The ultrastructural basis of alveolar-capillary membrane permeability to peroxidase used as a tracer

The permeability of the alveolar-capillary membrane to a small molecular weight protein, horseradish peroxidase (HRP), was investigated by means of ultrastructural cytochemistry. Mice were injected intravenously with HRP and sacrificed at varying intervals. Experiments with intranasally instilled HRP were also carried out. The tissue was fixed in formaldehyde-glutaraldehyde fixative. Frozen sections were cut, incubated in Graham and Karnovsky's medium for demonstrating HRP activity, postfixed in OsO4, and processed for electron microscopy. 90 sec after injection, HRP had passed through endothelial junctions into underlying basement membranes, but was stopped from entering the alveolar space by zonulae occludentes between epithelial cells. HRP was demonstrated in pinocytotic vesicles of both endothelial and epithelial cells, but the role of these vesicles in net protein transport appeared to be minimal. Intranasally instilled HRP was similarly prevented from permeating the underlying basement membrane by epithelial zonulae occludentes. Pulmonary endothelial intercellular clefts stained with uranyl acetate appeared to contain maculae occludentes rather than zonulae occludentes. HRP did not alter the ultrastructure of these junctions.     

The formation and distribution of intercellular junctions in the rhesus monkey optic cup: The early development of the cilio-iridic and sensory retinas

The eyes of prenatal monkeys from 30 to 102 ± 2 days old were examined by light microscopy, conventional electron microscopy, and the freeze-fracturing technique. At 30 days, invagination of the optic vesicle has begun, and the inner and outer walls of the forming optic cup are closely apposed anteriorly; invagination is complete at 45 days. By 58 days, the rudiment of the ciliary body and iris has appeared; at 71 days, primitive ciliary processes are present and retinal photoreceptors begin to differentiate. The distribution of intercellular junctions varies both in different regions of the optic cup and at different stages of development. At 30 days, adjacent ventricular and adjacent pigmented cells are joined throughout the optic cup by zonulae adhaerentes and gap junctions. The anterior region of the cup, however, contains two additional junctional specializations: (1) fasciae occludentes between ventricular cells and (2) intermediate and gap junctions between the apposing luminal surfaces of ventricular and pigmented cells. By 36 days the fasciae occludentes between ventricular cells in the anterior optic cup become zonular, signaling the morphological development of the blood-aqueous barrier. In the posterior optic cup, zonulae occludentes appear between adjacent pigmented cells at 36 days; furthermore, with the continuing obliteration of the optic ventricle, luminal junctions spread toward the optic stalk but do not reach the optic disc until 45 days, when invagination is complete. Between 58 and 102 days there are no further changes in the distribution of the junctions anteriorly between the primitive cilio-iridial epithelial cells, whereas in the posterior optic cup the luminal gap and intermediate junctions between pigmented cells and differentiating photoreceptors decrease in number and finally disappear. Two main conclusions can be drawn from this study. (1) In the optic cup, intermediate junctions are consistently present in regions of the plasma membrane which later contain junctional complexes. The temporal and spatial pattern of junctional development suggests that intermediate junctions are necessary for the establishment of tight and gap junctions. (2) Twenty days before the ciliary body-iris anlage becomes visible in the light microscope, the distribution of junctions in the anterior part of the optic cup is identical to that in the adult cilio-iridic retina. The time-honored view that the cilio-iridic retina appears late in development is, therefore, no longer tenable. In the monkey, the optic cup is divided into a cilio-iridic and a sensory region soon after the onset of invagination.     

