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.     

Studies on perineural junctional complexes and the sites of uptake of microperoxidase and lanthanum in the cockroach central nervous system

The perineurial junctional complexes in the nerve cord of Periplaneta americana have been shown to consist of septate desmosomes, extensive gap junctions and relatively limited regions of tight junctions. Microperoxidase (M.W. 1,900) undergoes limited intercellular penetration into the septate desmosomes. Lanthanum penetrates both the septate desmosomes and gap junctions. It is concluded that the restricted access of these substances to the underlying extracellular spaces results from the presence of the perineurial tight junctions. These results contrast with those for small peripheral nerves, which lack equivalent junctional complexes, and in which the extracellular spaces are found to be accessible to externally applied lanthanum. The results are discussed in relation to current concepts of the insect blood-brain barrier.     

Evidence for high molecular weight proteins in arthropod gap and smooth septate junctions

The proteins that make up arthropod gap and septate junctions have not been identified with any certainty. Several candidate proteins for both types of junctions have been proposed in the literature, but there has been no agreement on any of these. Arthropod gap junctions do not label with antibodies to vertebrate gap junction connexins; it thus appears that unrelated proteins form these rather similar structures. Gap junctions inManduca sextamidgut epithelium are unusual since they function only during the molt and are non-functioning during the larval instars. We have developed a preparation from this tissue that is highly enriched in both gap and smooth septate junctions when examined by electron microscopy. SDS-PAGE gels of this preparation have two major protein bands, at 75 and 90 kDa. The presence of gap junctions correlates best with the 75 kDa protein and smooth septate junctions with the 90 kDa protein. Further, the 75 kDa band is stained by an antibody to a putative gap junction protein fromC. elegans. We propose that the 75 kDa protein is a major structural component of gap junctions inManduca sextamidgut epithelium and that the 90 kDa protein forms the smooth septate junctions.     

COEXISTENCE OF GAP AND SEPTATE JUNCTIONS IN AN INVERTEBRATE EPITHELIUM

The intercellular junctions of the epithelium lining the hepatic caecum of Daphnia were examined. Electron microscope investigations involved both conventionally fixed material and tissue exposed to a lanthanum tracer of the extracellular space. Both septate junctions and gap junctions occur between the cells studied. The septate junctions lie apically and resemble those commonly discerned between cells of other invertebrates. They are atypical in that the high electron opacity of the extracellular space obscures septa in routine preparations. The gap junctions are characterized by a uniform 30 A space between apposed cell membranes. Lanthanum treatment of gap junctions reveals an array of particles of 95 A diameter and 120 A separation lying in the plane of the junction. As this pattern closely resembles that described previously in vertebrates, it appears that the gap junction is phylogenetically widespread. In view of evidence that the gap junction mediates intercellular electrotonic coupling, the assignment of a coupling role to other junctions, notably the septate junction, must be questioned wherever these junctions coexist.     

THE STRUCTURAL ORGANIZATION OF THE SEPTATE AND GAP JUNCTIONS OF HYDRA

The septate junctions and gap junctions of Hydra were studied utilizing the extracellular tracers lanthanum hydroxide and ruthenium red. Analysis of the septate junction from four perspectives has shown that each septum consists of a single row of hexagons sharing common sides of 50–60 A. Each hexagon is folded into chair configuration. Two sets of projections emanate from the corners of the hexagons. One set (A projections) attaches the hexagons to the cell membranes at 80–100-A intervals, while the other set (V projections) joins some adjacent septa to each other. The septate junctions generally contain a few large interseptal spaces and a few septa which do not extend the full length of the junction. Basal to the septate junctions the cells in each layer are joined by numerous gap junctions. Gap junctions also join the muscular processes in each layer as well as those which connect the layers across the mesoglea. The gap junctions of Hydra are composed of rounded plaques 0.15–0.5 µ in diameter which contain 85-A hexagonally packed subunits. Each plaque is delimited from the surrounding intercellular space by a single 40-A band. Large numbers of these plaques are tightly packed, often lying about 20 A apart. This en plaque configuration of the gap junctions of Hydra contrasts with their sparser, more widely separated distribution in many vertebrate tissues. These studies conclude that the septate junction may possess some barrier properties and that both junctions are important in intercellular adhesion. On a morphological basis, the gap junction appears to be more suitable for intercellular coupling than the septate junction.     

Intercellular communication in a positional field. Ultrastructural correlates and tracer analysis of communication between insect epidermal cells.

