Distribution of adherens junction mediated by VE-cadherin complex in rat spleen sinus endothelial cells

The splenic sinus endothelium regulates the passage of blood cells through the splenic cord. The goal of the present study was to assess the localization of vascular endothelial (VE)-cadherin, β-catenin, and p120-catenin in the sinus endothelial cells of rat spleen and to characterize the presence and distribution of adherens junction formation mediated by the cadherin-catenin complex. Immunofluorescent microscopy of tissue cryosections demonstrated that VE-cadherin, β-catenin, and p120-catenin were localized in the junctional regions of adjacent endothelial cells. Double-staining immunofluorescent microscopy for VE-cadherin and β-catenin revealed colocalization at junctional regions. Transmission electron microscopy of thin sections of sinus endothelial cells treated with Triton X-100 clearly showed adherens junctions within the plasma membrane. Adherens junctions were located at various levels in the lateral membranes of adjacent endothelial cells regardless of the presence or absence of underlying ring fibers. Immunogold electron microscopy revealed VE-cadherin, β-catenin, and p120-catenin in the juxtaposed junctional membranes of adjacent sinus endothelial cells. Double-staining immunogold microscopy for VE-cadherin and β-catenin and for VE-cadherin and p120-catenin demonstrated colocalization to the junctional membranes of adjacent endothelial cells. Immunolabeling was evident at various levels in the lateral junctional membranes and was intermittently observed in the sinus endothelium. These data suggest that adherens junctions, whose formation appears to be mediated by VE-cadherin-catenin complexes, probably regulate the passage of blood cells through the spleen. This work was supported by a Grant-in-Aid for Scientific Research (C), Japan     

The intercellular junctions of guinea-pig placental capillaries: a possible structural basis for endothelial solute permeability

The endothelial cell junction in guinea-pig placental capillaries consists of a continuous ribbon desmosome (zonula adherens) within which lies a particulate tight junction consisting of between one and five anastomosing strands. The intercellular space at these tight junctions is narrowed and is subdivided by junctional bars which are probably continuous with the intramembrane particle rows seen in freeze-fracture replicas of the junctions. Perfusion with lanthanum salts shows the gaps between the junctional bars to be lanthanum-filled and the entire junction to be lanthanum permeable. The estimated size of the spaces between the junctional bars is consistent with the junctional pore size indicated by previous ultrastructural tracer studies. The wider lateral intercellular space of the ribbon desmosome is spanned by more widely spaced "linkers" which may act as a coarser three-dimensional filter in series with size-limiting pores between the tight junctional bars.     

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.     

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.     

Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo

The peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12-13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types. The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.     

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: structure of the junctions assessed by freeze-fracture and lanthanum permeability

The organization of tight junctional complexes (TJs) was studied in cultured porcine thyroid cells during the inversion of polarity induced by collagen-embedding of inside-out follicles, using freeze-fracture replicas and lanthanum penetration. During the early steps of polarity reversal, freeze-fractures showed that TJs generally persisted. They increased in width and progressively branched out into the basolateral surfaces, towards the basal pole. Later, the number of TJ strands decreased and gap junctions inserted within TJ networks were found between cells in reversed follicles, in the same manner as in typically polarized follicles, embedded in collagen or in suspension. The de novo formation of TJ complexes was rarely found in the reversing structures. Despite the heterogeneity of TJs assessed by freeze-fracture, impermeability to lanthanum tracer was noted in inside-out structures. During the reversal process, some TJs remained unstained, whereas others displayed permeability to lanthanum. This heterogeneity might be due to the "opening" of a small number of junctions (perhaps only one by aggregate). When the process was achieved after 48 hr in collagen, the tightness of the junctions was complete, confirmed by the absence of lanthanum in luminal cavities of newly formed follicles.     

