Studies on transmembrane and paracellular phenomena in the foot of the slug Agriolimax reticulatus (Mü)

Summary Using the enzyme peroxidase and ionic lanthanum as tracers, paracellular uptake has been demonstrated in the foot of the slug Agriolimax reticulatus (Mü). Both tracers appeared to pass between adjacent foot epithelial cells and were demonstrated in the zonula adhaerens, the septate desmosomes, and the intercellular spaces which occur beneath the septate junctions. Ferritin, a somewhat larger tracer, was excluded from all these sites.Ionic lanthanum was not normally pinocytosed in short incubation times. The epithelial cells could be induced to endocytose this marker, however, when combined with a variety of proteins. The implications these findings have on the uptake of molluscicides is discussed.This research was supported by the Agricultural Research Council (G.B.) Grant No. AG 72/13     

X-ray microanalysis with transmission electron microscopy determined presence and movement of tracer (lanthanum chloride) at blood-neuron barrier of Drosophila melanogaster (Diptera : Drosophilidae) larva

In studying the larval Drosophila (Diptera : Drosophilidae) blood-brain barrier, it was important to determine if even minute amounts of tracer ultimately seeped through the septate junctions between perineurial cells to reach the neuronal region. Concurrent TEM with X-ray microanalysis was undertaken to resolve that issue. Ultrathin sections of Drosophila nervous tissue in LR White embedment were exposed to ionic tracer (lanthanum chloride) and assayed for presence or absence of lanthanum extracellular to the perineurium and glia making up the nerve sheath. Tracer filled the distal interseptal lattice of pleated sheet-septate junctions, but was contained prior to reaching the proximal paracellular space. No detectable tracer passed through septate junctions to enter the glial-neuronal domain. Based on our present data and the research of others, septate junctions in immature Drosophila are multifunctional structures that enforce spatial relationships between cells, seal intercellular spaces, and control cell proliferation in the epithelia. Septate junctions in Drosophila with the (dlg) gene also exhibit protein homologies to the Z0–1 human tight junction component.     

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.     

Septate junctions mediate the barrier to paracellular permeability in sea urchin embryos

In sea urchin embryos, blastula formation occurs between the seventh and tenth cleavage and is associated with changes in the permeability properties of the epithelium although the structures responsible for mediating these changes are not known. Tight junctions regulate the barrier to paracellular permeability in chordate epithelia; however, the sea urchin blastula epithelium lacks tight junctions and instead possesses septate junctions. Septate junctions are unique to non-chordate invertebrate cell layers and have a characteristic ladder-like appearance whereby adjacent cells are connected by septa. To determine the function of septate junctions in sea urchin embryos, the permeability characteristics of the embryonic sea urchin epithelia were assessed. First, the developmental stage at which a barrier to paracellular permeability arises was examined and found to be in place after the eighth cleavage division. The mature blastula epithelium is impermeable to macromolecules; however, brief depletion of divalent cations renders the epithelium permeable. The ability of the blastula epithelium to recover from depletion of divalent cations and re-establish a barrier to paracellular permeability using fluorescently labelled lectins was also examined. Finally, septate junction structure was examined in embryos in which the permeability status of the epithelium was known. The results provide evidence that septate junctions mediate the barrier to paracellular permeability in sea urchin embryos.     

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.     

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.     

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.     

Structure and permeability of goblet cell tight junctions in rat small intestine

Summary Two major cell types, goblet and absorptive cells, dominate the epithelial lining of small intestinal villi. We used freezefracture replicas of rat ileal mucosa to examine the possibility that tight junction structure, known to relate to transepithelial resistance, might vary with cell type. Tight junctions between absorptive cells were uniform in structure while those associated with villus goblet cells displayed structural variability. In 23% of villus goblet cell tight junctions the strand count was less than 4 and in 30% the depth was less than 200 nm. In contrast, only 4% of absorptive cell tight junctions had less than 4 strands and only 9% had depth measurements less than 200 nm. Other structural features commonly associated with villus goblet cell tight junctions but less commonly with absorptive cell tight junctions were: deficient strand cross-linking, free-ending abluminal strands, and highly fragmented strands. Bothin vivo ileal segments and everted loops were exposed to ionic lanthanum. Dense lanthanum precipitates in tight junctions and paracellular spaces were restricted to a subpopulation of villus goblet cells and were not found between villus absorptive cells. After exposure of prefixed ileal loops to lanthanum for 1 hour, faint precipitates of lanthanum were found in 14% of tight junctions and paracellular spaces between absorptive cells compared to 42% of tight junctions and paracellular spaces adjacent to villus goblet cells. When tested in Ussing chambers, the methods used for lanthanum exposure did not lower transepithelial resistance. Everted loops exposed to ionic barium and examined by light microscopy showed dense barium precipitates in the junctional zone and region of the paracellular space of villus goblet cells but not in these regions between absorptive cells. However, the macromolecular tracers, microperoxidase, cytochromec and horseradish peroxidase, were excluded from both villus goblet cell and absorptive cell paracellular spaces inin vivo segments. These findings suggest that a subpopulation of villus goblet cells may serve as focal sites of high ionic permeability and contribute to the relatively low resistance to ionic flow which characterizes the small intestinal epithelium.     

Effects of lanthanum on the heart sarcolemmal ATPase and calcium binding activities.

