Organization and function of septate junctions

In most cell types, distinct forms of intercellular junctions have been visualized at the ultrastructural level. Among these, the septate junctions are thought to seal the neighboring cells and thus to function as the paracellular barriers. The most extensively studied form of septate junctions, referred to as the pleated septate junctions, is ultrastructurally distinct with an electron-dense ladder-like arrangement of transverse septa present in invertebrates as well as vertebrates. In invertebrates, such as the fruit fly Drosophila melanogaster, septate junctions are present in all ectodermally derived epithelia, imaginal discs, and the nervous system. In vertebrates, septate junctions are present in the myelinated nerves at the paranodal interface between the myelin loops and the axonal membrane. In this review, we present an evolutionary perspective of septate junctions, especially their initial identification across phyla, and discuss many common features of their morphology, molecular organization, and functional similarities in invertebrates and vertebrates.     

Pleated septate junctions in leech photoreceptors: ultrastructure, arrangement of septa, gate and fence functions

The leech photoreceptor forms a unicellular epithelium: every cell surrounds an extracellular “vacuole” that is connected to the remaining extracellular space via narrow clefts containing pleated septate junctions. We analyzed the complete structural layout of all septa within the junctional complex in elastic brightfield stereo electron micrographs of semithin serial sections from photoreceptors infiltrated with colloidal lanthanum. The septa form tortuous interseptal corridors that are spatially continuous, and open ended basally and apically. Individual septa seem to be impermeable to lanthanum; interseptal corridors form the only diffusional pathway for this ion. The junctions form no diffusion barrier for the electron-dense tracer Ba²⁺, but they hinder the diffusion of various hydrophilic fluorescent dyes as demonstrated by confocal laser scanning microscopy (CLSM) of live cells. Even those dyes that penetrate gap junctions do not diffuse beyond the septate junctions. The aqueous diffusion pathway within the septal corridors is, therefore, less permeable than the gap-junctional pore. Our morphological results combined with published electrophysiological data suggest that the septa themselves are not completely tight for small physiologically relevant ions. We also examined, by CLSM, whether the septate junctions create a permeability barrier for the lateral diffusion of fluorescent lipophilic dyes incorporated into the peripheral membrane domain. AFC₁₆, claimed to remain in the outer membrane leaflet, does not diffuse beyond the junctional region, whereas DiIC₁₆, claimed to flip-flop, does. Thus, pleated septate junctions, like vertebrate tight junctions, contribute to the maintenance of cell polarity.     

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.     

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     

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.     

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.     

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.     

Sealing junctions in a number of arachnid tissues

The junctions present in the central nervous system (CNS), midgut, silk gland and venom gland of arachnids have been investigated. Special care was taken to try to locate tight junctions in tissue other than CNS but they were not found in any of the other tissues. The detailed structure of the junctions present are discussed. The tight junctions present in CNS are somewhat different in appearance and fracturing behaviour to most vertebrate tight junctions and closely resemble only those found in Urochordates (a non-vertebrate chordate). The two types of septate junctions found in the other tissues belong to the pleated septate and smooth septate classes but show some interesting differences. It appears probable that the septate junctions in Arachnida, Merostomata and Myriopoda have different fracturing properties from those found in other arthropods. The finding that only septate junctions are present in most arachnid tissues, although tight junctions are present in CNS, is discussed in the context of the sealing function of septate junctions in invertebrate tissues.     

Septate junctions in the cephalic epidermis of turbellarians (Bipalium)

Summary In the epidermis of turbellarians septate junctions of the pleated sheet type have been demonstrated in conventional thin sections and freeze fractured preparations. The structure of these junctions entirely agrees with that found in molluscs and arthropods.Financially supported by DFG (Sto 75/3,4; We 380/5)     

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.     

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.     

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.     

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.     

Relative humidity affects the intercellular spaces and cell contacts of the kidney epithelium of the terrestrial snail Helix aspersa müller

The effects of relative humidity on hemolymph osmolarity and on kidney ultrastructure are explored in Helix aspersa. The snails are active at 95% relative humidity and less active at 50% relative humidity. The hemolymph osmotic pressure increases with the decrease of relative humidity. Pericardial fluid and hemolymph collected from the heart contain similar amounts of total proteins, and both fluids display hemocyanin molecules in negatively stained preparations. When the snails are kept in an atmosphere of 95% relative humidity, numerous wide intercellular spaces are observed in the single-layered-kidney epithelium. The spaces are almost absent when the snails are kept at 50% relative humidity. It is suggested that prourine is formed through a paracellular junctional pathway across the single-layered kidney epithelium, and that the pericardial cavity is not the site of prourine formation. The septate junctions joining the kidney epithelial cells form a continuous belt of intimate contact in the paracellular pathway of prourine. Long septate junctions with many septa are present in the kidneys of snails from the atmosphere of 50% relative humidity, whereas short septate junctions with fewer septa are found in the kidneys of snails from the atmosphere of 95% relative humidity. It is possible that the longer septate junctions with many septa reduce prourine formation across the kidney sac epithelium.     

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.     

Cell junctions in early embryos of squid (Loligo pealei)

Summary Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7–8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12–13). During germinal layer establishment (stages 12–13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavagestage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.     

Analog of vertebrate anionic sites in blood-brain interface of larval Drosophila

The blood-brain barrier ensures brain function in vertebrates and in some invertebrates by maintaining ionic integrity of the extraneuronal bathing fluid. Recent studies have demonstrated that anionic sites on the luminal surface of vascular endothelial cells collaborate with tight junctions to effect this barrier in vertebrates. We characterize these two analogous barrier factors for the first time on Drosophila larva by an electron-dense tracer and cationic gold labeling. Ionic lanthanum entered into but not through the extracellular channels between perineurial cells. Tracer is ultimately excluded from neurons in the ventral ganglion mainly by an extensive series of (pleated sheet) septate junctions between perineurial cells. Continuous junctions, a variant of the septate junction, were not as efficient as the pleated sheet variety in blocking tracer. An anionic domain now is demonstrated in Drosophila central nervous system through the use of cationic colloidal gold in LR White embedment. Anionic domains are specifically stationed in the neural lamella and not noted in the other cell levels of the blood-brain interface. It is proposed that in the central nervous system of the Drosophila larva the array of septate junctions between perineurial cells is the physical barrier, while the anionic domains in neural lamella are a charge-selective barrier for cations. All of these results are discussed relative to analogous characteristics of the vertebrate blood-brain barrier.     

The olfactory epithelium of the lantern fish,Tarletonbeania crenularis (myctophidae)

Summary The permeability of the septate junction of the nephrocytes of Helix pomatia from the basal lamina to lumen has been studied with two electron opaque tracers, horse-radish peroxidase and lanthanum. Ultrathin sections of tissues from animals treated with these materials showed that the septa offer a considerable restraint to the movement of horse-radish peroxidase, but that the lanthanum permeates the septate region of the junction. Tangential sections of lanthanum-infiltrated junctions showed that the septa are corrugated sheets. The infiltration by lanthanum shows that in some areas there are discontinuities in these sheets which may allow the passive movement of solutes from the intercellular space to the kidney lumen.     

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.