The cell junctions of the notochord of Xenopus laevis tadpoles

The cell junctions of the notochord of Xenopus laevis tadpoles were examined with the electron microscope using thin sections, lanthanum tracer experiments, and freeze-fracture replicas. Both the peripheral and vacuolated cells of the notochord are connected by numerous spot desmosomes characterized by an intercellular desmogloea and intermediate filaments on the cytoplasmic sides. The peripheral cells also display numerous hemidesmosomes facing the underlying basal lamina. Staining with rhodamine-phalloidin for F-actin yielded negative results and suggested that adhaerens-type junctions are absent. Tracer experiments with lanthanum and freeze-fracture replicas clearly revealed the presence of gap junctions between both cell types but no indications of tight junctions were found and no intercellular barrier existed for tracer infiltration of the notochord.     

Tight junction of sinus endothelial cells of the rat spleen

The fine structure of the tight junctions between sinus endothelial cells of the rat spleen and the permeability of such sinus endothelial cells were examined by transmission electron microscopy, using freeze-fracture, triton extraction, and lanthanum-tracer techniques. In freeze-fracture replicas, the segmented strands and grooves of the tight junctions were frequently observed on the basolateral surfaces of the sinus endothelial cells irrespective of the location of the ring fiber. There were one or two wavy-strands or grooves which were, for the most part, oriented parallel to the long cell axis thus forming networks at places. In addition, some strands or grooves were discontinuous while some networks of the junctional strands were not closed. These strands also occasionally lacked intramembranous particles in the tight junctions. The junctional strands run apicobasically at certain sites. In the vertical sections of the sinus endothelial cells treated with lanthanum nitrate, although no tight junctions were observed wherever the endothelial cells were apposed, most of them were situated on the basal part of the lateral surfaces of the adjacent endothelial cells. Several fusions of the junctional membranes were observed in a vertical section of the lateral surfaces of the adjacent endothelial cells. The intercellular spaces of the adjacent endothelial cells except for the fusion of the junctional membranes, were electron dense and the infiltration of lanthanum nitrate was found not to be interrupted by these tight junctions. Based on these observations, the molecular 'fence' and paracellular 'gate' functions of the tight junctions in the sinus endothelial cells are discussed.     

The shape of caveolae is omega-like after glutaraldehyde fixation and cup-like after cryofixation

Caveolae were defined as flask- or omega-shaped plasma membrane invaginations, abundant in adipocytes, fibroblasts, endothelial and smooth muscle cells. The major protein component of caveolar membranes is an integral membrane protein named caveolin. We compared the freeze-fracture behavior of caveolae in glutaraldehyde-fixed and cryofixed mouse fibroblast cells and found distinct differences. In glutaraldehyde-fixed cells almost all caveolae were cross-fractured through their pore and only very few caveolar membranes were membrane-fractured. We found the reverse situation in rapid frozen cells without any chemical fixation where most of the caveolae were membrane-fractured, showing different degrees of invagination from nearly flat to deeply invaginated. In ultrathin sections of glutaraldehyde-fixed heart endothelial cells, caveolae exhibit the well known omega-like shape. In high-pressure frozen, freeze-substituted and low temperature embedded heart endothelial cells, the caveolae frequently exhibit a cup-like shape without any constriction or pore. The cup-like caveolar shape could also be shown by tilt series analysis of freeze-fracture replicas obtained from cryofixed cells. Freeze-fracture immunolabeling of caveolin-1 revealed a lateral belt-like caveolin alignment. These findings point out that the constricted “neck” region of caveolae in most cases is an effect that is caused and intensified by the glutaraldehyde fixation. Our data indicate that caveolae in vivo show all degrees of invagination from nearly flat via cup-like depressed to in a few cases omega-like.     