Structure and distribution of gap junctions in lens epithelium and fiber cells

Summary We report a comparative study of gap junctions in lens epithelia of frog, rabbit, rat and human, using a double mounting method for freeze-fracture electron microscopy. The gap junctions on the narrow sides of hexagonal cortical fiber cells of various species were also studied with the same technique. Gap junctions were commonly present between epithelial cells of the entire undifferentiated epithelium, between fiber cells on both wide and narrow sides, and between epithelial cells and fiber cells. Structural diversity of gap junctions, based on connexon arrangements, was evident in lens epithelia among the four species studied. Gap junctions with random arrays of connexons were found predominantly in frog lens epithelium, while the crystalline and striated configurations were mainly observed in the epithelia of human and rat, and of rabbit, respectively. On the other hand, there was no structural variation of gap junctions observed on either wide or narrow sides of lens fiber cells from any species studied. Only the random-type gap junction was found. However, the distribution of gap junctions was unique on the narrow sides. There was a single row of junctional plaques along the middle of the narrow sides, whereas the wide sides showed an uneven distribution pattern. The gap junctions between epithelial cells and fiber cells had a random packing of connexons.     

Isolation and protein composition of gap junctions from rabbit hearts.

We have modified a method for isolating gap-junctional membrane from mouse hearts [Kensler & Goodenough (1980) J. Cell Biol. 86, 755-764] to isolate gap junctions of comparable purity from rabbit hearts more rapidly, with better yield, and without resort to non-ionic detergents. Purification was monitored by electron microscopy of thin-sectioned membrane pellets and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gap junctions were obtained as vesicles whose mean surface area approximated that of junctions in intact myocardial cells. About 10-20% of the vesicles were ferritin-impermeable. Approx. 125 micrograms of membrane protein was obtained per 8 g of rabbit heart. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of purified gap junctions showed five major protein bands of mol.wts. 46 000, 44 000, 33 000, 30 000 and 28 500 that co-purified with the junctions. This protein composition was nearly identical with that published for gap junctions of mouse hearts, and differed markedly from the protein composition of gap junctions from non-excitable cells (lens and liver). The constancy of junctional protein composition between hearts of two different species and its non-identity with that from liver and lens suggest that, although gap-junctional structure in mammalian tissues seems to be remarkably similar by electron-microscopic techniques, junctional-channel protein composition actually varies from tissue to tissue and may be adapted to the permeability requirements of the tissue.     

Immunocytochemical localization of the main intrinsic polypeptide (MIP) in ultrathin frozen sections of rat lens

The in situ distribution of the 26-kdalton Main Intrinsic Polypeptide (MIP or MP 26), a putative gap junction protein in ocular lens fibers, was defined at the electron microscope level using indirect immunoferritin labeling of ultrathin frozen sections of rat lens. MIP was found distributed throughout the plasma membrane of the lens fiber cell, with no apparent distinction between junctional and nonjunctional membrane. MIP was not detectable in the basal or lateral plasma membrane of the lens epithelial cell, including the interepithelial cell gap junctions; nor was MIP detectable in the plasma membrane or gap junctions of the hepatocyte. Previous reports have indicated that the protein composition of the lens fiber cell junction differs from that of the hepatocyte gap junction. The evidence presented here suggests that the composition of the fiber cell junction and plasma membrane is also immunocytochemically distinct from that of its progenitor, the lens epithelial cell.     

Ultrastructure of traumatic cataractogenesis in the frog: a comparison with mouse and human lens.

Normal and needle-punctured lenses of Rana pipiens were examined with the electron microscope in order to characterize the sequence of ultrastructural changes that follow the injury over a 5-month period. Results were compared with those obtained previously in experimentally injured mouse and accidentally injured human lenses. The normal adult frog lens was found to have a morphology similar to that of mammalian lenses. As in the human, frog lens epithelial cells contained scattered microfilaments and were connected by desmosomes and gap junctions. They differed from mouse cells, which had been shown to lack desmosomes and to have microfilaments organized into dense bundles. These differences are postulated to be related to the degree of accommodative deformation of the lens displayed by these species. After injury, cellular debris and fibrin, accumulated in the wound, were phagocytized by extrinsic cells derived from the blood and ocular tissues. Leucocytes, pigmented cells and fibroblasts remained in the wound for eight weeks, along with epithelial cells which proliferated and migrated from the wound margins.Epithelial cells showed an increase in those organelles associated with protein synthesis and transport, and in microfilaments. In cataractous lenses, epithelial cells showed changes in matrix, and lens fibers became organized into smaller, denser compressed units. At five months, considerable healing had taken place, but localized opacities persisted in many frog lenses.     

