Schistocephalus solidus and Ligula intestinalis: Pinocytosis by the tegument

Using ruthenium red as a macromolecule, endocytosis was demonstrated in the plerocercoid of Ligula intestinalis and adult Schistocephalus solidus. Uptake, transport across the tegument, and exocytosis across the basal plasma membrane occurred within 6 min. The types of vesicles in the tegument of L. intestinalis are redescribed and their former classification is modified. The vertical and longitudinal distribution of pinosomes in the tegument of adult S. solidus and L. intestinalis plerocercoids were determined. The possible role of macromolecular uptake in the economy of pseudophyllidean tapeworms is discussed with particular reference to growth and defence of an unencysted larval stage in the tissues of the intermediate host.     

Taenia crassiceps: Basic mechanisms of endocytosis in the cysticercus

The effects of glucose, yeast extract, fetal bovine serum albumin, and ruthenium red on endocytosis of smooth micropinocytotic vesicles (pinosomes) in the tegument of the cysticercus of Taenia crassiceps have been investigated stereologically. Glucose has been shown to stimulate pinocytosis, whether it was used alone or in combination with yeast extract or bovine serum albumin. Yeast extract was a stimulant of endocytosis. Bovine serum albumin was the most potent stimulant of all the substances investigated in this study. Although the time of incubation in ruthenium red was the same for all incubation experiments, varied numbers of ruthenium red-containing pinosomes were observed in different experiments. The role of ruthenium red as a stimulant and/or initiator of endocytosis and the possible explanations for differences in ruthenium red uptake are discussed.     

Fasciola hepatica: ultrastructure and histochemistry of the glycocalyx of the tegument.

The morphology and histochemistry of the glycocalyx of the tegument was investigated by electron microscopy. The results showed that both the morphology and histochemistry of the glycocalyx varied depending on the environment immediately prior to fixation and also on postfixation treatments. Conventional electron microscope fixation appeared to preserve only about half the total thickness of the glycocalyx, and only histochemical tests applied en bloc gave a true morphological and histochemical picture. The glycocalyx, therefore, consists of two parts, an inner continuous layer which is tightly bound to the apical plasma membrane and is always preserved, and an outer fibrillar layer, which is not always preserved by conventional fixation. Both layers are anionic and carbohydrate-rich and therefore contain glycoproteins and sialic acids. The surface of Fasciola hepatica, therefore, has morphological and chemical features very like those proposed for the “greater membrane” by Lehninger.     

Morphogenesis of the photoreceptor outer segment during postnatal development in the mouse (BALB/c) retina

Summary Disc formation of rod photoreceptor cells in developing BALB/c mice retinas was studied by rapid freeze, freeze-substitution, freeze-etching, immunocytochemistry, and myosin S-1 decoration methods. Freeze-substituted photoreceptor cells contained variously shaped vesicles in the apical swelling of the connecting cilium or the base of the outer segment during postnatal development. Rapid freezing successfully arrested pinocytosis; the fusion of small vesicles to give large ones, and the compression of certain vesicles (0.3–0.6 m) appears to lead gradually to the formation of the so-called discs. We therefore propose that membranous discs are formed by the fusion of small pinocytotic vesicles and their subsequent compression. Discs formed in this way were partially stacked, but were ordered at random during the early developmental stages. During development, a partial stack of discs was progressively rearranged to a regular form as seen in mature outer segments. Cytoskeletal actin was expected to be involved in the disc formation; it was demonstrated in the distal axoneme of the connecting cilium during development and showed no change in its distribution. However, the polarity of the actin filaments, as revealed by myosin S-1 decoration in early developmental stages, was much more variable than in the adult. Barbed ends of actin filaments were associated with the plasma membrane or the membrane of vesicles. We also found actin filaments coiled up helically on ciliary microtubules.     

Mise en évidence par immunofluorescence des cellules sécrétrices de glucagon dans le pancréas endocrine du poisson téléostéen Xiphophorus helleri H.