Cell junctions in the rapidly moving edge of chick and quail blastoderms during gastrulation

Summary The intercellular contacts of the migrating edge of chick and quail blastoderms during gastrulation were studied by transmission electron microscopy of thin sections and of freeze-fracture replicas. Tight junctions and gap junctions as well as desmosomes were found. Tight junctions were organized as single junctional strands or as a complex of numerous junctional strands interposed between the lamellae and the bodies of the cells building up the margin of overgrowth. The function of these intercellular junctions is considered in relation to the locomotion of the margin of overgrowth cells.     

Freeze-fracture replication of junctional complexes in unincubated and incubated chick embryos

Junctional complexes have been investigated in the epiblast of young chick embryos by examination of freeze-fracture replicas and of sections of comparable specimens stained with lanthanum nitrate. By means of freeze-fracture, tight junctions were shown to be present in the unincubated embryo (stage 1 of Hamburger and Hamilton). The number of ridges or grooves was found to vary between 2 and 10 near the dorsal border, whereas isolated ridges were found more ventrally. Lanthanum was unable to penetrate between the cells in the region of the dorsally situated tight junctions. Similar tight junctions were found in incubated embryos (stage 3) examined by both techniques. Tight junctions were also seen in cleavage (pre-laying) embryos examined in section. Gap junctions were extremely uncommon in unincubated embryos, though occasional aggregates of gap junction particles were seen on the lateral cell membranes close to the dorsal surface. In only one instance were associated pits visible. By contrast, gap junctions were more frequently encountered by stage 3, and these junctions possessed both pits and particles. Desmosomes were never seen in the freeze-fracture replicas at either stages 1 or 3, though structures which might be developing desmosomes were visible in sections. The functions of both the tight and gap junctions in the young chick embryo are discussed. The results are also considered in relation to recent theories about the way in which gap junctions are formed.     

Tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen

The fine structure of plasmalemmal tubular invaginations with caveolae and coated pits in the sinus endothelial cells of the rat spleen has been demonstrated by scanning and transmission electron microscopy. In addition, the three-dimensional structure of the tubular invagination has been revealed by computer-aided reconstruction. The tubular invaginations of the plasma membrane plunged into the cytoplasm everywhere from the apical, lateral, and basal surfaces of the plasma membrane. The invaginations were tubular and branched away, and their plasma membranes were reinvaginated to form numerous caveolae and occasional coated pits. Numerous caveolae were found in clusters that looked similar to a bunch of grapes and the coated pits were present at the base of the clusters. The caveolae and coated pits derived from the tubular invaginations were almost ultrastructurally identical to those derived from the surface plasma membrane. From examination of the fractured surfaces of the endothelial cells treated with the aldehyde prefix osmium-dimethyl sulfoxide-osmium method and of ultrathin sections of those infiltrated by lanthanum nitrate, the tubular invaginations were found to not penetrate any endothelial cells. A computer-aided reconstruction revealed that the caveolae derived from the tubular invaginations were in close apposition to the surface-connected canaliculi. The reaction product of Concanavalin A conjugated to horseradish peroxidase was present on the outer leaflet of the membranes of the coated pits and coated vesicles and also in the contents of the endosomes, but it was absent from any caveolae. Based on our observations, the functional significance of the tubular invaginations in sinus endothelial cells is discussed. Accepted: 13 September 1999     

Intercellular junctions of antennal gland epithelial cells in the crayfish,Orconectes virilis

