Smooth Septate Junction Research Articles

Visualization of RNA Transcripts in Western Corn Rootworm (Diabrotica virgifera virgifera) and Plants by In Situ Hybridization.

In situ hybridization (ISH) is a methodology by which nucleic acids are detected within fixed tissue samples. Recent advances in detection technology and target recovery have greatly enhanced the technique's ability to detect single mRNA molecules. Here we detail the fixation, paraffin embedding, sectioning, target recovery, and chromogenic detection of an mRNA (DvSSJ1), encoding for a membrane protein associated with the smooth septate junction (SSJ) in Western corn rootworm [Diabrotica virgifera (Dv)]. Further, we demonstrate, the expression of dsRNA of DvSSJ1 in maize root tissues using signal amplification and background suppression technology.

Molecular basis for epithelial morphogenesis and ion transport in the Malpighian tubule.

During development, the insect Malpighian tubule undergoes several programmed morphogenetic events that give rise to the tubule's ability to transport ions and water at unparalleled speed. Studies in Diptera, in particular, have greatly increased our understanding of the molecular pathways underlying embryonic tubule development. In this review, we discuss recent work that has revealed new insights into the molecular players required for the development and maintenance of structurally and functionally intact adult Malpighian tubules. We highlight the contribution of the smooth septate junction (sSJ) proteins to the morphogenesis and transport function of the epithelial cells of the Drosophila melanogaster Malpighian tubule and also discuss new findings on the role of the GATAe transcription factor. We also consider the roles of sSJ proteins in the fly midgut, as compared to the Malpighian tubule, and the importance of cellular context for the functions of these proteins.

Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli

ABSTRACT RNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in Escherichia coli and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in E. coli embedded within the very stable scaffold of Eggplant latent viroid (ELVd), a small circular non-coding RNA. Stability in E. coli of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from Tetrahymena thermophila as a spacer. RNA circularization and large-scale accumulation in E. coli cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to smooth septate junction 1 (DvSSJ1) gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second T. thermophila self-splicing intron in a permuted form.

The Snakeskin-Mesh Complex of Smooth Septate Junction Restricts Yorkie to Regulate Intestinal Homeostasis in Drosophila.

SummaryTight junctions in mammals and septate junctions in insects are essential for epithelial integrity. We show here that, in the Drosophila intestine, smooth septate junction proteins provide barrier and signaling functions. During an RNAi screen for genes that regulate adult midgut tissue growth, we found that loss of two smooth septate junction components, Snakeskin and Mesh, caused a hyperproliferation phenotype. By examining epitope-tagged endogenous Snakeskin and Mesh, we demonstrate that the two proteins are present in the cytoplasm of differentiating enteroblasts and in cytoplasm and septate junctions of mature enterocytes. In both enteroblasts and enterocytes, loss of Snakeskin and Mesh causes Yorkie-dependent expression of the JAK-STAT pathway ligand Upd3, which in turn promotes proliferation of intestinal stem cells. Snakeskin and Mesh form a complex with each other, with other septate junction proteins and with Yorkie. Therefore, the Snakeskin-Mesh complex has both barrier and signaling function to maintain stem cell-mediated tissue homeostasis.

Molecular characterization of the insecticidal activity of double-stranded RNA targeting the smooth septate junction of western corn rootworm (Diabrotica virgifera virgifera).

The western corn rootworm (WCR, Diabrotica virgifera virgifera) gene, dvssj1, is a putative homolog of the Drosophila melanogaster gene, snakeskin (ssk). This gene encodes a membrane protein associated with the smooth septate junction (SSJ) which is required for the proper barrier function of the epithelial lining of insect intestines. Disruption of DVSSJ integrity by RNAi technique has been shown previously to be an effective approach for corn rootworm control, by apparent suppression of production of DVSSJ1 protein leading to growth inhibition and mortality. To understand the mechanism that leads to the death of WCR larvae by dvssj1 double-stranded RNA, we examined the molecular characteristics associated with SSJ functions during larval development. Dvssj1 dsRNA diet feeding results in dose-dependent suppression of mRNA and protein; this impairs SSJ formation and barrier function of the midgut and results in larval mortality. These findings suggest that the malfunctioning of the SSJ complex in midgut triggered by dvssj1 silencing is the principal cause of WCR death. This study also illustrates that dvssj1 is a midgut-specific gene in WCR and its functions are consistent with biological functions described for ssk.

