Ultrastractural features of the principal cell in the epididymis of the rhesus monkey

The ultrastructural features of the principal cell in the epididymal epithelium of the monkey epididymis are suggestive of the cell carrying out a dual function of absorption and secretion. Both these functions occur on the luminal surface of the cell as well as on the lateral and basal aspects of the cell which face the intercellular spaces. Transmision Electron Microscopic studies of epididymal tissues following their impregnation with lanthanum nitrate indicated that the intercellular spaces are effectively sealed-off from the luminal space by the apically situated tight junctions between adjoining principal cells. The intercellular spaces are contiguous with the perivascular spaces of the subepithelial blood capillaries. It is suggested that the absorptive and secretory functions occuring on the apical surface of cells may be related to the creation of an appropriate intraluminal milieu for the maturation of spermatozoa while the occurrence of these functions in the intercellular spaces may represent an exchange of substances between the principal cells and the subepithelial capillaries.     

Surface coat in steroid-secreting cells of the mouse ovary

Steroid-secreting cells (luteal, thecal and interstitial cells) of the mouse ovary have been studied with the ruthenium red method to stain the "cell coat". The results showed that a typical cell coat covers the entire surface of the plasma membrane except where the cells are connected by specific cell junctions. Further, particularly heavy concentration of ruthenium-red-reacting material was demonstrated in pericapillary and intercellular spaces. The differences in the thickness of the cell coat and its topographical modifications among different groups of steroidogenic cells may be related not only to intercellular adhesion and interactions but also concerned with a function of control in which the cell permeability is modified in relation with phenomena of cell recognition.     

Formation of the septate junction in regenerating planarian gastrodermis

Septate junctions develop initially just basad from apical junctional complexes at the apical ends of regenerating gastrodermal cells. The first morphological indication of differentiation of the junction is the appearance of gentle undulations of the plasma membranes of apposing cells. Subsequently dense dots develop at fairly regular intervals at the cytoplasmic surface of one cell, while SER cisternae become localized opposite them near the surface of the apposing cell. The dense dots are associated with bulges which narrow the intercellular space. Later the dense dots are replaced by filaments aligned along the inner leaflet of the parent cell. Strands of amorphous deposits form connections between SER cisternae and the sister membrane on the opposite side of the junction. Ruthenium red staining provides information on precursors which occupy the intercellular space between the apposed plasma membranes. As development of the junction progresses, ruthenium red stains only the newly formed septa but not the interseptal matrix. Regular arrangement of individual septa seems to be completed under the control of V-projections from both of their surfaces. Precursors for the structural material of the septa may be a secretory product derived from the SER. Dense dots and their derived filaments probably serve as reinforcing material for strengthening the cell membrane of the junction.     

Studies on the surface of chick blastoderm cells. II. Electron microscopy of surface binding characteristics

The surfaces of cells from the early embryo of the chick were examined with the electron microscope using histochemical techniques in order to determine the distribution of cell surface material. The use of lanthanum nitrate as a stain for protein-polysaccharide showed that in whole embryos the surface layer was present in areas of close membrane apposition, but relatively sparse where the membranes bounded large intercellular spaces. On cells freshly dissociated with EDTA, this technique demonstrated a very uniform layer over the surface, possibly indicating some redistribution. Close examination of the lanthanum stained material in whole embryos revealed the presence of periodicities about 180 Å in diameter. In correlation with a progressive reduction of electrophoretic mobility, demonstrated previously, from embryonic stage 1 to stage 5, some decrease in the affinity of the surface for lanthanum and colloidal iron was observed.     

Cell surface saccharides of Trypanosoma lewisi. I. Polycation-induced cell agglutination and fine-structure cytochemistry.