Summary Labyrinth and nephridial canal cells of the crayfish (Orconectes virilis) antennal gland possess two types of intercellular junctions revealed by freeze-fracture studies. Apical margins of the cells are connected by long septate junctions. In replicas, these junctions consist of many parallel rows of 80–140 Å intramembrane particles situated on the PF membrane face (EF and PF fracture faces of Branton et al., 1975). Rows of pits are found on the EF fracture face and are deemed complementary to the rows of particles. Moreover, lateral margins of basal regions of the epithelial cells are attached by many intercellular junctions. These contacts are characterized in thin plastic sections by a narrow dense cytoplasmic plaque located subjacent to the plasma membrane at sites of adjoined cells, and 5 to 12 fine strands of dense material that extend across the intercellular gap between adjoined cells. In freeze-fracture replicas, EF intramembrane faces basal to the region of the plasma membrane containing septate junctions exhibit numerous discoid clusters of particles. The particle aggregates, assumed to represent freeze-cleave images of adhering junctions, range from 900 to 3,700 Å in diameter, with individual particles about 185 Å in diameter. These junctions appear to connect epithelial cell processes formed by basal infoldings of the plasma-lemma, and occur between adjacent cells as well as adjacent processes of a single cell. The discrete aggregates of particles resemble replicated desmosomes (Shienvold and Kelly, 1974) and hemi-desmosomes (Shivers, 1976); therefore, they probably do not constitute a basis for electrical coupling between antennal gland epithelial cells.Supported by the National Research Council of Canada     

The ultrastructural organization of gap junctions between follicle cells and the oocyte in Xenopus laevis

The cellular contact sites between the full-grown oocyte of Xenopus laevis and the surface extensions of surrounding follicles cells were analysed by electron microscopy of ultrathin sections, freeze-fracture replicas and critical point-dried specimens. Evidence is given for the presence of clusters of intramembranous particles (IMPs) at the P-face which represent gap junctions in diverse forms. Most common are maculae (phi 0.2-0.5 micron) of densely packed IMPs (phi 12 +/- 2 nm) which represent focal gap junctions generally found at the tips of follicle cell surface extensions. Inside many maculae an IMP-free area occurs which appears as a smooth disk (phi 70-80 nm) at both fracture faces. Occasionally a few IMPs are trapped within the smooth disks. Beside the maculae, networks of arrayed IMPs occur that enclose several smooth disks. These latter gap junctions probably are more frequent in side-to-side contacts between surface extensions of the oocyte and the follicle cells. The possible function of these IMP networks is discussed as being related to similar membrane specializations in excitable cells. In addition, indirect evidence was found that the extensions of the follicle cells transport yolky material.     

Freeze-fracture study of the epidermal cells of a teleost with particular reference to intercellular junctions and permeability to tracer

The plasmatic membranes, the intercellular junctions and the intercellular spaces of the epidermis of the fish Pimelodus maculatus were studied by freeze-fracture and by lanthanum methods. The observations has confirmed the presence of desmosomes. Gap junctions were not found and the tight junctions can be seen very rarely, arranged to form small discrete maculae. The finger-print pattern due to the microridges of the apical plasma membrane of the superficial cells was studied by direct replicas. The tracer penetrates all the intercellular epidermal spaces but failed to penetrate the dermis, suggesting the presence of a barrier at the dermo-epidermal level.     

Cell junctions and other membrane specializations in the ocellus of the wasp,Paravespula Germanica L. (Hymenoptera : Vespidae)

Five types of cell contacts and other membrane specializations were found in the ocellus of the adult wasp, Paravespula germanica L. (Hymenoptera : Vespidae), based on freeze-fracture replicas and thin sections.Septate junctions alongside small gap junctions are present between iris cells and between corneagenous cells. Gap junctions are sometimes observed between glial cell processes. Photoreceptor cells and glial cells are frequently connected by scalariform junctions. Tight junction-like structures are found on receptor-cell membranes near rhabdomeric microvilli. Desmosomes are widespread in the ocellus, connecting iris cells, corneagenous cells, receptor cells, and glial cell processes. Desmosomes are found next to septate junctions.Glial membranes connected to receptor cells have a non-junctional type of membrane specializations, consisting of intramembraneous particles arranged in a rhombic pattern. Interestingly, both particle arrays and scalariform junctions are often adjacent to each other. Furthermore, a conspicuous modification of the cell surface in freeze cleaved cells is seen between adjacent glial cells intermediating two receptor cells.     