THE INTER-SERTOLI CELL TIGHT JUNCTIONS IN GERM CELL-FREE SEMINIFEROUS TUBULES FROM PRENATALLY IRRADIATED RATS: A FREEZE-FRACTURE STUDY

The tight junctions between Sertoli cells were examined by freeze-fracture in 3-month-old prenatally irradiated rats, whose seminiferous tubules are devoid of germ cells. The replicas from irradiated tubules show elaborate interdigitations of the lateral membranes of Sertoli cells and very extensive tight junctions. These junctions are characterized by a great number of continuous parallel or complex interweaving strands of intramembranous particles, preferentially associated with E fracture faces. The presence of highly cross-linked tight junctional strands is compatible with an epithelium deprived of germ cells, with a reduced need for flexibility. Anomalous ectoplasmic specializations, consisting of groups of cisternae arranged perpendicularly to the lateral surface, are found in the irradiated tubules. These structures may be involved in a storage mechanism of redundant lateral membrane resulting from the elimination of germ cells. Typical gap junctions, intercalated between the tight junctional strands, are larger and more frequently found in treated animals than in controls. These findings indicate that a very tight permeability barrier seems to be established in the irradiated testis even in the absence of germ cells. Thus, the formation and maintenance of Sertoli tight junctions do not appear to be directly dependent on the presence of germ cells. Nevertheless, the alterations detected in the tight junction architecture and in the ectoplasmic specializations indicate that maturing germ cells probably contribute to the functional organization of the blood—testis barrier in the normal testis.     

Relationship of sertoli-sertoli tight junctions to ectoplasmic specialization in conventional and en face views

Ectoplasmic specializations are actin filament-endoplasmic reticulum complexes that occur in Sertoli cells at sites of intercellular attachment. At sites between inter-Sertoli cell attachments, near the base of the cells, the sites are also related to tight junctions. We studied the characteristics of ectoplasmic specializations from six species using conventional views in which thin sections were perpendicular to the plane of the membranes, we used rare views in which the sections were in the plane of the membrane (en face views), and we also used the freeze-fracture technique. Tissues postfixed by osmium ferrocyanide showed junctional strands (fusion points between membranes) and actin bundles, actin sheets, or both, which could be visualized simultaneously. En face views demonstrated that the majority of tight junctional strands ran parallel to actin filament bundles. Usually, two tight junctional strands were associated with each actin filament bundle. Parallel tight junctions were occasionally extremely close together ( approximately 12 nm apart). Tight junctional strands were sometimes present without an apparent association with organized actin bundles or they were tangential to actin bundles. En face views showed that gap junctions were commonly observed intercalated with tight junction strands. The results taken together suggest a relationship of organized actin with tight junction complexes. However, the occasional examples of tight junction complexes being not perfectly aligned with actin filament bundles suggest that a precise and rigidly organized actin-tight junction relationship described above is not absolutely mandatory for the presence or maintenance of tight junctions. Species variations in tight junction organization are also presented.     

Tight junctions in the choroid plexus epithelium. A freeze-fracture study including complementary replicas

The tight junctions of the choroid plexus epithelium of rats were studied by freeze-fracture. In glutaraldehyde-fixed material, the junctions exhibited rows of aligned particles and short bars on P-faces, the E-faces showing grooves bearing relatively many particles. A particulate nature of the junctional strands could be established by using unfixed material. The mean values of junctional strands from the lateral, third, and fourth ventricles of Lewis rats were 7.5 +/- 2.6, 7.4 +/- 2.2, and 7.5 +/- 2.4; and of Sprague-Dawley rats 7.7 +/- 3.4, 7.4 +/- 2.3, and 7.3 +/- 1.6. Examination of complementary replicas (of fixed tissue) showed that discomtinuities are present in the junctional strands: 42.2 +/- 4.6% of the length of measured P-face ridges were discontinuities, and the total amount of complementary particles in E-face grooves constituted 17.8 +/- 4.4% of the total length of the grooves, thus approximately 25% of the junctional strands can be considered to be discontinuous. The average width of the discontinuities, when corrected for complementary particles in E-face grooves, was 7.7 +/- 4.5 nm. In control experiments with a "tighter" tight junction (small intestine), complementary replicas revealed that the junctional fibrils are rather continuous and that the very few particles in E-face grooves mostly filled out discontinuities in the P-face ridges. Approximately 5% of the strands were found to be discontinuous. These data support the notion that the presence of pores in the junctional strands of the choroid plexus epithelium may explain the high transepithelial conductance in a "leaky" epithelium having a high number of junctional strands. However, loss of junctional material during fracturing is also considered as an alternative explanation of the present results.     

