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.     

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.     

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 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).     

Junctional structures in digestive epithelia of a cephalopod

Intercellular junctions have been studied in the epithelia of digestive organs of Sepia officinalis (digestive gland, digestive duct appendages and caecum) by conventional staining, lanthanum tracer and freeze-fracturing techniques. In the three organs studied the same junctional complex occurs, consisting of a belt desmosome, a septate junction and gap junctions. The septate junction is of pleated-sheet type and the gap junction has its particles on the P face of the fracture. Circular structures have been found in the digestive gland septate junctions. Neither continuous nor tight junctions have been found. These results show that Cephalopods have junctional structures very close to those of other Molluscs and of Annelids. Some small differences between the septate junctions of the three organs could be related to their different physiology.     

The terminal bar in the larval midgut epithelium ofAeshna cyanea

Summary Thin section and freeze-fracture electron microscopy revealed that the terminal bars of the larval midgut epithelium ofAeshna cyanea consisted of extended smooth septate junctions (SSJ), multiple adhesive junctions and rare gap junctions. Freeze-fractures of native tissue suggested that the septal building units were anchored only in the external membrane leaflet by partially integrated proteins while the interseptal pegs were anchored partly in both leaflets by completely integrated proteins and partly by presumed peripheral proteins.Reversible depletion of the physiological Ca⁺⁺ concentration had no apparent structural effect on the SSJ of the terminal bars, but led to a reversible formation of junctional septa between the foot processes concomitant with a rearrangement of IMPs in the basolateral plasma membranes. The basolateral SSJ assembly and disassembly induced by reversible Ca⁺⁺ deprivation was interpreted as exaggerated response of an intrinsic capability normally related to the apical growth of regenerative cells and to the extrusion of degenerating cells. Lanthanum tracer ingested with hyperosmotic drinking solution was always found excluded from the basolateral intercellular spaces underneath the terminal bar, but there was a dual effect on the SSJ structure. Part of the junctions remained structurally intact, part was dissociated in the apical portion and invaded by tracer.Abbreviations EF exoplasmic fracture face - EGTA ethylenglycol-bis(2-aminoethylether)-N,N-tetraacetic acid - IMP intramembrane particle - PAS periodic acid Schiff reagent - PF protoplasmic fracture face - PSJ pleated septate junction - SDS sodium dodecyl sulphate - SSJ smooth septate junction Dedicated to Prof. Dr. E.Scholtyseck in honour of his 65th birthday.     

Different localizations of 21 and 27 kDa gap-junction proteins in rat salivary glands

Antibodies against 21 and 27 kDa gap-junction proteins from rat liver were used to examine the identification and localization of gap-junction proteins in rat salivary glands. Acinar cells of the submandibular glands and parotid glands stained well for the 27 kDa gap junction protein and less intensely for the 21 kDa protein. Acinar cells of the sublingual glands were stained heavily for the 27 kDa gap junction protein and stained well for 21 kDa gap junction protein. No 27 kDa protein was observed in the ducts of the salivary glands. The 21 kDa gap-junction protein was distributed in some of the intercalated ducts in the parotid and submandibular glands. Immunoblotting of an extract of parotid glands with antibodies against 21 and 27 kDa gap-junction proteins revealed the presence of 21 and 27 kDa proteins in the parotid glands. It is concluded that the 27 kDa gap-junction protein in tistributed as a major component of the gap junctions in the acinar cells of all the salivary glands; the 21 kDa protein is localized as a minor component in the acinar cells and some portions of the intercalated ducts in the salivary glands. It is possible that these gap-junction proteins might contribute to the regulation of function of the salivary glands.     

