Glio-neuronal and glio-glial syncytial cytoplasmic connections in peripheral nerve trunks of the crayfish Astacus leptodactylus

The paper considers various aspects of glial sheaths of neuritis in the crayfish peripheral nerve trunks and roots. There are revealed dotted glio-neurite tight junctions and a varicose deformation of the intercellular glio-neurite cleft. Rupture of membranes in the area of contact leads to formation of the glio-neurite pore (less than 10 nm) that is enlarged and forms wide (up to 240 nm) syncytial perforations. At the edge of perforation, either remnants of tight junctions are present or damaged membranes that fuse and are rounding. The lumen of perforations always contains residual membranous bodies in the form of vesicles. Their deviation from the median line can indicate a mutual translocation of substances of the glio- and neuroplasm. In the adjacent layers of the multilayer glial sheath there is noted a similar phenomenon of formation of the glio-glial syncytial connection terminating by fusion of neighbor glial layers, which is terminated by fusion of neighbor glial layers into the single lamina. The process begins from the varicose deformation of interglial clefts, which appears as a result of massive formation of dotted and expanded tight membranous contacts. As a result of transformation of ellipsoid varicose deformations into the spherical ones, syncytial pores (less than 10 nm) between them are formed, which are enlarged and break the paired gliolemmas into fragments. As a result, the adjacent glial layers are united. Since this process in intact animals occurs on the background of undamaged nerve structures, a suggestion is put forward about its reversibility and the functional nature.     

Glio-neuronal and glio-glial syncytial cytoplasmic connections in peripheral nerve trunks of the crayfish <Emphasis Type="Italic">Astacus leptodactylus</Emphasis>

The paper considers various aspects of glial sheaths of neurites in the crayfish peripheral nerve trunks and roots. There are revealed dotted glio-neuritic tight junctions and a varicose deformation of the intercellular glio-neuritic cleft. Rupture of membranes in the area of contact leads to formation of the glio-neuritic pore (less than 10 nm) that is enlarged and forms wide (up to 240 nm) syncytial perforations. At the edge of perforation, either remnants of tight junctions are present or damaged membranes that fuse and become rounded. The lumen of perforations always contains residual membranous bodies in the form of vesicles. Their deviation from the median line can indicate a mutual translocation of substances of the glio- and neuroplasm. In the adjacent layers of the multilayer glial sheath there is noted a similar phenomenon of formation of the glio-glial syncytial connection terminating by fusion of neighbor glial layers, which is terminated by fusion of neighbor glial layers into the single lamina. The process begins from the varicose deformation of interglial clefts, which appears as a result of massive formation of dotted and expanded tight membranous contacts. As a result of transformation of ellipsoid varicose deformations into the spherical ones, syncytial pores (less than 10 nm) between them are formed, which are enlarged and break the paired gliolemmae into fragments. As a result, the adjacent glial layers are united. Since this process in intact animals occurs on the background of undamaged nerve structures, a suggestion is put forward about its reversibility and the functional nature.     

Ultrastructural analysis of interneuronal syncytial perforations

The structural regularities of the organization of interneuronal syncytial cytoplasmic connections between neuronal bodies in gyrus dentatus and CA1 and CA2 (CA is cornu ammonis) of hippocampus, as well as between cell neurites of the caudal mesenteric ganglion were studied by transmission electron microscopy. The syncytial perforations are located only on the base of tight junctions. The perforations have rounded edges corresponding to the fusion edges of perforated membranes of adjacent neurons – or where their edges have a form of thinned plate – a remnant of the tight junction. In the lumen of the perforations, remnants of contact membranes – residual bodies – are revealed. On living neurons in tissue culture, the syncytial connection of two contacting processes of different neurons is found during the death of the body of one of them, but with preservation of viability of its processes that contact with other neurons.     

Syncytial perforations of human neuronal embryo membranes

An electron microscopy study of the anlage of cerebral cortex of human embryo has been carried with the aim of determining the presence of syncytial interneuronal connections in embryogenesis. It has been determined that, in part of the neurons, the glial embryo is absent and their external cell membranes are directly attached to each other by forming elongated or dotted tight junctions. Sometimes these junctions are perforated and, on their basis, the true syncytial interneuronal connections are formed. Natural structural properties of these connections are the following: formation of the base of tight membrane contacts, obligatory rounding of perforation edges, and the presence of residual particles in the form of spherical vesicles in the lumen of perforations. Results obtained allowed us to conclude that, in the anlage of cerebral cortex of embryos obtained during surgical abortion of pregnancy, apart from the formation of synaptic contacts, or until their formation, there is the possibility of syncytial interneuronal connections appearing. This should be considered during the transplantation of the developing brain.     