The junctional membrane in the epidermal cells of the larval beetle (Tenebrio molitor L.) is comprised of macular gap junctions embedded in septate junctions. Ultrastructural and morphometric analysis of the distribution of gap junctions within the segmental epidermis suggests that this junction alone could account for the high electrotonic coupling recorded for the epidermal sheet. Analysis of the lanthanum-impregnated septate junction makes it doubtful that this junction serves as a communicating channel between beetle cells. A new model for the septate junction is presented in which pleated septa, less than 30 A thick, connect adjacent plasma membranes; the septa themselves are interconnected by two interseptal platforms that are coplanar with the plasma membranes. Iontophoretic injection of organic tracers into single epidermal cells suggests that only molecules of less than MW 1000 can transfer between cells through low-resistance junctions.     

The Na+/K+ ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system

Although the correct architecture of epithelial tubes is crucial for the function of organs such as the lung, kidney and vascular system, little is known about the molecular mechanisms that control tube size. We show that mutations in the ATPalpha alpha and nrv2 beta subunits of the Na+/K+ ATPase cause Drosophila tracheal tubes to have increased lengths and expanded diameters. ATPalpha and nrv2 mutations also disrupt stable formation of septate junctions, structures with some functional and molecular similarities to vertebrate tight junctions. The Nrv2 beta subunit isoforms have unique tube size and junctional functions because Nrv2, but not other Drosophila Na+/K+ ATPase beta subunits, can rescue nrv2 mutant phenotypes. Mutations in known septate junctions genes cause the same tracheal tube-size defects as ATPalpha and nrv2 mutations, indicating that septate junctions have a previously unidentified role in epithelial tube-size control. Double mutant analyses suggest that tube-size control by septate junctions is mediated by at least two discernable pathways, although the paracellular diffusion barrier function does not appear to involved because tube-size control and diffusion barrier function are genetically separable. Together, our results demonstrate that specific isoforms of the Na+/K+ ATPase play a crucial role in septate junction function and that septate junctions have multiple distinct functions that regulate paracellular transport and epithelial tube size.     

The arthropod gap junction and pseudo-gap junction: Isolation and preliminary biochemical analysis

Summary The hepatopancreas of the crayfish, Procambarus clarkii, contains an unusual abundance of gap junctions, suggesting that this tissue might provide an ideal source from which to isolate the arthropod-type of gap junction. A membrane fraction obtained by subcellular fractionation of this organ contained smooth septate junctions, zonulae adhaerentes, gap junctions and pentalaminar membrane structures (pseudo-gap junctions) as determined by electron microscopy. A further enrichment of plasma membranes and gap junctions was achieved by the use of linear sucrose gradients and extraction with 5 mM NaOH. The enrichment of gap junctions correlated with the enrichment of a 31 Kd protein band on polyacrylamide gels. Extraction with 20 mM NaOH or 0.5% (w/v) Sarkosyl NL97 resulted in the disruption and/or solubilization of gap junctions. Negative staining revealed a uniform population of 9.6 nm diameter subunits within the gap junctions with an apparent sixfold symmetry. Using antisera to the major gap junctional protein of rat liver (32 Kd) and to the lens membrane protein (MP 26), we failed to detect any homologous antigenic components in the arthropod material by immunoblotting-enriched gap junction fractions or by immunofluorescence on tissue sections. The enrichment of another membrane structure (pseudo-gap junctions), closely resembling a gap junction, correlated with the enrichment of two protein bands, 17 and 16Kd, on polyacrylamide gels. These structures appeared to have originated from intracellular myelin-like figures in phagolysosomal structures. They could be distinguished from gap junctions on the basis of their thickness, detergent-alkali insolubility, and lack of association with other plasma membrane structures, such as the septate junction. Pseudo-gap junctions may be related to a class of pentalaminar contacts among membranes involved in intracellular fusion in many eukaryotic cell types. We conclude that pseudo-gap junctions and gap junctions are different cellular structures, and that gap junctions from this arthropod tissue are uniquely different from mammalian gap junctions of rat liver in their detergentalkali solubility, equilibrium density on sucrose gradients, and protein content (antigenic properties).     