Glial cell contacts in insects: Effects of feeding on intercellular junctions

The intercellular junctions associated with the modified glial cells of the perineurium have been examined in the ganglia and main abdominal nerves of the blood-sucking bug Rhodnius prolixus, both before and and after feeding, by means of freeze-fracture and tracer studies. It was found that the pleated septate junctions found in the main abdominal nerve have many fewer septa than those found in the ganglion. These junctions appear to provide the flexibility needed for the movement of cells which occurs to accommodate the tremendous increase in body size that takes place after a bloodmeal. On feeding and during the subsequent period of digestion the nerves stretch to double their length, yet the blood-brain barrier is maintained throughout. In the same manner as loosely interconnected tight junctions, septate junctions with fewer septa seem to form a junction which is able to respond readily to the stress of stretching. With feeding and afterwards the septate junctions become disorganized and disassemble, while the gap junctions and tight junctions remain intact. It is envisaged, therefore, that the primary function of the septate junction is adhesive.     

Analysis of the rat liver gap junction protein: clarification of anomalies in its molecular size

The major gap junction polypeptide in most tissues has an apparent molecular mass of 27 kDa with a 47 kDa dimer present in junction-enriched fractions. However, a 54 kDa protein recognized by gap junction-specific antibodies has been reported and a complementary DNA (cDNA) sequence for both human and rat liver gap junctions codes for a 32 kDa protein. In this paper we show that these are all forms of the same gap junction protein that can be observed on SDS-polyacrylamide gels simply by varying the concentration of acrylamide in the gels. A 64 kDa dimer is also obtainable. Antibodies to the gap junction protein or to a synthetic peptide constructed to match the rat liver gap junction amino-terminal sequence recognize all of these forms. Under some conditions a 54 kDa dimer is 'preferred', explaining the presence of this species in whole tissue homogenate Western blots. These results clarify several controversies and indicate that the protein forming the gap junction channel probably undergoes no major post-translational modification as the cDNA sequence codes for a protein of molecular mass 32 kDa and this protein species and its 64 kDa dimer are demonstrable on SDS-polyacrylamide gels under appropriate conditions.     

Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila

Iron is an essential element in many biological processes. In vertebrates, serum transferrin is the major supplier of iron to tissues, but the function of additional transferrin-like proteins remains poorly understood. Melanotransferrin (MTf) is a phylogenetically conserved, iron-binding epithelial protein. Elevated MTf levels have been implicated in melanoma pathogenesis. Here, we present a functional analysis of MTf in Drosophila melanogaster. Similarly to its human homologue, Drosophila MTf is a lipid-modified, iron-binding protein attached to epithelial cell membranes, and is a component of the septate junctions that form the paracellular permeability barrier in epithelial tissues. We demonstrate that septate junction assembly during epithelial maturation relies on endocytosis and apicolateral recycling of iron-bound MTf. Mouse MTf complements the defects of Drosophila MTf mutants. Drosophila provides the first genetic model for the functional dissection of MTf in epithelial junction assembly and morphogenesis.     

A variation of the smooth septate junction in sea spiders (Pycnogonida)

A new type of septate junction considered to be a variation of the arthropod smooth septate junction is described in pycnogonid (sea spider) endothermal tissue based on the use of conventional thin-section, lanthanum tracer and freeze-fracture techniques. This new type of septate junction is apparently unique to the Pycnogonida but closely resembles septate junctions previously described in the Merostomata and Collembola. This work in conjunction with previous work suggests that the septa of smooth septate junctions may not be as ‘smooth’ as generally thought and probably have a complex substructure.     

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.     

Junctional specializations in the seminiferous epithelium of an insect (Triatoma infestans): a freeze fracture and lanthanum tracer study

Intercellular junctions in the seminiferous epithelium of the testis of Triatoma infestans were examined by conventional electron microscopy, tannic acid fixation, electron-opaque tracers, and freeze-fracture techniques. Distinctive aspects of the intercellular junctions are described in different regions of the testis follicles. In the basal region, close to the basal lamina, smooth septate junctions intermingled with gap junctions were observed between sustentacular cells. In the parabasal regions of the seminiferous epithelium, plated septate junctions, 'molluscous and arthropod' type (according to the classification of Green, 1981), were observed. Over the above junctions, in the central regions, also located between sustentacular cell membranes, parallel rows of intramembrane particles form successive belts of widely spaced septate junctions in an atypical configuration. Invertebrate gap junctions were also observed between adjacent spermatocyte membranes.     

