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.     

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.     

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.     

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.     

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.     

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.     

Septate junctions in imaginal disks of Drosophila: a model for the redistribution of septa during cell rearrangement

The organization of septate junctions during morphogenesis of imaginal disks is described from freeze-fracture replicas and thin sections with a view to understanding junction modulation during rearrangements of cells in epithelia. The septate junctions of each epithelial cell of the disk are distributed in a number of discrete domains equal to the number of neighboring cells. Individual septa traverse domains of contact between pairs of adjacent cells, turn downwards at the lateral boundary of the domain and run parallel to the intersection with a third cell. This arrangement leaves small channels at three-cell intersections that are occupied by specialized structures termed "tricellular plugs." Cell rearrangement involves a progressive change in the width of contact domains between adjacent cells, until old contacts are broken and new ones established. It is proposed that the septate junction adjusts to the changing width of domains by the compaction or extension of existing septa. This redistribution of septa theoretically allows a transepithelial barrier to be maintained during cell rearrangements. The applicability of this model to other epithelial tissues is discussed.     

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 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.     

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/μm²), 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.     

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.     

A clarification of the two types of invertebrate pleated septate junction

It is confirmed that there are two distinct variations of invertebrate septate junction. The first of these, the ‘lower invertebrate pleated septate junction’, is described fully using conventional thin section, lanthanum tracer and freeze-fracture techniques. The second type, the well-known pleated septate junction characteristic of the molluscs and athropods, is renamed the ‘mollusc-arthropod pleated septate junction’, and is described briefly to allow easier comparison between the two variations. As both types have now been studied in a range of invertebrate phyla the results can be used as a basis for discussing their respective phylogenetic positions. The lower invertebrate pleated septate junction occurs in several groups in the minor phyla immediately above the Coelenterata and in the lower phyla of both the deuterostome and proterostome lineages. The mollusc-arthropod pleated septate junction is restricted to the Mollusca and Arthropoda as its name implies.     

Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control

Epithelial tubes of the correct size and shape are vital for the function of the lungs, kidneys, and vascular system, yet little is known about epithelial tube size regulation. Mutations in the Drosophila gene sinuous have previously been shown to cause tracheal tubes to be elongated and have diameter increases. Our genetic analysis using a sinuous null mutation suggests that sinuous functions in the same pathway as the septate junction genes neurexin and scribble, but that nervana 2, convoluted, varicose, and cystic have functions not shared by sinuous. Our molecular analyses reveal that sinuous encodes a claudin that localizes to septate junctions and is required for septate junction organization and paracellular barrier function. These results provide important evidence that the paracellular barriers formed by arthropod septate junctions and vertebrate tight junctions have a common molecular basis despite their otherwise different molecular compositions, morphologies, and subcellular localizations.     

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.     

Role of the septate junction in the regulation of paracellular transepithelial flow

A comparison of the distribution of septate junctions in invertebrate epithelia and tight junctions in vertebrate systems suggests that these structures may be functionally analogous. This proposition is supported by the internal design of each junction which constitutes a serial arrangement of structures crossing the intercellular space between cells to effectively provide resistance to the paracellular flow of water and small molecules. We have tested the validity of such an analogy by examining whether the osmotic sensitivity of the septate junctions of planarian epidermis follow the rather striking pattern observed for the junctions of very tight vertebrate epithelia (e.g. toad urinary bladder). It has been found that the septate junctions in this system respond in similar fashion to their vertebrate counterparts, blistering with accumulated fluid when the medium outside the epidermis is made hypertonic with small, water-soluble molecules. We conclude that the two types of junction probably are functionally analogous and that, in each case, this rectified structural response to transepithelial osmotic gradients may be indicative of the role of such structures in the transport function of epithelia.     

SPECIALIZED MEMBRANE JUNCTIONS BETWEEN NEURONS IN THE VERTEBRATE CEREBELLAR CORTEX

"Gap" junctions, the morphological correlate for low-resistance junctions, are demonstrated between some mossy fiber terminals and granule cell dendrites in some lower vertebrate cerebella (gymnotid and frog). Most of the gap junctions (GJs) seen in the gymnotid-fish cerebellum exhibit an asymmetrical configuration, the electron-opaque cytoplasmic material underlying the junction being more extensive in the dendritic than in the axonal side. In the frog cerebellum, the GJs have a symmetrical distribution of such electron-opaque material. In both species the GJs are encountered at the same synaptic interface as the conventional synaptic zone (CSZ), constituting "mixed synapses" in a morphological sense. The axonal surface covered by CSZs is larger than that covered by GJs. In mammalian cerebellum, GJs are observed only in the molecular layer, between perikarya, dendrites, or perikarya and dendrites of the inhibitory interneurons. These GJs are intermixed with attachment plates and intermediary junctions interpreted as simply adhesive. In the mammalian cerebellum, a new type of junction which resembles the septate junctions (SJs) of invertebrate epithelia is observed between axonal branches forming the tip of the brush of basket fibers around the initial segment of the Purkinje cell axon. It is suggested that such junctions may be modified forms of septate junctions. The physiological implications of the possible existence of high-resistance cross-bridges between basket cell terminals, which may compartmentalize the extracellular space and thus regulate extracellular current flow, must be considered.     

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 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 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.     

Junctional complexes between the Sertoli cells in the testis of the crayfish,Procambarus paeninsulanus

The testis of the crayfish,Procambarus paeninsulanus, was prepared for light and electron microscopic study. It is composed of tubules containing germ-spermatogenic and somatic-Sertoli cells. In sections of tubules lacking sperm, the Sertoli cells rest on the basement membrane. A desmosome-like junction is found near the luminal surface between two adjacent Sertoli cells. It is closely associated with a long, septate junction. Between Sertoli cells which have surrounded numerous spermatids, the undulating membranes exhibit profiles of pleated septate junctions in tangential sections. The morphology of the pleated septate junctions between adjacent Sertoli cells suggests a possible role as a permeability barrier.