Fine structure of the rectum in cockroaches (Dictyoptera): General organization and intercellular junctions

The organization of the rectal pads is described in cockroaches belonging to the Groups Blattoidea (Periplaneta americana, Blatta orientalis) and Blaberoidea (Supella supellectilium, Blaberus craniifer). In the Blattoidea, each pad is composed of two layers (principal and basal cells) and is surrounded by very narrow junctional cells supporting the sclerotized cuticle of the pad frame; basally, the junctional cells abut on to the basal cells. In the Blaberoidea, the basal cell layer is discontinuous, the basal cells being interspersed between extensions of the junctional cells beneath the pad. The ultrastructural features of each cell type is described, with special reference to the intercellular junctions, which exhibit unusual complexity. Four types of junction are recognized: desmosomes (belt and spot desmosomes), gap junctions, septate junctions and scalariform (ladder-like) junctions. The last are usually closely associated with mitochondria, forming mitochondrial-scalariform junction complexes (MS). The distribution of these junctions is examined in relation to the partitioning of extracellular spaces, and to the problem of fluid transport.     

Cell contacts between follicle cells and the oocyte of Helisoma (Mollusca,Pulmonata)

The cell contacts between follicle cells, and follicle cells and oocytes of egg-laying populations of Helisoma duryi and non-egg-laying populations of H. trivcolvis have been studied. Scanning electron microscopy reveals that four to six follicle cells envelop a single developing oocyte. Thin sections and lanthanum impregnations demonstrate apical zonulae adherentes followed by winding pleated-type septate junctions between follicle cells. Gap junctions and septate junctions have been found between follicle cells and vitellogenic oocytes. Freeze-fracture replicas show relatively wide sinuous rows of septate junctional particles, and nemerous large gap junctional particle aggregates on the P-face between vitellogenic oocytes and follicle cells. Septate and gap junctions between immature or nonvitellogenic oocytes and follicle cells are fewer compared to those in vitellogenic oocytes. Similarly, the junctional complexes are less developed in non-egg-laying H. trivolvis compared to those in egg-laying H. duryi. It is possible that intimate interaction between follicle cells and a developing oocyte is necessary for the maturation of the oocyte. The junctional complexes could be involved in the interaction of the follicle cells and the oocyte, and they must disassemble at the onset of ovulation. Rhombic particle arrays and nonjunctional ridges of particles have been found in the basal part of the oolemma.     

Freeze-fracture analysis of gap and septate junctions in embryos of a moth

Gap and septate junctions were examined in embryos of Manduca sexta (tobacco hornworm). The junctions observed were similar in structure to those reported for adult insect tissues. In the epidermis typical pleated septate junctions were found. Associated with the pleated septate junctions were inverted gap junctions which had irregularly arranged particles and pits. In the midgut typical smooth septate junctions were found. Associated with these septate junctions were gap junctions which had a regular hexagonal packing pattern. This codistribution of gap and septate junction types is discussed in light of current theories that the gap junction types are alternative forms of the same structure in different metabolic environments. In addition to these gap and septate junctions a new junction, perhaps a modified pleated septate junction, is described.     

Topological analysis of the major protein in isolated intact rat liver gap junctions and gap junction-derived single membrane structures

The topological organization of the major rat liver gap junction protein has been examined in intact gap junctions and gap junction-derived single membrane structures. Two methods, low pH and urea at alkaline pH, were used to "transform" or "split" double membrane gap junctions into single membrane structures. Low pH treatment "transforms" rat liver gap junctions into small single membrane vesicles which have an altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after digestion with L-1-to-sylamido-2-phenylethylchloromethyl ketone-trypsin. Alkaline pH treatment in the presence of 8 M urea can split isolated rat liver gap junctions into single membrane sheets which have no detectable structural alteration or altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile after proteolytic digestion, suggesting that these single membrane sheets may be useful for topological studies of the gap junction protein. Proteolytic digestion studies have been used to localize the carboxyl terminus of the molecule on the cytoplasmic surface of the intact gap junction. However, the amino terminus does not appear to be accessible to proteases or to interaction with an antibody that is specific for the amino-terminal region of the molecule in intact or split gap junctions. Binding of antibodies, that block junctional channel conductance, can be eliminated by proteolytic digestion of intact gap junctions, suggesting that all antigenic sites for these antibodies are located on the cytoplasmic surface of the intact gap junction. In addition, calmodulin gel overlays indicate that at least two calmodulin binding sites exist on the cytoplasmic surface of the junctional protein. The information generated from these studies has been used to develop a low resolution two-dimensional model for the organization of the major rat liver gap junctional protein in the junctional membrane.     

Freeze-fracture studies of gap junctions in the developing and adult amphibian cardiac muscle.

