal ocelli of the honeybee Apis mellifera have been studied with conventional and freeze-fracture electron microscopy. Four types of junctions are found in the lentigenic and retinogenic part during development. These are desmosomes, septate junctions, tight junctions, and gap junctions. Gap junctions and septate junctions are found between differentiating photoreceptor cells only as long as the rhabdoms are beginning to form. Their disappearance after differentiation indicates that they could play a part in cell determination. Desmosomes connect photoreceptor cells into the early imaginai stage and then disappear. Other junctions, once they have formed, remain for the life of the animal, but can change considerably in structure, distribution and frequency. The cells of the perineurium surrounding the ocellus are connected by septate and gap junctions, which may be the basis of the blood-eye barrier. Rhombic particle arrays on the E-face of the glial membrane attached to the photoreceptor cell membrane first appear in small groups one day before emergence. In the further course of life these arrays become more extensive and apparent. Their significance may be to play some role in receptor function.     

Permeability barrier in the mantle epithelium lining the testis in the apple-snail Pomacea canaliculata (Gastropoda: Ampullariidae)

Intercellular junctions are studied in the epithelium lining the testis of the freshwater snail Pomacea canaliculata by conventional staining and lanthanum tracer techniques. The junctional complex consists of belt desmosomes and septate junctions. Septate junctions are of the pleated-sheet type and they are constantly associated with mitochondria. Gap and tight junctions appear to be absent. These septate junctions seem to be the structural correlate of an epithelial permeability barrier that separate the testis from the extrapallial space where the shell elements are deposited. These junctions may contribute to a functional barrier in the male gonad of Pomacea canaliculata. The results indicate that freshwater prosobranchs have junctional structures very close to those found in other molluscs.     

Intercellular junctions in the corpora cardiaca of locusts

Summary The intercellular junctions in the corpora cardiaca of the locusts Schistocerca gregaria and Locusta migratoria were investigated by transmission electron microscopy. In the glandular lobes, complexes consisting of scalariform junctions and associated mitochondria, comparable to those previously observed in ion transporting epithelia, are formed between gland cells, and more rarely between gland cells and the neurons innervating them. Their structure and abundance are apparently unaffected by the stage of development or by the various experimental conditions employed. In the neural lobe, scalariform junctions form between glial cells and show close association with the endoplasmic reticulum. Gap junctions are present among glandular, neural and glial elements, and are formed between cells of the same type and of different types. Contacts resembling punctate tight junctions are widely distributed in the gland, but would be unlikely to form a barrier to diffusion. Septate junctions are formed exclusively between glial cells.     

Role of Gap junctions in bone tissue

Gap junctions are transmembrane channels, that connect the membranes of adjacent cells and are involved in the direct signal transmission between the cells. The intercellular communication involving this type of channels provides the proper functioning of tissues and organs. Gap junctions formation and synthesis of connexins is regulated by hormones, growth factors and signaling molecules. Gap junctions, located between osteoblasts, osteoclasts, and osteocytes, play a key role in the process of bone turnover, and therefore in the modeling and bone tissue regeneration. They also mediate the regulation of proliferation, differentiation and maturation of osteoblasts, as well as formation and activity of osteoclasts. This paper presents the current state of knowledge on the role of intercellular connections via Gap junctions in bone cells, with particular emphasis on the involvement of connexin 43, in the regulation of osteogenesis.     

Freeze-fracture study of the epidermal cells of a teleost with particular reference to intercellular junctions and permeability to tracer

The plasmatic membranes, the intercellular junctions and the intercellular spaces of the epidermis of the fish Pimelodus maculatus were studied by freeze-fracture and by lanthanum methods. The observations has confirmed the presence of desmosomes. Gap junctions were not found and the tight junctions can be seen very rarely, arranged to form small discrete maculae. The finger-print pattern due to the microridges of the apical plasma membrane of the superficial cells was studied by direct replicas. The tracer penetrates all the intercellular epidermal spaces but failed to penetrate the dermis, suggesting the presence of a barrier at the dermo-epidermal level.     