Biochemical and genetic investigations on gap junctions from mammalian cells

Gap junction protein (26K) in mouse or rat liver has been studied using a rabbit antiserum directed against the sodium dodecylsulfate denatured 26K protein from mouse liver. The liver 26K protein has been localized in gap junction plaques of hepatic plasma membranes by immuno electron microscopy. Affinity purified anti-26K antiserum showed weak cross reactivity with mouse or bovine lens gap junction protein (MIP26). This result suggests some structural homology between the different gap junction proteins in liver and lens. After partial hepatectomy of young rats the liver 26K protein appears to be degraded and later resynthesized. A variant of established Chinese hamster fibroblastoid cells has been isolated and shown to be defective in metabolic cooperation via gap junctions.     

Freeze-fracture study of the junctional complexes of human and rabbit thyroid follicles

Summary Zonulae occludentes, gap junctions and desmosomes have been demonstrated in replicas of freeze-fractured follicular cells of normal human and rabbit thyroid glands. The zonulae occludentes between the human follicular cells are composed of two to eight strands, which completely separate the intercellular space from the follicular lumen. Four to twelve or more strands are visible between the follicular cells of the rabbit thyroid gland.In the meshes of the zonulae occludentes as well as below them, gap junctions are present. They are numerous on the fracture faces of the human follicular cell membranes, but infrequent in those of the rabbit.Aggregates of particles related to desmosomes are found in the deeper meshes of the zonulae occludentes or close to them.     

家兔晶状体纤维的超微结构:纤维细胞的间隙连接

本文报道晶状体纤维细胞间间隙连接的形态结构。我们利用冰冻断裂技术,在不同部位的球-和-凹连结的头部以及在纤维细胞和纤维细胞之间都观察到间隙连接的存在。通过极其丰富的上述连接,可实现细胞间代谢物和离子的传递。作者认为:对正常晶状体纤维细胞之间的间隙连接的深入了解,将会为晶状体发病机制的研究提供新的线索。     

Effects of extracellular calcium depletion on membrane topography and occluding junctions of mammary epithelial cells in culture

Ca2+ dependence of occluding junction structure and permeability, well documented in explanted or cultured epithelial sheets, presumably reflects inherent control mechanisms. As an approach to identification of these mechanisms, we induced disassembly of zonulae occludentes in confluent monolayers of mouse mammary epithelial cells by exposure to low concentrations of the chelators, EGTA or sodium citrate. Stages in disassembly were monitored during treatment by phase-contrast microscopy and prepared for transmission and scanning electron microscopy. Cellular response included several events affecting occluding junctions: (a) Centripetal cytoplasmic contraction created tension on junction membranes and displaced intramembrane strands along lines determined by the axis of tension. (b) Destabilization of junction position, probably through increased membrane fluidity, augmented tension-induced movement of strands, resulting in fragmentation of the junction belt. (c) Active ruffling and retraction of freed peripheral membranes remodeled cell borders to produce many filopodia, distally attached by occluding-junction fragments to neighboring cell membranes. Filopodia generally persisted until mechanically ruptured, when endocytosis of the junction and adhering cytoplasmic bleb ensued. Junction disassembly thus resulted from mechanical tensions generated by initial centripetal contraction and subsequent peripheral cytoskeletal activity, combined with destabilization of the junction's intramembrane strand pattern.     

Cell junctions in the epithelium of Bowman's capsule

Summary The intercellular connections between the epithelial cells of Bowman's capsule were investigated. It could be demonstrated that typical zonulae occludentes (tight junctions) are present in the species (rat, hamster, and Tupaia) studied. Freeze-fracturing shows a network of anastomizing strands; some species variations are described. In the rat two strands are common. In the golden hamster mostly two to four and occasionally five strands occur. In Tupaia regularly three tight junction strands are found and also gap junctions associated with the zonulae occludentes. In thin sections the goniometric analysis confirms the freeze-fracturing results and reveals attachment zones of macular shape, which are classified as intermediate junctions and desmosomes. The functional role of these cell junctions observed in the epithelium of Bowman's capsule is discussed.     