Syncytial perforations of human embryo membranes

An electron microscopy study of the anlage of cerebral cortex of human embryo has been carried with the aim of determining the presence of syncytial interneuronal connections in embryogenesis. It has been determined that, in part of the neurons, the glial embryo is absent and their external cell membranes are directly attached to each other by forming elongated or dotted tight junctions. Sometimes these junctions are perforated and, on their basis, the true syncytial interneuronal connections are formed. Natural structural properties of these connections are the following: formation of the base of tight membrane contacts, obligatory rounding of perforation edges, and the presence of residual particles in the form of spherical vesicles in the lumen of perforations. Results obtained allowed us to conclude that, in the anlage of cerebral cortex of embryos obtained during surgical abortion of pregnancy, apart from the formation of synaptic contacts, or until their formation, there is the possibility of syncytial interneuronal connections appearing. This should be considered during the transplantation of the developing brain.     

A Distinct Mechanism of Vascular Lumen Formation in Xenopus Requires EGFL7

During vertebrate blood vessel development, lumen formation is the critical process by which cords of endothelial cells transition into functional tubular vessels. Here, we use Xenopus embryos to explore the cellular and molecular mechanisms underlying lumen formation of the dorsal aorta and the posterior cardinal veins, the primary major vessels that arise via vasculogenesis within the first 48 hours of life. We demonstrate that endothelial cells are initially found in close association with one another through the formation of tight junctions expressing ZO-1. The emergence of vascular lumens is characterized by elongation of endothelial cell shape, reorganization of junctions away from the cord center to the periphery of the vessel, and onset of Claudin-5 expression within tight junctions. Furthermore, unlike most vertebrate vessels that exhibit specialized apical and basal domains, we show that early Xenopus vessels are not polarized. Moreover, we demonstrate that in embryos depleted of the extracellular matrix factor Epidermal Growth Factor-Like Domain 7 (EGFL7), an evolutionarily conserved factor associated with vertebrate vessel development, vascular lumens fail to form. While Claudin-5 localizes to endothelial tight junctions of EGFL7-depleted embryos in a timely manner, endothelial cells of the aorta and veins fail to undergo appropriate cell shape changes or clear junctions from the cell-cell contact. Taken together, we demonstrate for the first time the mechanisms by which lumens are generated within the major vessels in Xenopus and implicate EGFL7 in modulating cell shape and cell-cell junctions to drive proper lumen morphogenesis.     

Tight junction formation is closely linked to the polar redistribution of intramembranous particles in aggregating MDCK epithelia

We have used freeze-fracture electron microscopy to investigate the relationship between the formation of the tight junction in the establishment of a differential distribution of intramembranous particles (IMPs) between the luminal and basolateral membranes of a canine kidney cell line (MDCK). This involves a characterization of the IMP distribution in these membranes in confluent monolayers of MDCK cells, in EGTA-dissociated cells, and in cells at various stages of reassociation. While normal confluent MDCK monolayer cultures exhibit tight junctions and an IMP differential distribution between the luminal and basolateral membranes, cultures dissociated with EGTA lose both formed tight junctional elements and the differential IMP distribution. We have also found that as tight junctions reform between reaggregating MDCK cells, intramembranous particles appear to rapidly redistribute with respect to them. An asymmetric distribution of these particles in the luminal and basolateral membranes is eventually achieved. Tight junction formation appears so closely linked to the genesis of IMP polarity that at early time points when only a string of tight junctional components spans the junctional zone, differential IMP distributions are seen. Thus, our dissociation studies suggest a close relationship between the integrity of the tight junction and the maintenance of IMP polarity between the luminal and basolateral membranes, while cell reassociation studies suggest that the tight junction may be functionally linked to the genesis of IMP polarity.     

Fatty acid transport into the brain: of fatty acid fables and lipid tails

The blood-brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the central nervous system. Brain capillaries are a continuous layer of endothelial cells with highly developed tight junctional complexes and a lack of fenestrations. The presence of these tight junctions in the cerebral microvessel endothelial cells aids in the restriction of movement of molecules and solutes into the brain. Fatty acids are important components of biological membranes, are precursors for the biosynthesis of phospholipids and sphingolipids and are utilized for mitochondrial β-oxidation. The brain is capable of synthesizing only a few fatty acids. Hence, most fatty acids must enter into the brain from the blood. Here we review current mechanisms of transport of free fatty acids into cells and describe how free fatty acids move from the blood into the brain. We discuss both diffusional as well as protein-mediated movement of fatty acids across biological membranes.     