Cell junctions in the early chick embryo--a freeze etch study

Cell junctions in the early chick embryo have been examined in freeze-etch specimen. Well developed zonulae occludentes are found in the epiblast as early as stage 4. Large gap junctions are also found in the epiblast at this stage. In those cells which have left the surface to form mesenchymal structures (Hensen's node, juxtanodal mesenchyme, primitive streak mesenchyme), one finds not only gap, but also tight, junctions. These junctions do not form continuous belts, but appear as fragments, often reduced to single strands, of typical tight junctions. They probably correspond to the focal tight junctions described earlier in sectioned material. The origin and possible significance of these contacts is discussed, and it is suggested that they represent remnants of junctions between neighboring cells in the epiblast. These junctional remnants slowly disappear by “dilution,” either through cell division and/or cell movement. The appearance of newly formed gap junctions is also described.     

C. elegans Enabled exhibits novel interactions with N-WASP, Abl, and cell-cell junctions

Ena/VASP proteins are associated with cell-cell junctions in cultured mammalian cells [1] and Drosophila epithelia [2, 3], but they have only been extensively studied at the leading edges of migratory fibroblasts, where they modulate the protrusion of the leading edge [4]. They act by regulating actin-filament geometry, antagonizing the effects of actin-capping protein [5]. Embryos lacking the C. elegans Ena/VASP, UNC-34, display subtle defects in the leading edges of migrating epidermal cells but undergo normal epidermal morphogenesis. In contrast, embryos lacking both UNC-34 and the C. elegans N-WASP homolog have severe defects in epidermal morphogenesis, suggesting that they have parallel roles in coordinating cell behavior. GFP-tagged UNC-34 localizes to the leading edges of migrating epidermal cells, becoming redistributed to new junctions that form during epidermal-sheet sealing. Consistent with this, UNC-34 contributes to the formation of cadherin-based junctions. The junctional localization of UNC-34 is independent of proteins involved in Ena/VASP localization in other experimental systems; instead, junctional distribution depends upon the junctional protein AJM-1. We also show that Abelson tyrosine kinase, a major regulator of Enabled in Drosophila, is not required for UNC-34/Ena function in epithelia. Instead, our data suggest that Abelson kinase acts in parallel to UNC-34/Ena, antagonizing its function.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

Major involvement of connexin 43 in seminiferous epithelial junction dynamics and male fertility

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, β-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO^−/−) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO^−/− as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO^−/− testis. Occludin, N-cadherin and β-catenin levels were enhanced in SCCx43KO^−/− mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.     

Intercellular contact in the unincubated chick embryo

Summary The unincubated chick blastoderm, which consists of a complete upper epithelial layer of one cell thickness (epiblast) and an incomplete lower layer (hypoblast), was examined with the electron microscope in order to define the types of cell contact present. The terminal contacts between the cells of the epiblast invariably involved several focal tight junctions, but only occasionally involved tight junctions. Desmosomes were not observed in these areas, but were encountered in various phases of development in the deeper contact regions between epiblast cells. This deeper region also showed sporadic focal tight junctions and frequent micropapillae. These micropapillae were also common on the surfaces of hypoblast cells. Intercellular spaces between epiblast and hypoblast cells and within the hypoblast were often wide, narrowing to occasional focal tight junctions. Tight junctions and desmosomes were not observed in association with hypoblast cells. Gap junctions were not observed in any region of the embryo.These observations are discussed in relation to the morphogenetic movements occurring in the forming hypoblast and also the influence of this layer on the subsequent development of the embryo. Comparisons are drawn between the contact morphology in the unincubated blastoderm and that in later stages of development.Supported by the Medical Research Council of Canada.     

Functional analysis of tight junctions

Epithelial and endothelial cells are joined to each other via a set of intercellular junctions that differ in their morphological appearance, composition, and function. The tight junction or zonula occludens is the intercellular junction that regulates diffusion between cells and therefore allows endothelia and epithelia to form cellular barriers that separate compartments of different composition. This intercellular gate formed by tight junctions is not only highly regulated but is size- and ion-selective and, hence, represents a semipermeable diffusion barrier. In epithelia, tight junctions form a morphological and functional border between the apical and basolateral cell surface domains. They directly contribute to the maintenance of cell surface polarity by forming a fence that prevents apical/basolateral diffusion of lipids in the outer leaflet of the plasma membrane. Here we describe a set of assays that allow the analysis of tight junctions to determine their integrity and functional state.     