Intercellular junctions and transfer of small molecules in primary vascular endothelial cultures

The ultrastructure of gap and tight junctions and the cell-to-cell transfer of small molecules were studied in primary cultures and freshly isolated sheets of endothelial cells from calf aortae and umbilical veins. In thin sections and in freeze-fracture replicas, the gap and tight junctions in the freshly isolated cells from both sources appeared similar to those found in the intimal endothelium. Most of the interfaces in replicas had complex arrays of multiple gap junctions either intercalated within tight junction networks or interconnected by linear particle strands. The particle density in the center of most gap junctions was noticeably reduced. In confluent monolayers, after 3-5 days in culture, gap and tight junctions were present, although reduced in complexity and apparent extent. Despite the relative simplicity of the junctions, the cell-to-cell transfer of potential changes, dye (Lucifer Yellow CH), and nucleotides was readily detectable in cultures of both endothelial cell types. The extent and rapidity of dye transfer in culture was only slightly less than that in sheets of freshly isolated cells, perhaps reflecting a reduced gap junctional area combined with an increase in cell size in vitro.     

CELL CONTACTS IN THE MOUSE MAMMARY GLAND : I. Normal Gland in Postnatal Development and the Secretory Cycle

The nature and distribution of cell contacts have been examined in thin sections and freeze-fracture replicas of mammary gland samples from female C3H/Crgl mice at stages from birth through pregnancy, lactation, and postweaning involution. Epithelial cells of major mammary ducts at all stages examined are linked at their luminal borders by junctional complexes consisting of tight junctions, variable intermediate junctions, occasional small gap junctions, and one or more series of desmosomes. Scattered desmosomes and gap junctions link ductal epithelial and myoepithelial cells in all combinations; hemidesmosomes attach myoepithelial cells to the basal lamina. Freeze-fracture replicas confirm the erratic distribution of gap junctions and reveal a loose, irregular network of ridges comprising the continuous tight-junctional belts. Alveoli develop early in gestation and initially resemble ducts. Later, as alveoli and small ducts become actively secretory, they lose all desmosomes and most intermediate junctions, whereas tight and gap junctions persist, The tight-junctional network becomes compact and orderly, its undulating ridges oriented predominantly parallel to the luminal surface. It is suggested that these changes in junctional morphology, occurring in secretory cells around parturition, may be related to the greatly enhanced rate of movement of milk precursors and products through the lactating epithelium, or to the profound and recurrent changes in shape of secretory cells that occur in relation to myoepithelial cell contraction, or to both.     

Cell junctions in explanted tissues from early chick embryos

Summary Hypoblast and definitive endoblast derived from young chick embryos were explanted and grown for 24 h in culture. The junctional complexes which characterise these tissues were studied on freeze-fracture replicas and thin sections. Cell membranes of the hypoblast displayed tight junctions only, disposed in randomly arranged strands or narrow belts which included many discontinuous strands. The definitive endoblast showed tight and gap junctions as well as desmosomes in close association with the tight junctions. It is suggested that the differences between the two types of tissue may be related to cell cohesiveness, which appears to be relatively low in the hypoblast and high in the definitive endoblast.     

Localization of caveolin-3 in the sinus endothelial cells of the rat spleen

The localization of caveolins in the sinus endothelial cells of the rat spleen has been demonstrated by confocal laser scanning and electron microscopy. Caveolin-3, a muscle-specific caveolin, was detected by Western blot analysis and immunofluorescence microscopy of isolated sinus endothelial cells and tissue cryosections of the spleen. During the immunofluorescence microscopy of isolated endothelial cells, both caveolin-3 and caveolin-1 were found. In tissue cryosections of the spleen, caveolin-3, as well as caveolin-1 and -2, was present in the contours and cytoplasm of the cells. Immunogold electron microscopy of tissue cryosections revealed caveolin-3, -1, and -2 to be present in caveolae in the apical, lateral, and basal plasma membranes and some vesicular profiles in the cytoplasm of sinus endothelial cells. Furthermore, caveolin-3 was colocalized with caveolin-1 in the same caveolae in the apical, lateral, and basal plasma membranes. Stress fibers and tubulovesicular structures were situated in the vicinity of caveolae labeled with anti-caveolin-3, anti-caveolin-1, and anti-caveolin-2 antibodies. It is speculated that caveolae in sinus endothelial cells play an important role in the constriction of stress fibers.     