Freeze-fracture replica studies of tight junctions in normal human bronchial epithelium

Using freeze-fracture techniques, tight junctional networks were observed in the human normal bronchial epithelium. They were morphologically classified into three types: type I was a loosely interconnected, most complicated network consisting of 7-11 roughly parallel wavy strands and situated between ciliated cells; type II was a randomly anastomosing, simple network made up of 2-4 strands and present between goblet cells; type III was an irregularly anastomosing network composed of 4-7 strands and located between a ciliated cell and a goblet cell. Type III junctions, when a goblet cell was strongly bulged, were located on the swollen ridge, the upper surface of which was separated by a deep groove from the bulged apical surface, around the lateral surface of the cell at the level of the luminal surface. The possible relation between the orientation of strands of these networks and extra- or intracellular stress was discussed.     

Cell junctions in amphioxus (Cephalochordata): a thin section and freeze-fracture study

Tissues from the epidermis, alimentary tract and notochord of the cephalochordate Branchiostoma lanceolatum have been examined in both thin sections and freeze-fracture replicas to ascertain the nature of the intercellular junctions that characterize their cell borders. The columnar epithelial cells from the branchial chamber (pharynx), as well as from the anterior and posterior intestine, all feature cilia and microvilli on their luminal surfaces. However, their lateral surfaces exhibit zonulae adhaerentes only. No gap junctions have been observed, nor any tight junctions (as are a feature of the gut of urochordates and higher vertebrates), nor unequivocal septate junctions (as are typical of the gut of invertebrates). The basal intercellular borders are likewise held together by zonulae adhaerentes while hemidesmosomes occur along the basal surface where the cells abut against the basal lamina. The lateral cell surfaces, where the adhesive junctions occur, at both luminal and basal borders, do not exhibit any specialized arrangement of intramembrane particles (IMPs), as visualized by freeze-fracture. The IMPs are scattered at random over the cell membranes, being particularly prevalent on the P-face. The only distinctive IMPs arrays are those found on the ciliary shafts in the form of ciliary necklaces and IMP clusters. With regard to these ciliary modifications, cephalochordates closely resemble the cells of the branchial tract of ascidians (urochordates). However, the absence of distinct junctions other than zonulae adhaerentes makes them exceptions to the situation generally encountered in both vertebrates and urochordates, as well as in the invertebrates. Infiltration with tracers such as lanthanum corroborates this finding; the lanthanum fills the extracellular spaces between the cells of the intestine since there are no junctions present to restrict its entry or to act even as a partial barrier. Junctions are likewise absent from the membranes of the notochord; the membranes of its lamellae and vesicles exhibit irregular clusters of IMPs which may be related to the association between the membranes and the notochordal filaments. Epidermis and glial cells from the nervous system possess extensive desmosomal-like associations or zonulae adhaerentes, but no other junctional type is obvious in thin sections, apart from very occasional cross-striations deemed by some previous investigators to represent 'poorly developed' septate junctions.     