Evidence for two physiologically distinct gap junctions expressed by the chick lens epithelial cell

Lens epithelial cells communicate with two different cell types. They communicate with other epithelial cells via gap junctions on their lateral membranes, and with fiber cells via junctions on their apices. We tested independently these two routes of cell-cell communication to determine if treatment with a 90% CO2-equilibrated medium caused a decrease in junctional permeability; the transfer of fluorescent dye was used as the assay. We found that the high-CO2 treatment blocked intraepithelial dye transfer but not fiber-to-epithelium dye transfer. The lens epithelial cell thus forms at least two physiologically distinct classes of gap junctions.     

Adherens junctions in the ocular lens of various species: ultrastructural analysis with an improved fixation

Summary The ultrastructure and distribution of adherens junctions in the intact adult lens of human, chicken, dove, rat, and rainbow trout were studied with thin-section electron microscopy, using an improved fixation containing a mixture of glutaraldehyde, lysine, and tannic acid. The nature of adherens junctions in the fiber-cells of the lens was also verified by immunofluorescence and rhodamine-phalloidin labelings for vinculin and actin. Electron microscopy revealed that adherens junctions of the lens were different ultrastructurally from the desmosomes found only between the lateral epithelial cells of the lens. The adherens junctions had the same structural characteristics as the zonulae adherentes, except that they were macular contacts, not belts. However, cross bridges were evident within the interspace of the junctions. Adherens junctions were located between the fiber-cells, between the epithelial cells and fiber-cells, and between the epithelial cells. They had a characteristic distribution in the intersections where three hexagonal fiber-cells met, as seen in cross-sections in all species studied. In addition, adherens junctions and associated actin were found distributed randomly along the entire cell membranes of both wide and narrow sides of cortical fiber-cells in the human, chicken, and dove lenses which have good accomodating capability. However, in the poorly-accomodating lenses of rat and fish, these junctions were seen predominantly on the narrow sides and at the regions of the wide sides that were very close to the intersections. It is suggested that adherens junctions and associated actin microfilaments are involved in stabilizing the structural integrity of lens cells during accomodation and in preserving a specific lens shape.     

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.     

The fine structure of the hypostome and mouth of hydra

Summary The normal morphology of the hypostome and mouth of hydra were examined by transmission electron microscopy with conventional thin sections and freeze-fracture replicas. Myonemes of the hypostome are small in diameter, have gap and intermediate-type cell junctions within each epithelial layer and are associated with the opposite epithelial layer by transmesogleal processes and gap junctions. Nematocysts and sensory cells are aggregated in the circumoral region. The fine structure of adherent flagella arising from gastrodermal gland cells, and the transition region at the mouth between epidermis and gastrodermis are described in detail for the first time. The possible functional significance of the findings is discussed.     

Identification of a 70,000-D protein in lens membrane junctional domains

A 70,000-D membrane protein (MP70), which is restricted to the eye lens fibers and is present in immunologically homologous form in many vertebrate species, has been identified. By use of anti-MP70 monoclonal antibodies for immunofluorescence microscopy and electron microscopy, this polypeptide was localized in lens membrane junctional domains. Both immunofluorescence microscopy and SDS PAGE reveal an abundance of MP70 in the lens outer cortex that coincides with a high frequency of fiber gap junctions in the same region.     

Lens fiber organization in four avian species: a scanning electron microscopic study