Structure and function of claudins

Claudins are tetraspan transmembrane proteins of tight junctions. They determine the barrier properties of this type of cell-cell contact existing between the plasma membranes of two neighbouring cells, such as occurring in endothelia or epithelia. Claudins can completely tighten the paracellular cleft for solutes, and they can form paracellular ion pores. It is assumed that the extracellular loops specify these claudin functions. It is hypothesised that the larger first extracellular loop is critical for determining the paracellular tightness and the selective ion permeability. The shorter second extracellular loop may cause narrowing of the paracellular cleft and have a holding function between the opposing cell membranes. Sequence analysis of claudins has led to differentiation into two groups, designated as classic claudins (1-10, 14, 15, 17, 19) and non-classic claudins (11-13, 16, 18, 20-24), according to their degree of sequence similarity. This is also reflected in the derived sequence-structure function relationships for extracellular loops 1 and 2. The concepts evolved from these findings and first tentative molecular models for homophilic interactions may explain the different functional contribution of the two extracellular loops at tight junctions.     

Structure and function of claudins

Claudins are tetraspan transmembrane proteins of tight junctions. They determine the barrier properties of this type of cell-cell contact existing between the plasma membranes of two neighbouring cells, such as occurring in endothelia or epithelia. Claudins can completely tighten the paracellular cleft for solutes, and they can form paracellular ion pores. It is assumed that the extracellular loops specify these claudin functions. It is hypothesised that the larger first extracellular loop is critical for determining the paracellular tightness and the selective ion permeability. The shorter second extracellular loop may cause narrowing of the paracellular cleft and have a holding function between the opposing cell membranes. Sequence analysis of claudins has led to differentiation into two groups, designated as classic claudins (1-10, 14, 15, 17, 19) and non-classic claudins (11-13, 16, 18, 20-24), according to their degree of sequence similarity. This is also reflected in the derived sequence-structure function relationships for extracellular loops 1 and 2. The concepts evolved from these findings and first tentative molecular models for homophilic interactions may explain the different functional contribution of the two extracellular loops at tight junctions.     

Modulation of cell junctions during differentiation of the chicken otocyst sensory epithelium.

The differentiation of sensory and support cells within the embryonic chick otocyst is accompanied by alterations in the distribution of preexisting intercellular junctions. Prior to innervation of this epithelium, tight, gap and adhering junctions exist between all cells. Upon differentiation of the epithelium, apical bands of tight and adhering junctions are maintained throughout, while gap junctions and desmosomes are found only between support cells. Thus, some of the gap junctions that join homogeneous epithelial cells prior to innervation are removed as sensory cells differentiate, and a separate population of very large gap junctions is formed between differentiating support cells. Morphological evidence suggests two possible mechanisms which may be responsible for the observed changes in gap junctional distribution: removal of gap junctions by internalization, and formation of gap junctions by aggregation of precursor particles. The temporal correlation between junctional modulation, cytological differentiation of sensory and support cells, and ingrowth of nerve fibers makes the latter event a likely developmental cue for differentiation of this epithelium.     

The supramolecular architecture of junctional microdomains in native lens membranes

Gap junctions formed by connexons and thin junctions formed by lens-specific aquaporin 0 (AQP0) mediate the tight packing of fibre cells necessary for lens transparency. Gap junctions conduct water, ions and metabolites between cells, whereas junctional AQP0 seems to be involved in cell adhesion. High-resolution atomic force microscopy (AFM) showed the supramolecular organization of these proteins in native lens core membranes, in which AQP0 forms two-dimensional arrays that are surrounded by densely packed gap junction channels. These junctional microdomains simultaneously provide adhesion and communication between fibre cells. The AFM topographs also showed that the extracellular loops of AQP0 in junctional microdomains adopt a conformation that closely resembles the structure of junctional AQP0, in which the water pore is thought to be closed. Finally, time-lapse AFM imaging provided insights into AQP0 array formation. This first high-resolution view of a multicomponent eukaryotic membrane shows how membrane proteins self-assemble into functional microdomains.     