Salinity alters snakeskin and mesh transcript abundance and permeability in midgut and Malpighian tubules of larval mosquito, Aedes aegypti

This study examined the distribution and localization of the septate junction (SJ) proteins snakeskin (Ssk) and mesh in osmoregulatory organs of larval mosquito (Aedes aegypti), as well as their response to altered environmental salt levels. Ssk and mesh transcripts and immunoreactivity were detected in tissues of endodermal origin such as the midgut and Malpighian tubules of A. aegypti larvae, but not in ectodermally derived hindgut and anal papillae. Immunolocalization of Ssk and mesh in the midgut and Malpighian tubules indicated that both proteins are concentrated at regions of cell-cell contact between epithelial cells. Transcript abundance of ssk and mesh was higher in the midgut and Malpighian tubules of brackish water (BW, 30% SW) reared A. aegypti larvae when compared with freshwater (FW) reared animals. Therefore, [3H]polyethylene glycol (MW 400Da, PEG-400) flux was examined across isolated midgut and Malpighian tubule preparations as a measure of their paracellular permeability. It was found that PEG-400 flux was greater across the midgut of BW versus FW larvae while the Malpighian tubules of BW-reared larvae had reduced PEG-400 permeability in conjunction with increased Cl− secretion compared to FW animals. Taken together, data suggest that Ssk and mesh are found in smooth SJs (sSJs) of larval A. aegypti and that their abundance alters in association with changes in epithelial permeability when larvae reside in water of differing salt content. This latter observation suggests that Ssk and mesh play a role in the homeostatic control of salt and water balance in larval A. aegypti.

Discovery of midgut genes for the RNA interference control of corn rootworm.

RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets - dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by “blebbing” of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality.

Additional Ultrastructural Observations of the First Segments of Malpighian Tubules in Cenocorixa bifida (Hemiptera: Corixidae) in Relation to Reabsorption of Solutes

Abstract The ultrastructure of the first segment of the four-segmented Malpighian tubules of Cenocorixa bifida Hungerford (Hemiptera: Corixidae) is described. The first segment of each of the four tubules empties proximally into the midgut—hindgut junction. Its squamous epithelium is composed of a single layer of binucleated cells containing two types of mitochondria. The cells rest on a prominent basal lamina and possess short, shallow, widely spaced basal plasma membrane infoldings closely associated with large mitochondria and a few short blunt microvilli devoid of mitochondria projecting into the lumen. In sharp contrast, the fourth segment cells that are secretory in function, display numerous, long basal membrane infoldings that penetrate deeply into the cytoplasm and numerous long microvilli at the luminal border. The cytoplasm of the first segment cells contains dense bodies, small mitochondria, microtubules, vesicles, and vacuoles and is rich in smooth endoplasmic reticulum, giving rise to clear vacuoles, but rough endoplasmic reticula and Golgi apparati are sparse. The apical plasma membrane is covered with glycocalyx and displays coated pits pinching off coated vesicles. The apical cytoplasm displays numerous vesicles containing fine granular material, smooth vesicles, dense tubules, large vacuoles, primary lysosomes, and multivesicular bodies. The lateral plasma membranes are linked by extensive junctional complexes each composed of an apical smooth septate junction, intercalated gap junctions, and a basal heterocellular scalariform junction. The contrast in ultrastructural features between of the first segment and fourth segment cells suggests that the first segment is involved in reabsorption than secretion of inorganic and organic solutes.