Trypanosoma lewisi bloodstream and culture forms were agglutinated differentially with low concentrations of the cationic compounds: ruthenium red, ruthenium violet, Alcian blue chloride, 1-hexadecylpyridinium chloride, lanthanum chloride, and cationized ferritin. The bloodstream form trypanosomes gave the highest agglutination levels with each of the compounds tested. Ruthenium red was the most effective inducer of cell agglutination among the several cations used. Trypsin-treated bloodstream forms were agglutinated less in the presence of ruthenium red than untreated controls. Ruthenium red-induced cell agglutination also was lowered with chondroitin sulphate and dextran sulphate, but not with alpha-D-glucose, alpha-D-mannose or with several methyl glycosides. Treatment of the bloodstream trypanosomes with alpha-amylase, dextranase, or neuraminidase had little effect on agglutination levels obtained with ruthenium red. Fine-structure cytochemical staining with ruthenium red, ruthenium violet, and Alcian blue-lanthanum nitrate was used to ascertain the presence and distribution of presumptive carbohydrates in the trypanosome cell surface. The extracellular surface coat of the bloodstream forms stained densely with each of the polycationic dyes. Trypsin treatment removed the surface coat from bloodstream trypanosomes; however, the surface membranes of the organisms were stained densely with the several dyes. Similar surface-membrane staining was obtained with the cationic compounds and the culture forms, which lack a cell surface coat. Cationized ferrin was used at the fine-structure level to visualize the negative surface charge present in the cell surface coat and external membrane of the several trypanosome stages. Results obrained from the agglutination and cytochemistry experiments indicate that complex polysaccharides are present in the surface membranes and cell surface coat of T. lewisi bloodstream forms. Similar conclusions also pertain to the surface membranes of the T. lewisi culture from trypanosomes. The carbohydrates probably represent glycopeptide and glycoprotein structural components of the surface membrane of this organism.     

Cell Communication in the Basal Cells of the Human Epidermis

Electrotonic spread can be measured in the basal cells of the human epidermis. The communication between neighboring cells is high, whereas no leak to the intercellular spaces could be detected. The specific resistance of the membranes between the cells is about 10 Ωcm². This finding suggests that for those particles that are able to pass the cell membrane the intracellular path through the epidermis is at least as suitable as the path through the intercellular spaces.     

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.     

FINE STRUCTURE OF THE OCTOPUS RETINA

The fine structure of the visual and the supporting cells and of the blood capillaries in the octopus retina is described. Lamellated structures contained in the proximal segment of the visual cell consist of compact arrays of dense membranes each of which is quintuple-layered and divides at its margins into two thinner sheets or membranes which are connected directly with the agranular or granular endoplasmic reticulum. Proximal to the deeper extremities of the rhabdomeres, the lateral plasma membranes of two adjoining visual cells contact each other forming a quintuple-layered compound membrane, which results in occlusion of the intercellular space. The central layer of the compound membrane is of high density, so that the membrane, as a whole, appears to be a single thick layer at low magnifications. The supporting cells are connected with the neighboring visual cells by two types of junctions. Long slender processes extend from the supporting cells to the surface of the retina through narrow spaces among the distal segments of the visual cells. The capillary endothelial cells are characterized by luminal surfaces irregularly contoured and by lateral surfaces elaborately interdigitated. The functional significance of the close contact between adjoining visual cells is discussed.     

Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Notophthalmus viridescens

The ultrastructure of the epidermal cells which migrate over the wound surface of the amputated limb of the adult newt, Notophthalmus viridescens, was observed with transmission (TEM) and scanning (SEM) electron microscopy. In order to aid in the visualization of polyanionic surface materials on the wound epithelium and wound surface with TEM, the basic dye, ruthenium red, was introduced into the fixatives and buffer. Control limbs were processed without ruthenium red. Shortly after amputation, basal cells at the wound margin possessed elongated, flattened profiles with long pseudopodial projections (lamellipodia and filopodia) that appeared to make contact with the fibrin exudate covering the stump tissues. Epidermal cells proximal to the site of amputation were also in a state of mobilization. Large intercellular spaces and a reduction in the number of desmosomes were observed in the migrating cells. Epidermal cell nuclei became characteristically euchromatic with well-developed nucleoli. Microfilaments were seen within the cytoplasm, extending toward the plasma membrane of cellular processes. Phagocytosed material was also present in the migrating cells. By approximately 9 hours post-amputation, wound closure was complete, and the wound epithelium consisted of three to four cell layers of a non-cornified epidermis. Generally, the amount of extracellular material present on the surface and in the enlarged intercellular spaces of migrating epidermal cells remained the same throughout the period of wound closure. A layer of polyanionic material was observed consistently over the fibrin meshwork covering the wound surface with TEM.     