Cell junctions in the cyst envelope in the silkworm testis,Bombyx mori linné

Summary The cell junctions of the cyst envelope in the testes of Bombyx mori were examined by electron microscopy utilizing a thin-sectioning technique following conventional fixation, tannic acid fixation and lanthanum tracer study, and also using a freeze-fracture technique. There are three kinds of junctions; septate junctions, gap junctions and tight junctions. Septate junctions are of the pleated type. Gap junctions are characterized by four electron-dense lines and three electronlucent lines in the reduced intercellular spaces seen by thin-sectioning. They are of the E type, having clusters of intramembraneous particles on the E-fracture face. The most striking finding is the frequent presence of tight junctions on the fracture planes, while focally fused outer leaflets of the junctional unit membranes are rarely detected on thin-sectioned preparations. Tight junctions are characterized by branching zigzag ridges on the P-fracture face and complementary grooves on the E-fracture face. It is proposed that tight junctions are new morphological evidence of blood-germ cell barrier in an insect. Acknowledgements: For helpful assistance the authors are indebted to their colleagues Miss N. Minemoto, Miss H. Kiyotake and Mr. Y. Goto     

Fine structure of the paracellular junctions of terminal villous capillaries in the perfused human placeta

Summary Selected lobules of human term placentae were extracorporeally perfused for a recovery period of 20 min, fixed by perfusion and mordanted with ferrocyanide prior to processing for transmission electron microscopy. The lateral membranes of the endothelial cells of the terminal villous capillaries were found to be separated by paracellular clefts of mean width 15.6 nm. At tight junctional regions (1–4 sites per cleft) the two membranes approached each other more closely and frequently appeared to fuse. However, tilting of the sections in the electron microscope stage showed that the membranes were separated by a gap of mean width 4.1 nm in at least 94% of tight junctional profiles. When individual tight junctions were studied by a combination of serial sectioning and goniometric tilting, they were seen to widen abruptly within a distance of three to seven consecutive thin sections, indicating they were not continuous throughout the axial length of the capillaries. The wide regions of the clefts usually showed linkers, strands of glycocalyx-like material spanning the gap. Linkers may contribute to cell adhesion and possibly form part of a filter within the tortuous paracellular pathway provided by the discontinuous network of tight junctional strands. Human term placental capillaries appear to resemble closely other continuous non-brain capillaries.     

Glial cells in peripheral nerves of the cockroach, Periplaneta americana

The ultrastructural organization and the junctional complexes of peripheral nerves have been investigated in the cockroach Periplaneta americana. Nerve 5 is surrounded by a layer of connective tissue, the neural lamella, beneath which is a layer of perineurial glial cells wrapping the axons. Adjacent perineurial cells are joined to one another by septate, gap and tight junctions. Septate and gap junctions were observed in freeze-fracture replicas of main trunk nerve 5. Septate junctions were found as rows of PF particles mainly in perineurial cell membranes. Gap junctions exhibited EF macular aggregates in perineurial and subperineurial glial cells. During incubations in vivo with extracellularly applied ionic lanthanum, the lanthanum did not penetrate beyond the perineurium. Where nerve 5 branches and contacts the muscle, lanthanum penetrated freely between the muscle fibres and the nerve branches. In small peripheral branches where the axons are surrounded by single a glial layer, lanthanum is unable to penetrate to the axolemma.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

The structure and function of the smooth septate junction in a transporting epithelium: The malpighian tubules of the new zealand glow-worm Arachnocampa luminosa