Freeze-fracture replicas of rat ameloblasts reveal unusual membrane domains around gap junctions

Specimens of aldehyde-fixed and glycerol-impregnated tooth germs obtained from 1-2 day old rats were prepared for ultrathin section studies and for freeze-fracture, with the purpose of studying the structural organization of membranes of developing ameloblasts. In this report we describe unusual membrane domains which were found surrounding several ameloblast gap junctions. Developing ameloblasts - when examined in ultrathin sections - exhibit gap junctions which appear straight, curved or invaginated. In freeze-fracture replicas, in addition to their typical appearance, several gap junctions were found to be surrounded by a membrane margin which was undulating and devoid of intramembrane particles (IMP's). We believe that these hitherto unreported particle-free membrane margins are associated with the formation of curved or invaginated gap junctions. It is possible that these membrane margins are particle-free because plasma membrane proteins (presumably IMP's) become transiently detached from the cytoskeleton and move laterally. It is therefore likely that these margins are pure lipid domains which are more flexible, thus providing a transient hinge-like mechanism which facilitates the movement required for the formation of the curved or invaginated ameloblast gap junctions.     

Localization of TRPC1 channel in the sinus endothelial cells of rat spleen

The ultrastructural localization of transient receptor potential C1 (TRPC1) channels in the sinus endothelial cells of rat spleen was examined by confocal laser scanning and electron microscopy. In addition, the localization of the closely associated proteins and channels, VE-cadherin, calreticulin, inositol-1,4,5-trisphosphate receptors type 1 (IP₃R1), and ryanodine receptor (RyR), was also examined. Immunofluorescence microscopy of tissue cryosections revealed TRPC1 channels to be localized within the cytoplasm, in the superficial layer of the apical and basal parts of the cells, and in the junctional area of the adjacent endothelial cells. The distribution of Ca²⁺-storing tubulovesicular structures within endothelial cells was established by using tissue sections treated with osmium ferricyanide. Electron microscopy revealed densely stained tubulovesicular structures closely apposed to the plasma membrane and that occasionally ran closely parallel to the plasma membrane and near the caveolae and junctional apparatus. Immunolocalization analysis at the electron microscopy level using immunogold bound to the secondary antibody confirmed that TRPC1 channels were localized in the plasma membrane, caveolae, and vesicular structures in the subplasmalemmal cytoplasm of sinus endothelial cells. Calreticulin was predominantly localized in endoplasmic reticulum. IP₃R1 and RyR, considered to be type 3, were colocalized in endoplasmic reticulum in proximity to the plasma membrane and caveolae. Thus, TRPC1 channels in sinus endothelial cells of the spleen might play an important role in controlling blood cell passage through phenomena including cytoskeletal reorganization, cell retraction, and disassembly of adherens junctions.This work was supported by a Grant-in-Aid for Scientific Research (C), Japan.     

The glomeruli of the human and the rat kidney studied by freeze-fracturing

Glomeruli isolated from rat and human kidneys were studied using the freeze-fracture technique. Discontinuous zonulae occludentes and gap junctions were found in the replicas of the split plasma membrane of the endothelial cells. A diaphragm across the endothelial pores was not demonstrated. The central layer of the basement membrane, corresponding to the lamina densa described in thin sections, revealed a coarse substructure. A slit membrane between the pedicles of the podocytes was not detectable; however, its position was indicated by the different texture of the replica, which abruptly changed at the transition of the basement membrane to the primary urinary space. Furthermore, at the level of the slit membrane arrays of particles were present within the cleaved membrane of the pedicles, probably representing the attachment points of the slit membrane. Isolated strands of a zonula occludens as well as gap junctions were seen on the split plasma membrane of the podocytes. The mesangial cells could be identified by their contiguity to the endothelial cells and by their numerous gap junctions.     