Immunocytological studies on an estradiol sensitive antigen in the cervicovaginal epithelium of neonatal mice

Summary Cells of the cervicovaginal epithelium of neonatal mice underwent morphological changes in response to estradiol injection. On the luminal border, estradiol treatment caused development of distinct microvilli and a prominent surface coat of delicate filamentous material. Very deep nuclear folds appeared, and the border between adjacent cells became strongly interdigitated. The cells developed a pronounced smooth and rough endoplasmic reticulum, and dark-stained membrane-bounded granules accumulated in the apical part of the cells.Estradiol promoted increased production of an antigenic material specific for the cervicovaginal epithelium (CVA). Immunofluorescence studies demonstrated CVA in the most apical part of the cells, in the extracellular material on the epithelial surface, and in the intercellular spaces between adjacent epithelial cells. This was confirmed by immunoferritin methods, which revealed that the antigen was localized to the surface coat and to material adhering closely to the exterior of the cell membrane, the part facing the lumen and also the part facing intercellular spaces. Within the cells, ferritin tagging was recognized around the membranes enclosing the dark-stained granules in the apical part of the cells and also on the inside of the luminal cell membrane. This is so interpreted that CVA acquires its antigenic properties when passing out from the dark-stained granules through the surrounding envelope. CVA apparently forms part of the glycocalyx of the cervicovaginal cells.This investigation was supported by grants from the Norwegian Cancer Society (Landsforeningen mot Kreft) and the Norwegian Research Council.     

Membrane contacts of the endoplasmic reticulum and their possible functions in the plant cell

Close contacts of the endoplasmic reticulum membrane and plasmalemma have been visualized inside plant cells by means of electron microscopy. The qualitative similarity of these contacts to high-permeable intercellular contacts in animals has been shown. New data confirming the hypothesis of the identity of stromules, i.e., dynamic tubular protuberances of the plastid membrane of the plant cell, and tubular elements of the endoplasmic reticulum have been presented. New possible functions of the contacts of the endoplasmic reticulum membrane with other membranes inside the cell have been discussed on the basis of this hypothesis.     

FINE STRUCTURE AND MORPHOGENIC MOVEMENTS IN THE GASTRULA OF THE TREEFROG, HYLA REGILLA

The blastoporal groove of the early gastrula of the treefrog, Hyla regilla, was examined with the electron microscope. The innermost extension of the groove is lined with invaginating flask- and wedge-shaped cells of entoderm and mesoderm. The distal surfaces of these cells bear microvilli which are underlain with an electron-opaque layer composed of fine granular material and fibrils. The dense layer and masses of vesicles proximal to it fill the necks of the cells. In flask cells bordering the forming archenteron the vesicles are replaced by large vacuoles surrounded by layers of membranes. The cells lining the groove are tightly joined at their distal ends in the region of the dense layer. Proximally, the cell bodies are separated by wide intercellular spaces. The cell body, which is migrating toward the interior of the gastrula, contains the nucleus plus other organalles and inclusions common to amphibian gastrular cells. A dense layer of granular material, vesicles, and membranes lies beneath the surface of the cell body and extends into pseudopodium-like processes and surface undulations which cross the intercellular spaces. A special mesodermal cell observed in the dorsal lining of the groove is smaller and denser than the surrounding presumptive chordamesodermal cells. A long finger of cytoplasm, filled with a dense layer, vesicles and membranes, extends from its distal surface along the edge of the groove, ending in a tight interlocking with another mesodermal cell. Some correlations between fine structure and the mechanics of gastrulation are discussed, and a theory of invagination is proposed, based on contraction and expansion of the dense layer and the tight junctions at distal cell surfaces.     