The Development of Cellular Junctions in the Drosophila Embryo

The pattern and development of cellular junctions in the different tissues of the Drosophila embryo from the blastoderm stage until hatching were analyzed. The cellular junctions found include: gap junctions, two types of septate junctions, and several types of cell-cell and cell-substrate adherens junctions. During early and mid embryogenesis (stages 4 to 13) only spot adherens junctions, gap junctions, and zonulae adherentes prevail. Scattered spot adherens junctions are already formed at the blastoderm stage. During and shortly after gastrulation, spot adherens junctions become concentrated at the apical pole and fuse into continuous zonulae adherentes in the posterior endoderm and the ectoderm. In addition to the zonulae adherentes, ectodermally derived epithelia possess scattered gap junctions and form pleated septate junctions and hemiadherens junctions during late embryogenesis (stages 14 to 17). Mesenchymal tissues (i.e., all nonepithelial tissues of the embryo, including the neural primordium and, transiently, the mesoderm and endoderm) possess both spot adherens junctions and gap junctions at a low frequency. Initially, the midgut epithelium does not establish a junctional complex and possess only gap junctions and spot adherens junctions. Only late in development does a circumferential smooth septate junction develop; zonulae adherentes are missing. The various derivatives of the mesoderm express spot adherens junctions, hemiadherens junctions, and gap junctions, but never zonulae adherentes or septate junctions. After organogenesis, several different types of tissue-specific adherens junctions are formed, among them connecting hemiadherens junctions (between gut epithelium and visceral muscle and early during the formation of the muscle tendon junction), muscle tendon junctions (between somatic muscle and tendon cells), fasciae adherentes (between the cells of both the visceral muscle and the dorsal vessel), and autocellular nephrocyte junctions (in nephrocytes). Interesting exceptions to the general pattern of junctional development are provided by the outer epithelial layer of the proventriculus and the Malpighian tubules. Both tissues develop as typical ectodermal epithelia and possess zonulae adherentes. During late embryogenesis, both epithelia lose the zonulae adherentes and form smooth rather then pleated septate junctions, thereby expressing a junctional complex similar to that of the endodermally derived midgut epithelium.

Unusual cytoskeletal association with the intercellular septate junction in the midgut of Collembola (Insecta : Apterygota)

Isotomurus palustris (Apterygota : Collembola) has a midgut epithelium with a discontinuous apical fibrous belt-like structure formed by filaments, 7–10 nm in diameter. This web structure receives the microvillar core rootlets, and is anchored to the lateral plasma membrane at junctional level. The filamentous material of the belt structure is not affected by Triton X-100 and cytochalasin B treatments; immunoelectronmicroscopy with an anti-actin monoclonal antibody did not reveal actin at the web level, while it specifically labelled the microvillar content. The microvillar filamentous core, but not the filamentous material of the web, is decorated by S 1 fragment of rabbit heavy meromysin. Comparative SDS-gel electrophoresis of Triton-resistant extracts of midguts from Isotomurus palustris and Orchesella villosa, a species devoid of the terminal web, showed similar electrophoretic patterns, with actin as a major component; however, an additional prominent band at 40 kDa is selectively expressed only in I. palustris, and it has been therefore considered as the putative component of the terminal web. While the intercellular septa of the smooth septate junction present in midgut cells of I. palustris do not seem altered by the presence of the web structure, the intramembrane organization of the junction is modified and the typical appearance of the smooth septate junction is not visible. Many additional intramembranous particles are present on the P-fracture face of the membrane. All the results suggest that the filamentous material of the web may be made up of proteins related to intermediate filaments; in addition, the adhesion of the web structure to the plasma membrane substantially alters the appearance of the smooth septate junction.

Isolation and characterization of invertebrate smooth septate junctions.