Effects of lanthanum on Ca2+-ATPase, Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities were studied in rat heart sarcolemma. Ten to 100 micrometers lanthanum depressed significantly the Ca2+-ATPase activity and 50--200 micrometers lanthanum inhibited the calcium binding activity. Lineweaver-Burk plots of the Ca2+-ATPase activity showed that the inhibition by lanthanum was competitive with calcium concentration. Neither Mg2+-ATPase nor Na+-K+-ATPase activities were affected by lanthanum when the assay medium contained 1 mM EDTA; however, in the absence of EDTA, these enzyme activities were significantly decreased by 10--100 micrometers lanthanum. Rat hearts perfused with HEPES buffer containing 0.5 mM lanthanum showed electron-dense deposits restricted to the outer cell surface and the sarcolemma obtained from these hearts also had the deposits, indicating that the membrane fraction isolated by the hypotonic shock--LiBr treatment method is of sarcolemmal origin. The Ca2+-ATPase activity of the sarcolemma isolated from lanthanum-perfused hearts, unlike the Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities, was significantly less than the control value. From these observations it is suggested that lanthanum may influence calcium movement across the sarcolemma by affecting sarcolemmal ATPase and calcium binding activities.     

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 lateral mobility of cell adhesion molecules is highly restricted at septate junctions in <Emphasis Type="Italic">Drosophila</Emphasis>

Background  

A complex of three cell adhesion molecules (CAMs) Neurexin IV(Nrx IV), Contactin (Cont) and Neuroglian (Nrg) is implicated in the formation of septate junctions between epithelial cells in Drosophila. These CAMs are interdependent for their localization at septate junctions and e.g. null mutation of nrx IV or cont induces the mislocalization of Nrg to the baso-lateral membrane. These mutations also result in ultrastructural alteration of the strands of septate junctions and breakdown of the paracellular barrier. Varicose (Vari) and Coracle (Cora), that both interact with the cytoplasmic tail of Nrx IV, are scaffolding molecules required for the formation of septate junctions.     

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.     

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.     

Blood-eye barrier of the developing drosophila melanogaster (Diptera : Drosophilidae)

For the past quarter century, it has been known that a blood-eye barrier exists in adult insects. However, the life stage at which the barrier arises and the anatomical correlate of the barrier were not known. Compound eye development in Drosophila melanogaster (Diptera : Drosophilidae) is essentially complete at approximately 140 h after pupariation ; or about 20 h prior to eclosion. A search for a blood-eye barrier spanned late third-instar larvae, through late pupal life sampled at 40 and 140 h post-pupariation. No blood-eye barrier is present in the eye discs of last-instar larvae, based on the presence of lanthanum tracer among ommatidial cells and their processes. Pleated-sheet septate junctions, which link larval ommatidial cells, are not yet capable of totally blocking tracer from paracellular passage. The blood-eye barrier is constructed in the early phase (0–60 h) of pupal development in the wake of apoptosis and new cellular reorganization. In the developing compound eye, mature photoreceptor neurons must then be protected from the ionic vagaries of hemolymph to become electrophysically competent. First vestiges of a barrier with occluding septate junctions are seen in the 40-h old pupa, and by 140 h, the barrier is complete. The barrier prevails throughout adult life.     

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.     

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.     

Ca2+-dependent deposits at the plasmalemma of Chara internodal cells

Electron dense deposits (EDD) were observed on the extracellular side of the plasmalemma of Chara internodal cells by a calcium-glutaraldehyde fixation technique. The number and size of EDD were greatly increased when cells had been preincubated in Ca2+-enriched medium before fixation. The addition of Na+, Mg2+, or La3+ instead of Ca2+ in incubation and fixation media produced no deposits at all, Sr2+ caused deposits with similar distribution to those formed by Ca2+, and Ba2+ addition resulted in deposits localized at different sites within the cell. Microprobe analysis of single EDD from Ca2+ incubated cells ascertained the presence of calcium in these deposits. Possible functions of the Ca2+-binding sites at the plasma membrane of Chara cells are discussed.     

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.     

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 release of slow-reacting substance of anaphylaxis from guinea pig lung: effects of calcium antagonists

The effects of three calcium antagonists, verapamil, lanthanum, and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) were studied on the release of slow-reacting substance of anaphylaxis (SRS-A) from ovalbumin-sensitized chopped guinea pig lung parenchyma in calcium-containing and calcium-free media. The SRS-A levels (mean +/- SEM) obtained from tissues incubated in normal and calcium-free Krebs-bicarbonate buffer were 51 +/- 8 (N = 19) and 21 +/- 4 (N = 14) U/mL, respectively. TMB-8 (0.1-10 microM), a reported intracellular calcium antagonist, reduced antigen-stimulated SRS-A release from lung tissue incubated in calcium-containing, but not calcium-free, medium; A23187-induced SRS-A release from normal guinea pig lung was not significantly altered by TMB-8 at concentrations up to 10 microM. Verapamil and lanthanum consistently reduced SRS-A release only at high concentrations (100 microM and 1mM, respectively). The quantities of SRS-A released from lung tissue incubated in the presence of verapamil in normal medium were similar to those obtained in calcium-free medium. Tissues incubated in the presence of potassium chloride (60 and 100 mM) did not release significant quantities of SRS-A, and release which did occur was not blocked by verapamil, suggesting that antigen-induced SRS-A release is not dependent on membrane depolarization and that verapamil was not exerting inhibition via blockade of voltage-dependent calcium channels. These data suggest that although intracellular calcium is important for the regulation of SRS-A secretion from guinea pig lung tissue, extracellular calcium is necessary for optimal release of SRS-A.