Oxidation of ruthenium red for use as an intercellular tracer

Summary When ruthenium red (RR) is combined with OsO₄, an electronopaque complex forms which readily binds to the cell surface coat. However, the RR-OsO₄ complex is often excluded from intercellular spaces in many cell types, and thus is not dependable as a tracer of regions continuous with the extracellular space. Postfixation of erythrocytes agglutinated by the lectin helix (Helix promatia) and intact carotid artery endothelium with a freshly prepared mixture of 1% OsO₄ containing 0.1% ruthenium red (RR) resulted in a dense surface deposit of these cells, but intercellular regions were penetrated to a minimal degree by the stain. When a similar mixture of RR-OsO₄ was allowed to stand 3 h before use, RR is oxidized by OsO₄ to yield a ruthenium compound that has a spectrophotometric absorbance maximum at 365 nm. This RR molecule has a reduced number of cationic sites due to binding with osmium dioxide OsO ₂ ⁼ . Postfixation of agglutinated RBCs and carotid artery endothelium with this oxidized ruthenium-OsO₄ mixture resulted in a 50–80% decrease in surface deposition but markedly enhanced penetration into intercellular regions. The enhanced penetration is attributed to decreased binding affinity of the oxidized ruthenium for anionic surface membrane components, permitting effective stain penetration into regions of cell-to-cell contact rather than extensive surface deposition. These studies indicate that the ruthenium compound formed by OsO₄ oxidation of ruthenium red may be a useful tracer for ultrastructural visualization of intercellular spaces and junctions.     

Structural organization of ovarian follicle cells in the cotton bug (Dysdercus intermedius) and the honeybee (Apis mellifera)

Summary The somatic epithelia of Dysdercus and Apis follicles were analyzed by electron microscopy, and the patterns of F-actin and microtubules were studied by fluorescence microscopy. The epithelia in both species differ considerably in shape and in the organization of the cytoskeleton. During previtellogenic stages, the epithelium consists of columnar-shaped cells with small (Dysdercus) or no (Apis) lateral intercellular spaces. During vitellogenesis, the follicle cells round up; the intercellular spaces increase in size in Dysdercus follicles, whereas in Apis follicles they remain small. Along the basal surface of the follicle cells, there are conspicuous parallel bundles of microfilaments perpendicular to the anteroposterior axis of the follicles. In the honeybee, these microfilament bundles are present in long filopodia, most of which are embedded in thickenings of the basement membrane and extend over the surfaces of neighbouring cells. In the cotton bug, the basal surface of the follicle cells is thrown into parallel folds. The microfilament bundles are located just underneath the cell membrane where the folds contact the basement membrane. In the polar regions of the Dysdercus follicle, the epithelial cells become flat and adhere to each other without forming intercellular spaces. The basement membrane is particularly thick in the polar areas; this has also been observed in Apis follicles around the intercellular bridge connecting oocyte and nurse cells.     

A DISTINCTIVE CELL CONTACT IN THE RAT ADRENAL CORTEX

Extensive cell contacts which resemble septate junctions occur between cells in the three major zones of the rat adrenal cortex. Characteristically, they extend between small intercellular canaliculi and the periendothelial space, frequently interrupted by gap junctions and rarely by desmosomes. Zonulae occludentes have not been identified in the adrenal cortex. Along this distinctive cell contact, the cell membranes of apposing cells are separated by 210–300 a bisected by irregularly spaced 100–150-A extracellular particles which are often circular in profile. In lanthanum preparations, these particles appear to form a continuous chain throughout the intercellular space and are visualized as an alveolate structure in sections parallel to the plane of the cell membrane. The cell membrane in the area of septate-like contact does not differ from nonjunctional areas of the cell membrane in freeze-fracture replicas. The cell contact retains its integrity after cell dispersion and after the separation of cell membranes from disrupted cells. The intercellular particles also persist after brief extraction in lipid solvents. Besides adherence, possible functions of this adrenal contact include maintenance of the width of the extracellular space, the provision of channels between intercellular canaliculi and the bloodstream, and utilization as cation depots. Similar structures are also present between adrenal cortical cells of several other species and between interstitial cells of the testis. This type of cell contact may, in fact, be a typical feature of steroid-hormone-secreting tissues in vertebrates.     