Using modifications of techniques used for the isolation of macula type intercellular junctions (gap junctions and desmosomes) the arthropod smooth septate junction has been isolated from insect midgut tissue. Midguts from cockroaches or mealworms were used and membrane fractions were obtained by sucrose gradient and ultracentrifugation techniques. Preparations with reasonable concentrations of septate junction were obtained and have been studied by thin-section, negative-stain and freeze-fracture electron microscopy. The junctions appeared to be well preserved, although there was evidence that the junction strands were able to slide within the plane of the membrane. Septa were seen to have a cross-striated appearance when viewed after negative staining but their exact structure remained difficult to determine. Polyacrylamide gel electrophoretic studies demonstrated the reproducibility of the isolation procedure and showed that septa may have a 47 000 molecular weight glycoprotein component. Gel electrophoresis also gave some indication of the intramembrane biochemistry of the smooth septate junction, with proteins of 31 000 and 32 000 molecular weight always occurring in the junction fractions. The junctions were, however, very sensitive to both mechanical and chemical treatments, the septa were destroyed by rough homogenization or by treatment with urea at a concentration as low as 1 M. Freeze-fracture of untreated, isolated junctions demonstrated no differences from junctions in intact tissue, while replicas of urea-treated material were more difficult to interpret as the component parts of the junctions became separated once the septa had been destroyed. Gap junctions were also obtained and resisted both mechanical and chemical treatment, which destroyed the septate junctions. Their major protein component appeared to have a molecular weight of 36 000. Attempts to isolate pleated septate junctions (from insects, molluscs and annelids) by the same techniques failed, implying a significant difference in the structures of the two types of septate junction.

A freeze fracture and thin section study of intestinal cell membranes and intercellular junctions of a nematode, Ascaris

The microvillar and lumenal plasma membrane P-face of Ascaris intestinal cells is shown to be covered by relatively large (13 nm) particles at a fairly high density (1000/μm 2), while the E-face has virtually none. The P-face of the lateral cell membranes, those separating the cells, have fewer and smaller (8 nm) particles. The intestinal cells are also shown to be connected by an apical complex of smooth septate and tricellular junctions similar to those found between some insect midgut cells. A periodic layer of tannic acid staining material is found on the cytoplasmic sides of the smooth septate junction, and when the intercellular space is filled with lanthanum, smoothly curved, 10 nm wide septal walls can be seen. Below the belt of septate junctions are a large number of gap junctions. These have closely packed arrays of particles on the P-face with some particle aggregates adhering to the closely packed pit arrays on the E-face.

Phylogenetic relationships within the invertebrata in relation to the structure of septate junctions and the development of ‘occluding’ junctional types

ABSTRACT The structures of 13 variants of invertebrate septate junction are reviewed on the basis of freeze-fracture, lanthanum tracer and thin-section studies. In addition, a simple type of occluding junction in the phylum Porifera, a variation of tight junction in the phylum Tunicata and the vertebrate tight junction are covered. All the junctions considered form a belt around the apical circumference of cells lining a lumen or an exterior surface. The large number of these junctions now recognized permits discussion relating to invertebrate classification and suggested phylogenetic relationships, and to the development of intercellular junctions. The relationships revealed are discussed under three headings: Coelenterates and lower invertebrates, Proterostomia (the annelid, molluscan and arthropod lineage) and the Deuterostomia (the echinoderm and chordate lineage). It is proposed that the pleated septate junction of the lower invertebrates resembles that of the hydrozoan rather than anthozoan Coelenterates. This lower invertebrate pleated septate junction occurs in several lower invertebrate phyla including the Annelida (of the proterostome lineage), but also occurs in the Sipunculoidea, a group supposedly on the deuterostome lineage. The proterostome line includes the molluscs and the arthropods, which have the molluscarthropod pleated septate junction. Several variations of the smooth septate junction are also seen in Arthropoda. Among the deuterostomes the Chaetognatha have both a paired septate junction and a pleated junction and are therefore considered to be not very far removed from the Sipunculoidea. The echinoderms and hemichordates also have double-septum septate junctions. In addition however, these two phyla have anastomosing septate junctions that are very similar, varying only in their final configuration. Of the two, the echinoderm anastomosing septate junction most closely resembles the tight junction seen in the tunicates, and the Hemichordata are therefore considered to be a lateral development from the main line of chordate evolution. The tunicates have a tight junction similar to that seen in vertebrates; it is however more ‘leaky’ and has distinctive freeze-fracture characteristics. In the phylum Porifera a form of simple parallel membrane junction appears to serve an occluding function. This junction has regular intercellular spacing in the absence of any septa and it is suggested that the spacing in septate junctions is probably not dictated by the septa. This interpretation is reasonable particularly when the diversity of septal types in conjunction with stable intercellular spacing is considered. Finally, a theory is put forward suggesting that in evolution a change from the septate to the tight junction could simply involve a modification of a ‘membrane spacing factor’, which allows the membranes of adjacent cells to come together at intervals, in the normal tight junction pattern.