The freeze-fracture technique has been used to characterize the junctional devices involved in the electrical coupling of Ambystoma cardiac tissue. These cells are connected by junctions formed by either linear or circular arrays of particles. Such structures can be interpreted as a special type of gap junction. Gap junctions have also been investigated during the growth and differentiation of two amphibians, Rana and Xenopus. In both genera the earliest stage of junctional assembly is characterized by linear rows of particles. Later, a gradual transformation of these linear rows into circles was found. Finally, in the fully formed gap junctions, these circles appeared to join together into clusters. In summary, in the adult amphibian myocardial cells, three different types of gap junctions can be described. The first type, which has been observed in all embryonic stages and in adults in all three genera, consists of linear or circular arrays of particles: this is the only type of gap junction seen at any age in Xenopus. The second type, consisting of a variable number of anastomosing circles forming regular networks, is never observed in embryonic cells. It is typical of the adult frog heart and may also be seen in Ambystoma. The third type is characteristic only of adult Ambystoma heart and consists of geometrically packed particles identifiable with classic communicating macula. The fact that only the first class of structure is observed in Xenopus heart strongly supports the conclusion that such linear arrays of intramembranous particles really represent true functional electrical junctions.     

The role of cytoskeletal components in the maintenance of intercellular junctions in an insect

Summary Accessory glands of the cockroach are composed of secretory and supportive cells, the latter providing a skeleton-like framework of attentuated cytoplasmic processes into which the former are positioned. These two cell types are associated with one another laterally by adhaering, pleated septate, and gap junctions. Hemi-adhaerens junctions are also found on both luminal and basal surfaces of the gland; the former are associated with the cuticular lining of the lumen and the latter with extracellular matrix. The adhering and septate junctions are flanked by both filaments and microtubules; the former insert into the junctional membranes and are actin-like, binding both rhodamine-conjugated phalloidin and the S₁ subfragment of rabbit heavy meromyosin. The role of this cytoskeletal protein with the cellular junctions has been explored by treatment with a disruptive agent, cytochalasin D. Dissociation of actin leads to changes in septate junctions and in microtubular distribution. This suggests that the latter act as anchors for the actin filaments which, in turn, appear bound to certain of the intramembranous junctional components.Supported by a Conicet/Royal Society Visiting Fellowship     

Change of form of septate and gap junctions during development of the insect midgut

In insects, smooth septate junctions join cells derived from the embryonic midgut, and pleated septate junctions are found in all other tissues. Relatively little is known about either type of septate junction or the relationship between them, but they have been treated as two different junctions in the literature. The gap junctions which are associated with these septate junctions also differ. Crystalline gap junctions are found in the midgut, associated with smooth septate junctions, and irregular gap junctions are found in tissues where pleated septate junctions are located. We have examined the development of smooth septate junctions and crystalline gap junctions and the relationship between them, by studying the embryogenesis of the midgut in Manduca sexta (tobacco hornworm). At 56 hr of development (hatching is at 104 hr) pleated septate junctions and irregular gap junctions joined the midgut epithelial cells. At 65 hr, the septate junctions had disappeared, but gap junctions persisted. At 70 hr, smooth septate junctions had replaced the earlier pleated septate junctions and gap junctions associated with these smooth septate junctions were often of the crystalline form. In later embryos, the smooth septate junctions matured and enlarged, while all gap junctions became crystalline in form.     

The Ly6 protein coiled is required for septate junction and blood brain barrier organisation in Drosophila

Background

Genetic analysis of the Drosophila septate junctions has greatly contributed to our understanding of the mechanisms controlling the assembly of these adhesion structures, which bear strong similarities with the vertebrate tight junctions and the paranodal septate junctions. These adhesion complexes share conserved molecular components and have a common function: the formation of paracellular barriers restraining the diffusion of solutes through epithelial and glial envelopes.

Methodology/Principal Findings

In this work we characterise the function of the Drosophila cold gene, that codes for a protein belonging to the Ly6 superfamily of extracellular ligands. Analysis of cold mutants shows that this gene is specifically required for the organisation of the septate junctions in epithelial tissues and in the nervous system, where its contribution is essential for the maintenance of the blood-brain barrier. We show that cold acts in a cell autonomous way, and we present evidence indicating that this protein could act as a septate junction component.

Conclusion/Significance

We discuss the specific roles of cold and three other Drosophila members of the Ly6 superfamily that have been shown to participate in a non-redundant way in the process of septate junction assembly. We propose that vertebrate Ly6 proteins could fulfill analogous roles in tight junctions and/or paranodal septate junctions.     