Effects of ovarian hormones on cell membranes in the rat uterus

Freeze-fracture techniques have been used to study tight junctions on the lateral plasma membrane of cells of the luminal epithelium of the rat uterus under various hormonal regimes. Tight junctions from ovariectomized control rats extended some 0.5 μm down the lateral membrane and the junctional strands often formed a network of closely packed, circular compartments. Following treatment of rats with estrogen for 3 days the tight junctional regional still extended 0.5 μm down the lateral membrane, but the strands ran more parallel to the apical surface. They did not enclose circular compartments. After treatment with progesterone, either alone or with estrogen in such a way as to condition the ovariectomized uterus for implantation, a third pattern of junctional organization emerged. In these animals the junctional region extended 1.1 μm down the lateral membrane and the strands frequently crosslinked, enclosing compartments of varying and irregular size and shape. Our observations suggest that ovarian hormones could regulate the contents of the uterine lumen by altering the structure extent of the tight junctions which connect the epithelial cells enclosing the lumen.     

Development of an apical plasma membrane domain and tight junctions during histogenesis of the mammalian pancreas

The role of tight junctions (zonula occludens) in the formation of apical plasma membrane (PM) domains was investigated in the embryonic rat pancreas. In the present study, lectin-rhodamine (WGA-TRITC and RCAII-TRITC) and lectin-gold (WGA-Au and RCAII-Au) conjugates were used to monitor apical PM domain formation and freeze-fracture analysis was used to monitor tight junction formation in the pancreatic epithelium of embryonic, neonatal, and adult rats. Fluorescent and TEM analysis of WGA and RCAII binding indicated that an apical PM domain is formed as early as Day 13 of gestation in the pancreatic epithelium. While apical WGA binding remained into adult life, RCAII binding was lost by 1 day after birth. In contrast, tight junctions were not observed until Day 14 of gestation. At this time, tight junctions were found to be incomplete in formation and typically consisted of linear arrays of IMPs or discontinuous arrays of sealing strands (focal adherens). Continuous tight junctions were not completely formed until Day 15 of gestation. Continued development of tight junctions during gestation was characterized by (1) an increase in the number of sealing strands and (2) a more parallel arrangement of sealing strands within each junctional complex. By 8 weeks after birth, tight junctions were more loosely organized and contained fewer sealing strands as compared to that observed in the fetus. These results suggest that lateral diffusion of apical PM glycoconjugates may be restricted even in the absence of complete tight junctional complexes during development of the rat pancreas.     

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.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

Tight junction development between cultured hepatoma cells: Possible stages in assembly and enhancement with dexamethasone

Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9–11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10^?6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.     

Stability and fusion of lipid layers on polyelectrolyte multilayer supports studied by colloidal force spectroscopy

The interaction between lipid layers supported by polyelectrolyte multilayer cushions has been studied by means of colloidal force spectroscopy. In a typical experiment, a colloidal probe engineered with a layer-by-layer film and a lipid bilayer on top is approached to a planar surface coated in a symmetrical way. Kinks of a few nanometres in width appear when lipid layers are pressed together—reflecting either fusion processes between lipid layers or membranes, or the penetration of polymer blobs into or through the lipid layers. Retracting curves show a stepwise shape, which results from lipid tether formation or from polymer stretching, the latter suggesting that polyelectrolyte multilayers make contact as a result of penetration or lipid fusion. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Localization of claudin-5 and ZO-1 in rat spleen sinus endothelial cells

Splenic sinus endothelial cells, which adhere through tight and adherens junctions, regulate the passage of blood cells through the splenic cord. The objective of this study was to assess the localization of tight junctional proteins, claudin-5 and ZO-1 in the sinus endothelial cells of rat spleen and to characterize spatial and functional relationships between tight and adherens junctions. Immunofluorescence microscopy of tissue cryosections demonstrated that claudin-5, ZO-1, and α-catenin were distinctly localized in the junctional regions of adjacent endothelial cells. Immunogold electron microscopy demonstrated claudin-5 localized in the tight-junctional fused membranes of adjacent endothelial cells. Immunogold labeling for ZO-1 was localized not only in the tight-junctional-fused membranes of endothelial cells but also in the junctional membrane. α-Catenin was intermittently localized along the juxtaposed junctional membranes of adjacent endothelial cells. Double-staining immunogold microscopy for claudin-5 and ZO-1, claudin-5 and VE-cadherin, ZO-1 and VE-cadherin, and ZO-1 and α-catenin demonstrated that ZO-1 was closely localized to VE-cadherin and α-catenin in their juxtaposed membranes of endothelial cells. Thus, ZO-1 might play an important role in regulating the cell–cell junctions of sinus endothelial cells for blood–cell passage through splenic cords. This work was supported by a Grant-in-Aid for Scientific Research (C), Japan.