The tight junction protein ZO-1 is concentrated along slit diaphragms of the glomerular epithelium

The foot processes of glomerular epithelial cells of the mammalian kidney are firmly attached to one another by shallow intercellular junctions or slit diaphragms of unknown composition. We have investigated the molecular nature of these junctions using an antibody that recognizes ZO-1, a protein that is specific for the tight junction or zonula occludens. By immunoblotting the affinity purified anti-ZO-1 IgG recognizes a single 225-kD band in kidney cortex and in slit diaphragm-enriched fractions as in other tissues. When ZO-1 was localized by immunofluorescence in kidney tissue of adult rats, the protein was detected in epithelia of all segments of the nephron, but the glomerular epithelium was much more intensely stained than any other epithelium. Among tubule epithelia the signal for ZO-1 correlated with the known fibril content and physiologic tightness of the junctions, i.e., it was highest in distal and collecting tubules and lowest in the proximal tubule. By immunoelectron microscopy ZO-1 was found to be concentrated on the cytoplasmic surface of the tight junctional membrane. Within the glomerulus ZO-1 was localized predominantly in the epithelial foot processes where it was concentrated precisely at the points of insertion of the slit diaphragms into the lateral cell membrane. Its distribution appeared to be continuous along the continuous slit membrane junction. When ZO-1 was localized in differentiating glomeruli in the newborn rat kidney, it was present early in development when the apical junctional complexes between presumptive podocytes are composed of typical tight and adhering junctions. It remained associated with these junctions during the time they migrate down the lateral cell surface, disappear and are replaced by slit diaphragms. The distribution of ZO-1 and the close developmental relationship between the two junctions suggest that the slit diaphragm is a variant of the tight junction that shares with it at least one structural protein and the functional property of defining distinctive plasmalemmal domains. The glomerular epithelium is unique among renal epithelia in that ZO-1 is present, but the intercellular spaces are wide open and no fibrils are seen by freeze fracture. The presence of ZO-1 along slit membranes indicates that expression of ZO-1 alone does not lead to tight junction assembly.     

Junctional types in the tissues of an onychophoran: the apparent lack of gap and tight junctions in Peripatus

The Onychophora are a rare group of primitive invertebrates, relatively little investigated. Tissues from a range of their digestive, secretory and excretory organs have been examined to establish the features of their intercellular junctions. Glutaraldehyde-fixed cells from the midgut and rectum, as well as the renal organ, mucous gland, salivary gland, epidermis, CNS and testis from specimens of Peripatus acacioi, have been studied by thin section and freeze-fracture electron microscopy. Adjacent cells in the epithelia of all these tissues are joined by apical zonulae adhaerentes, associated with a thick band of cytoskeletal fibrils. These are followed by regular intercellular junctional clefts, which, in thin sections, have the dense, relatively unstriated, appearance of smooth septate junctions (SSJ). However, freeze-fracture reveals that only the midgut has what appear to be characteristic SSJs with parallel alignments of closely-packed rows of intramembranous particles (IMPs); these IMPs are much lower in profile than is common in such junctions elsewhere. The mucous gland, testis, rectal and renal tissues exhibit, after freeze-fracture, the characteristic features of pleated septate junctions (PSJ) with undulating rows of aligned but separated junctional particles. Suggestions of tricellular septate junctions are found in replicas at the interfaces between 3 cells. In addition, renal tissues exhibit scalariform junctions in the basal regions of their cells. Between these basal scalariform and apical septate junctions, other junctions with reduced intercellular clefts are observed in these renal tissues as well as the rectum, but these appear not to be gap junctions. Such have not been unequivocally observed in any of the tissues studied from this primitive organism; the same is true of tight junctions.