Structure of coupled and uncoupled cell junctions

Cells of Chironomus salivary glands and Malpighian tubules have junctions of the "septate" kind. This is the only kind of junction discerned which is large enough to effect the existing degree of intercellular communication. The electron microscopic observations of the "septate" junction conform to a honeycomb structure, with 80-A-thick electron-opaque walls and 90-A-wide transparent cores, connecting the cellular surface membranes. A projection pattern of light and dark bands (the "septa") with a 150-A periodicity results when the electron beam is directed normal to any set of honeycomb walls. Treatment of the salivary gland cells with media, which interrupt cellular communication (without noticeable alteration of cellular adhesion) by reducing junctional membrane permeability or perijunctional insulation, produces no alterations in the junctional structure discernible in electron micrographs of glutaraldehyde-fixed cell material.     

Gap junction channel protein innexin 2 is essential for epithelial morphogenesis in the Drosophila embryo

Direct communication of neighboring cells by gap junction channels is essential for the development of tissues and organs in the body. Whereas vertebrate gap junctions are composed of members of the connexin family of transmembrane proteins, in invertebrates gap junctions consist of Innexin channel proteins. Innexins display very low sequence homology to connexins. In addition, very little is known about their cellular role during developmental processes. In this report, we examined the function and the distribution of Drosophila Innexin 2 protein in embryonic epithelia. Both loss-of-function and gain-of-function innexin 2 mutants display severe developmental defects due to cell death and a failure of proper epithelial morphogenesis. Furthermore, immunohistochemical analyses using antibodies against the Innexins 1 and 2 indicate that the distribution of Innexin gap junction proteins to specific membrane domains is regulated by tissue specific factors. Finally, biochemical interaction studies together with genetic loss- and gain-of-function experiments provide evidence that Innexin 2 interacts with core proteins of adherens and septate junctions. This is the first study, to our knowledge, of cellular distribution and protein-protein interactions of an Innexin gap junctional channel protein in the developing epithelia of Drosophila.     

THE CELL JUNCTION IN A LAMELLIBRANCH GILL CILIATED EPITHELIUM : Localization of Pyroantimonate Precipitate

The junctional complex in the gill epithelium of the freshwater mussel (Elliptio complanatus) consists of an intermediary junction followed by a 2–3 µ long septate junction. Homologous and heterologous cell pairs are connected by this junction. After fixation with 1% OsO₄ containing 1% potassium pyroantimonate, electron microscopy of the gill reveals deposits of electron-opaque precipitate, specifically and consistently localized along cellular membranes. In both junctional and nonjunctional membrane regions, the precipitate usefully outlines the convolutions without obliterating the 150 A intercellular space, which suggests the rarity or absence of either vertebrate-type gap or tight junctions along the entire cell border. The precipitate appears on the cytoplasmic side of the limiting unit membranes of frontal (F), laterofrontal (LF), intermediate (I), lateral (L), and postlateral (PL) cells. The membrane surfaces of certain vesicles of the smooth endoplasmic reticulum, of multivesicular bodies, and of mitochondrial cristae contain precipitate, as does the nucleolus. In other portions of the cell, precipitate is largely absent. The amount of over-all deposition is variable and depends on the treatment of the tissue prior to fixation. Deposition is usually enhanced by pretreatment with 40 mM NaCl as opposed to 40 mM KCl, which suggests that the precipitate is in part sodium pyroantimonate. Treatment with 0.2 mM ouabain does not enhance deposition. Regional differentiation of cell membranes with respect to their ability to precipitate pyroantimonate is found in at least three instances: (a) between the ciliary membranes and other portions of the cell membrane: the precipitate terminates abruptly at the ciliary base, (b) between the LF and I cell borders: the precipitate is asymmetric, favoring the LF side of the junction, and (c) between the septate junctional membrane and adjacent membrane: the precipitate occurs periodically throughout the septate junction region with the periodicity corresponding to the spacing of the septa. This suggests that different regions of the cell membrane may have differing ion permeability properties and, in particular, that the septa may be the regions of high ion permeability in the septate junction.     

Further studies on the junctional complex in the intestine of Sagitta setosa

Summary The intramembrane structures of the pleated septate junction which occur in the junctional complex of the intestine of the chaetognath Sagitta setosa have been investigated.The pleated septate junction is made up of linear rows of irregularly shaped and sized particles, often fused into short rods, and pits which can be fused into furrows. The distribution of these structures on E and P faces depends upon the preparative methods used. Many of the morphological characteristics are the same as those of the lower invertebrate pleated septate junction type defined by Green (1981a). The physiological significance of this junction is obscure.On the basis of the presence of septate junctions (both of the paired septate junction and pleated septate junction types) which have mainly morphological characteristics of the lower invertebrate pleated septate junction we can add to the hypothesis that chaetognaths are not related to the molluscs and arthropods.