An ultrastructural study and stereological analysis of the colon wall in the cockroach Periplaneta americana

The colonic epithelium has been examined for ultrastructural evidence of physiological activity. The cells show extensive folding of the apical plasma membrane, associated with mitochondria and an internal coating of particles about 120 Å diameter. Anteriorly many apical infoldings are dilated at the tip to form substantial extracellular spaces up to 0.8 μm wide. Narrow intercellular channels are present, opening to the haemolymph side of the epithelium. Pinocytosis is seen frequently at the basal surface. The surface densities of apical plasma membranes were not significantly different in the posterior mid-gut and colon. Similarly the volume densities of mitochondria were equal in the colonic epithelium and rectal pads, but the surface density of outer mitochondrial membranes was greater in the colon. It is suggested that the colon may absorb organic solutes from the gut lumen.     

PATHS OF TRANSTUBULAR WATER FLOW IN ISOLATED RENAL COLLECTING TUBULES

The cells of perfused rabbit collecting tubules swell and the intercellular spaces widen during osmotic flow of water from lumen to bath induced by antidiuretic hormone (ADH). Ouabain had no influence on these changes. In the absence of net water flow intercellular width was unaffected when tubules were swollen in hypotonic external media. Therefore, during ADH-induced flow widening of intercellular spaces is not a consequence of osmotic swelling of a closed intercellular compartment containing trapped solutes, but rather is due to flow of solution through the channel. Direct evidence of intercellular flow was obtained. Nonperfused tubules swollen in hypotonic media were reimmersed in isotonic solution with resultant entry of water into intercellular spaces. The widened spaces gradually collapsed completely. Spaces enlarged in this manner could be emptied more rapidly by increasing the transtubular hydrostatic pressure difference. In electron micrographs a path of exit of sufficient width to accommodate the observed rate of fluid flow was seen at the base of the intercellular channel. It is concluded that the intercellular spaces communicate with the external extracellular fluid and that water, having entered the cells across the luminal plasma membrane in response in ADH, leaves the cells by osmosis across both the lateral and basilar surface membranes.     

Plasmodium lophurae: membrane proteins of erythrocyte-free plasmodia and malaria-infected red cells

Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitro-en decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm3, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm3 and 1.158 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230, 000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

Exocytosis of vacuolar apical compartment (VAC): a cell-cell contact controlled mechanism for the establishment of the apical plasma membrane domain in epithelial cells

The vacuolar apical compartment (VAC) is an organelle found in Madin-Darby canine kidney (MDCK) cells with incomplete intercellular contacts by incubation in 5 microM Ca++ and in cells without contacts (single cells in subconfluent culture); characteristically, it displays apical biochemical markers and microvilli and excludes basolateral markers (Vega-Salas, D. E., P. J. I. Salas, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:1249-1259). The apical surface of cells kept under these culture conditions is immature, with reduced numbers of microvilli and decreased levels of an apical biochemical marker (184 kD), which is, however, still highly polarized (Vega-Salas, D. E., P. J. I. Salas, D. Gundersen, and E. Rodriguez-Boulan. 1987. J. Cell Biol. 104:905-916). We describe here the morphological stages of VAC exocytosis which ultimately lead to the establishment of a differentiated apical domain. Addition of 1.8 mM Ca++ to monolayers developed in 5 microM Ca++ causes the rapid (20-40 min) fusion of VACs with the plasma membrane and their accessibility to external antibodies, as demonstrated by immunofluorescence, immunoperoxidase EM, and RIA with antibodies against the 184-kD apical plasma membrane marker. Exocytosis occurs towards areas of cell-cell contact in the developing lateral surface where they form intercellular pockets; fusion images are always observed immediately adjacent to the incomplete junctional bands detected by the ZO-1 antibody (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). Blocks of newly incorporated VAC microvilli and 184-kD protein progressively move from intercellular ("primitive" lateral) spaces towards the microvilli-poor free cell surface. The definitive lateral domain is sealed behind these blocks by the growing tight junctional fence. These results demonstrate a fundamental role of cell-cell contact-mediated VAC exocytosis in the establishment of epithelial surface polarity. Because isolated stages (intercellular pockets) of the stereotyped sequence of events triggered by the establishment of intercellular contacts in MDCK cells have been reported during normal differentiation of intestine epithelium (Colony, P. C., and M. R. Neutra. 1983. Dev. Biol. 97:349-363), we speculate that the mechanism we describe here plays an important role in the establishment of epithelial cell polarity in vivo.