Arthropod immune system. III. Septate junctions in the hemocytic capsule of the german cockroach, Blattella germanica

Granulocytes (GRs) and/or plasmatocytes (PLs), the two major immunocytes in arthropods, participate in cellular encapsulation of foreign tissue. Although gap and desmosome junctions have been reported in insect capsules, smooth septate junctions are being reported for the first time by both thin section and freeze-fracture techniques in Blattella germanica. In 7-day-old capsules, the septa are 23 nm thick, faintly 'scalloped' and slightly curved in appearance; the interseptal space has a periodicity of about 5 nm. In freeze-fractured capsules, the septa are associated on both sides with the corresponding intramembranous structures, belonging to the plasma membranes of the two junction-forming GRs. The intercellular space is 27 nm wide. There are 36-40 septa/1 mum junctional length. The junctions show furrows on the extracellular fracture face (E) and the complementary regular rows of intramembranous particles on the cytoplasmic face (P). The septate junctions often occur in the region of the capsule that also shows the presence of gap junctions, but only rarely that of desmosomes. The septate junctions are in close proximity with mitochondria. It is suggested that the function of these junctions is to produce compact capsules.     

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.     

Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia

The Discs large (Dlg) protein of Drosophila is the prototypic member of a growing family of proteins termed membrane-associated guanylate kinase homologs (MAGUKs). The MAGUKs are composed of a series of peptide domains that include one or three DHR/PDZs, an SH3, and a region homologous to guanylate kinase (GUK). We have previously shown that the product of this gene, the Dlg protein, is localized at the septate junctions between epithelial cells, and that mutations in the gene cause neoplastic overgrowth of the imaginal discs. The dlg locus is therefore defined as a tumor suppressor gene. In this paper, we show that the Dlg protein is localized on the cytoplasmic face of the septate junction and is required for the maintenance of this structure. It is also required for proper organization of the cytoskeleton, for the differential localization of membrane proteins, and for apicobasal polarity of epithelial cells. However, these other functions can be uncoupled from Dlg's role as a tumor suppressor since mutations in two domains of the protein, the SH3 and GUK, cause loss of normal cell proliferation control without affecting the other functions of the protein. These results suggest that, besides regulating cellular proliferation, the Dlg protein is a critical component of the septate junctions and is required for maintaining apicobasal polarity in Drosophila epithelium.     

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

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

Expression of connexin43 gap functions between cultured vascular smooth muscle cells is dependent upon phenotype

The smooth muscle cell is the predominant cell type of the arterial media. In the adult vascular system, smooth muscle cells are found primarily in the contractile phenotype, but following injury or during atherosclerotic plaque formation the secretory synthetic phenotype is expressed. Recently it has been shown that gap junction connexin43 messenger RNA levels are six times higher in cultured smooth muscle cells in the synthetic phenotype than in intact aorta. We have modulated rabbit aortic smooth muscle cells in culture between the synthetic phenotype and one resembling the contractile phenotype, and correlated gap junction expression with phenotype. A dual labelling technique with antibodies against smooth muscle myosin and a synthetic peptide constructed to match a portion of the connexin43 gap junction protein was used for these experiments. Gap junctions are numerous between synthetic phenotype cells but few are observed between contractile cells. Rat aortic smooth muscle cells were also cultured and the growth and structure of gap junctions followed in the synthetic phenotype by use of freeze-fracture electron microscopy and immunohistochemical techniques. Junctional plaques are similar in structure to those observed in cardiac muscle, their size and number increasing with time in culture. The increased numbers of gap junctions between synthetic phenotype smooth muscle cells may be important during vessel development, following injury, or in atherosclerotic plaque formation.     

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.     

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.