Interdigitating reticulum cells in lymph nodes of Sézary syndrome. Freeze-fracture and ultrathin-section morphology

Lymph nodes with extensive leukemic infiltration from three patients with the Sézary syndrome were examined in ultrathin sections and in freeze-fracture replicas. Sézary cells (SC) and interdigitating reticulum cells (IDC) were the predominant cell types in the lymph nodes. Both were closely connected with each other by apparently interdigitating cytoplasmic processes. The projections between these cells were, in the main, processes from the IDC. In freeze-fracture replicas these cellular processes did not appear as interdigitations but were more bubble-like, and for this reason these cells are imprecisely described by the term "interdigitating." The SC were seen to possess only short cytoplasmic processes. The frequent polar grouping of cell organelles in SC in the region of the contact zone with IDC and the high organelle content of IDC ('activated IDC') could be the morphologic expression of intense interaction between IDC and SC. IDC displayed three features in freeze-fracture which are not specific to the Sézary syndrome, but should be applicable to IDC in general: (1) they exhibited an approximately equal density of intramembrane particles in both the E-face and the P-face, (2) some of the intramembrane particles in the P-face were assembled in clusters and (3) the surface showed bubble-like formations of the cytoplasmic processes. On the basis of these properties it was possible to distinguish IDC from macrophages and lymphocytes in freeze-fracture replicas.     

Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord

The historical development of concepts of gap junctions as sites for electrical, ionic, and metabolic coupling is reviewed, from the initial discovery of gap junctions linking heart cells, to the current concepts that gap junctions represent 'electrotonic synapses' between neurons. The ultrastructure and immunocytochemistry of gap junctions in heart, brain, and spinal cord of adult rats is examined using conventional thin sections, negative staining, grid-mapped freeze-fracture replicas, and immunogold-labeled freeze-fracture replicas. We review evidence for neuronal gap junctions at 'mixed' (combined electrical and chemical) synapses throughout adult rat spinal cord. We also show immunogold labeling of connexin43 in astrocyte and ependymocyte gap junctions and of connexin32 in oligodendrocyte gap junctions. Ultrastructural and freeze-fracture immunocytochemical methods have provided for definitive determination of the number, size, histological distribution, and connexin composition of gap junctions between neurons in all regions of the central nervous systems of vertebrate species.     

Cell junctions in the gut of Protura

The main cell junctions in the intestinal tract of a small group of apterygotan insects, Protura, were examined in conventional thin sections, tracer-infiltrated sections and freeze-fracture replicas. The smooth septate junctions in the midgut of collembolan Tomocerus minor were also studied for comparison. Pleated septate junctions are found in foregut, hindgut and Malpighian papillae. They exhibit regular septa crossing the intercellular clefts in thin sections; and the septa with a pronounced zig-zag appearance run parallel to form a honeycomb structure in tracer-impregnated sections. After freeze-fracture undulating rows of intramembranous particles (IMPs) are visible on the P face. Smooth septate juncions are observed in the midgut. The interceullar septa often run in pairs for long tracts and exhibit a wavy course in lanthanum impregnated sections. The IMPs exhibited on the E face are usually separated one from another. Twin arrangement of particle rows is also apparent on the replicas. Gap junctions are frequent in both the midgut and hindgut and possess the conventional characteristics of 'inverted gap junction' with E face connexons. These results provide further evidence relating Protura closely to Collembola as well as to primitive arthropods.