The three-dimensional organization of the eye lenses of the chicken, the canary, the song-thrush and the kestrel was studied using light and scanning electron microscopy. The lenses of birds are characterized by the presence of two distinct compartments: the annular pad and the main lens body, separated by a cavum lenticuli. The annular pad fibers had a hexagonal circumference all contained a round nucleus and except for the canary were smooth-surfaced and lacking anchoring devices. In the canary, however, the annular pad fibers were studded with edge protrusions and ball-and-socket junctions. The semicircular main lens body fibers of all four species were studded with ball-and-socket junctions and edge protrusions. In contrast with mammals these anchoring devices were present throughout the lens up to the embryonal nucleus. Superficially the main lens body fibers were extremely flat. Additionally membrane elevations and depressions and globular elements were found on these central fibers in three species, the kestrel being the exception. At the transition between annular pad and main lens body the fibers turned their course and the nuclei became oval and disappeared in the deeper aspect of the main lens body. The cavum lenticuli was filled with globules tied off from the annular pad fibers. It seems attractive to assume that the presence of a separated annular pad, a cavum lenticuli filled with globular elements, the extreme flatness of the superficial central fibers and the studding of these central fibers with anchoring devices up to the embryonal nucleus are morphological expressions of the mouldability of the bird's eye lenses and consequently would explain their efficient accommodative mechanism including formation of a lenticonus. The presence of nuclei in the annular pad fibers and their typical change at the transitional zone between annular pad and main lens body are suggestive for a two-phased differentiation in bird's lens fibers: differentiation of the germinative epithelial cells to annular pad fibers which migrate to the main lens body after which they differentiate further to main lens body fibers.     

Structure of zonulae occludentes and the permeability of the epithelium to short-chain fatty acids in the proximal and the distal colon of guinea pig

Summary Absorption of short-chain fatty acids has been studied in the proximal and the distal colon of anaesthetized guinea pigs. Segments were perfused with a solution similar in chemical composition to that of normal colonic fluids. In the proximal colon the permeability of the mucosa was similar for acetate, propionate and butyrate. For acetate the permeability was significantly higher in the proximal than in the distal colon, and the reverse was seen for butyrate. In the distal colon the short-chain fatty acids seem to be absorbed mainly in the undissociated form due to their lipid solubility: a paracellular pathway for the dissociated molecules is of no major importance. In the proximal colon, on the other hand, a considerable portion of acetate and propionate disappears in the ionized form. Light microscopy (semithin sections) and electron microscopy (freeze-fracture replicas) showed remarkable morphological differences between the proximal and the distal colon. Leaky spots with only few strands were present in the zonulae occludentes between the epithelial cells at the surface of the proximal colon. In the distal colon the junctions between the cells were more compact, and significantly more strands separated the lumen from the intercellular space. These results suggest that short-chain fatty acids could be absorbed by a paracellular pathway in the proximal colon, and not in the distal colon. In the proximal colon the number of strands of the zonulae occludentes between surface cells and that between cryptal cells was similar. On the contrary, in the distal colon significantly more strands were present between surface cells than between cryptal cells. Morphological and physiological considerations suggest that absorption of short-chain fatty acids in the crypts is negligible.     

Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning,ultrastructural localization,and post-translational phosphorylation

Summary Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with³²P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.     

Square arrays and their role in ridge formation in human lens fibers

Square arrays in human lens fibers were studied with freeze-fracture and thin-section TEM. In superficial fibers a number of patches of square array particles in the P face and pits in the E face are found in the smooth membrane. In the deeper cortex and the nucleus, fiber cells have undulating membranes and many ridges. Numerous patches of the particles (P face) are distributed in the concave regions, and the pits (E face) in the convex areas of the bumpy membrane. In most ridges, patches of the particles occur at regular intervals in the "valley" portion, while the pits are on the "crest" portion of ridges. Also, continuous square arrays having the same "valley" location as the regularly arranged patches are found in areas with extensive ridge patterns. The overlapping of the outer portions of two adjacent square arrays is found on the sides between the "crest" and the "valley" of the ridges. Structurally, square arrays are located in a nonjunctional part of the membrane; in an orthogonal crystalline arrangement; and with a particle size of about 6 nm and center-center spacing about 6.4 nm. They are structurally different from gap junctions found in the lens fibers. Thin-section studies reveal two types of cellular contacts: thin pentalamellar structures (about 12-13 nm in overall thickness) associated with the ridge patterns are believed to be square arrays; thick heptalamellar structures (about 16-17 nm in overall thickness) with a narrow gap in between the two central laminae are believed to be gap junctions. This study strongly suggests that square arrays are specifically involved in ridge formation in human lens fibers.