Junctional complexes between the epithelial cells in the four distinct regions of the glow-worm Malpighian tubule were investigated by electron microscopy using thin sectioning, freeze-fracturing, osmotic disruption and tracer techniques. The lateral plasma membranes of all four cell types are joined by smooth septate junctions but the extent of the complex across the cell depth varies in the four different regions. The width of the septa, the interseptal spacing and the separation between the outer leaflets of the adjacent plasma membranes are different for each cell type. Gap junctions were identified only in the junctional complex between Type IV cells and were intercalated amongst large lateral sinuses. In oblique sections of lanthanum infiltrated tissue, the electron-lucent septa at the basal side of the junction are outlined by the tracer as it penetrates. In the Junctional complexes of all four regions the septa appear as short, distinct, linear bars. In tangential sections of gap junctions between Type IV cells, the junctions appear as a hexagonal array of intermembrane particles with a centre to centre spacing of 18 nm. Horseradish peroxidase did not penetrate the junctional complexes very far but readily passed through the basal lamina into the spaces between extracellular invaginations of the basement membrane of the cells. Junctional complexes in all four areas of the tubule have similar freeze-fracture faces. In freeze-fracture replicas of fixed tissue continuous ridges of fused particles are seen on the P face and complementary furrows are found on the E face. Junctional response to osmotically adjusted Ringer solutions was similar in all four cell types. Distortion or ‘blistering’ of the intercellular space between the septa of the junction occurred when the tissue was bathed in or injected with a hypertonic Ringer solution. The structure of these junctions, visualized by the different techniques, and the role of the septate junction in a transporting epithelium, are discussed.     

Differentiation of the plasma membrane of hepatic cells in monolayer cultures

Hepatocytes from rats were isolated by treatment with trypsin and cultured. Plasma membranes at different culture stages were observed by electron microscopy. The activities of 5' nucleotidase and adenosinetriphosphatase on the plasma membranes were examined. The cell coat was also studied by use of the concanavalin A-peroxidase technique. The surfaces of single cells, covered with microvilli, are the site of adenosinetriphosphatase activity only and are devoid of 5'-nucleotidase activity. After a few h of culture, the cells are grouped together in tight clusters or long trails and are separated by an intercellular space of 250 A, partially permeable to lanthanum nitrate. The juxtaposed plasma membranes on which 5'-nucleotidase and adenosinetriphosphatase activities occur also delimit spaces similar to bile canaliculi. The formation of junction complexes and their permeability to lanthanum nitrate was also studied. No enzymatic activity is observed at the junctions. The numerous tight junctions, impervious to the tracer, are always accompanied by a profusion of microfilaments. Mature desmosomes are rare, and are present only in the form of "maculae adhaerentes diminutae." The gap junctions, nearly always permeable to the tracer, form rapidly and assume a variety of shapes (trail, bulge and ring-like), the significance of which is open to discussion. The use of concanavalin A permits localization of the free sugar sites on the surface of the cells, in the pinocytotic vesicles and in the internal space of the gap junctions.     

Freeze-fracture investigation of the red pulp of human spleen

This study concerns the morphology of the human spleen in freeze-fracture replicas and compares this with the findings in ultrathin sections. The material investigated consisted of two spleens resected at gastrectomy and one resected because of splenomegaly in a case of hairy cell leukaemia. The current concepts concerning the ultrastructure of the spleen were generally confirmed with the freeze-fracture technique. The sinus walls were found, as expected, to consist of closely fitting endothelial cells, which were identifiable in freeze-fracture replicas by numerous caveolae of the cell membrane. Contrary to the opinion upheld in the literature, the sinus endothelial cells were occasionally found to be connected by desmosomes or maculae adhaerentes. Corresponding to the finding of desmosomes in ultrathin sections, focal collections of intramembranous particles were observed in freeze-fracture replicas and a positive immunohistochemical reaction for desmoplakin in the sinuses was found at the light microscopic level. The view generally held in the literature that sinus endothelial cells can exhibit tight junctions was not confirmed. However, such junctions were found between vascular endothelial cells. The ring fibres of the sinuses, which are closely connected to the sinus endothelial cells through contractile fibres, apparently have various functions. Firstly, they contribute towards maintaining mechanical stability. Secondly, they represent basement membranes through which exchange occurs between the sinus endothelial cells and their surroundings. This is indicated by the caveolae and vesicles that are often found here in large numbers and in focal collections. Hairy cells exhibit no features in freeze-fracture replicas to suggest a cytogenetic relationship to interdigitating reticulum cells.