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.

The structure and function of the smooth septate junction in a transporting epithelium: The malpighian tubules of the new zealand glow-worm Arachnocampa luminosa

Junctional complexes between the epithelial cells in the four distinct regions of the glow-worm Malpighian tubule were investigated by electron microscopy using thin sectioning, freeze-fracturing, osmotic disruption and tracer techniques. The lateral plasma membranes of all four cell types are joined by smooth septate junctions but the extent of the complex across the cell depth varies in the four different regions. The width of the septa, the interseptal spacing and the separation between the outer leaflets of the adjacent plasma membranes are different for each cell type. Gap junctions were identified only in the junctional complex between Type IV cells and were intercalated amongst large lateral sinuses. In oblique sections of lanthanum infiltrated tissue, the electron-lucent septa at the basal side of the junction are outlined by the tracer as it penetrates. In the Junctional complexes of all four regions the septa appear as short, distinct, linear bars. In tangential sections of gap junctions between Type IV cells, the junctions appear as a hexagonal array of intermembrane particles with a centre to centre spacing of 18 nm. Horseradish peroxidase did not penetrate the junctional complexes very far but readily passed through the basal lamina into the spaces between extracellular invaginations of the basement membrane of the cells. Junctional complexes in all four areas of the tubule have similar freeze-fracture faces. In freeze-fracture replicas of fixed tissue continuous ridges of fused particles are seen on the P face and complementary furrows are found on the E face. Junctional response to osmotically adjusted Ringer solutions was similar in all four cell types. Distortion or ‘blistering’ of the intercellular space between the septa of the junction occurred when the tissue was bathed in or injected with a hypertonic Ringer solution. The structure of these junctions, visualized by the different techniques, and the role of the septate junction in a transporting epithelium, are discussed.

Topographical variations in the structure of the smooth septate junction.

Smooth septate junctions in the midgut of Musca domestica and in Malpighian tubules of both Musca and Rhodnius prolixus are described. Details of the structures revealed after standard fixation, fixation in the presence of the stain, lanthanum hydroxide, and after freeze-fracture are discussed in the light of models previously put forward to explain the interrelations of the images obtained by these different methods. The organization of the junction between cells of the midgut varies in the apical-to-basal axis. At the apical border the septa (or ridges in freeze-fracture replicas) are packed tightly and follow an undulating but strictly parallel course. This packing loosens towards the middle of the junction until, at its basal extremity, the septa (ridges in replicas) are widely separated and follow independent meandering courses. That these features are found both in lanthanum-infiltrated specimens and freeze-fracture replicas allows a correlation to be made between the septa and the freeze-fracture ridges. The functional significance of these smooth septate junctions is discussed.

Effect of osmotic gradients on intercellular junctions of the toad bladder

Effect of osmotic gradients on intercellular junctions of the toad bladder