Summary The ruthenium red staining of the surface coats was studied in the adrenal medullary cells of golden hamster. Both immersion and perfusion fixation was used with the ruthenium red containing fixative, however, only the perfusion fixation gave positive results. A rather thick electron dense ruthenium red positive layer was found on the plasma membrane of the endothelial cells, around the capillaries in the basal lamina, in the basement membrane of the chromaffin cells as well as on the apical and lateral cell surfaces of the adrenomedullary cells. Coated pits and coated vesicles usually showed an intensive ruthenium red staining, but the other cell components in the cytoplasm did not. On the basis of these observations author suggests that the ruthenium red positive material corresponds to acidic mucopolysaccharides in the hamster adrenal medulla, and its wide-ranging occurrence is indicative of its significance in the secretion process of catecholamines.Wellcome Research Fellow.     

Hymenolepis diminuta: ultrastructural observations on complement-mediated tegumental lysis and destrobilation of 4-day-old worms in vitro

Changes in the ultrastructure of the tegument and subtegumental cells of 4-day-old Hymenolepis diminuta were studied in vitro in 50% fresh normal rat serum over a 5-h period and compared with heat-inactivated serum and saline controls. First, membrane-bound vesicles accumulate above the microthrix-border. After 30–40 min large vacuoles, which may contain membranous elements, appear in the tegument at a time when the surface of the young strobila is virtually denuded of the microthrix-border. With prolonged incubations there are subtegumental secretory inclusions with dark, enveloping cytoplasm in the tegument and finally the apical plasma membrane, together with the majority of the matrix, is lost. The disrupted portion of the worm is abruptly demarcated from the comparatively intact scolex/anterior neck region by a constriction. Even after 5 h incubation there is no evidence of loss of tegumental matrix components from regions anterior to the constriction but the neck region shows a significant denudation of the microthrix layer and the tegument contains numerous inclusions. The scolex tegument only showed little evidence of loss of membrane from the surface. Possible mechanisms for the avoidance of complement-mediated lysis in the anterior region are discussed.     

Differentiation of epithelial cells in the urinary tract

Uroplakins, cytokeratins and the apical plasma membrane were studied in the epithelia of mouse urinary tract. In the simple epithelium covering the inner medulla of the renal pelvis, no uroplakins or cytokeratin 20 were detected and cells had microvilli on their apical surface. The epithelium covering the inner band of the outer medulla became pseudostratified, with the upper layer consisting of large cells with stalks connecting them to the basal lamina. Uroplakins and cytokeratin 20 were not expressed in these cells. However, some superficial cells appeared without connections to the basal lamina; these cells expressed uroplakins Ia, Ib, II and III and cytokeratin 20, they contained sparse small uroplakin-positive cytoplasmic vesicles and their apical surface showed both microvilli and ridges. Cytokeratin 20 was seen as dots in the cytoplasm. This epithelium therefore showed partial urothelial differentiation. The epithelium covering the outer band of the outer medulla gradually changed from a two-layered to a three-layered urothelium with typical umbrella cells that contained all four uroplakins. Cytokeratin 20 was organized into a complex network. The epithelium possessed an asymmetric unit membrane at the apical cell surface and fusiform vesicles. Umbrella cells were also observed in the ureter and urinary bladder. In males and females, the urothelium ended in the bladder neck and was continued by a non-keratinized stratified epithelium in the urethra in which no urothelial cell differentiation markers were detected. We thus show here the expression, distribution and organization of specific proteins associated with the various cell types in the urinary tract epithelium.W. Mello Jr. thanks FAPESP, São Paulo, Brazil for financial support.     

Internalization of ferritin-concanavalin A by the lactating mammary cell in vivo

Summary Ferritin-concanavalin A (Fer-Con A) was used to label the apical plasma membrane of the lactating cell to determine whether membrane internalization takes place. Rat glands were infused in vivo via the teat with 0.2 mg of Fer-Con A in 0.2 ml tris buffer (pH 7.0) containing 0.1% trypan blue, the latter acting as a marker of the infusate. Tissues were obtained from separate animals 5, 10 and 60 min postinfusion. Fer-Con A was seen in alveolar lumina bound to the outer surfaces of apical plasma membrane, microvilli and milk fat globules. It was observed within lactating cells on the inner membrane surfaces of endocytotic vesicles, Golgi cisternae, and secretory vesicles containing casein micelles, and in multivesicular bodies and lysosomes. Internalization of the ferritin-lectin conjugate into casein-containing secretory vesicles was detectable in the 5-min postinfusion tissue. Lysosomes were the only structures in control tissue that contained particles bearing some resemblance to Fer-Con A. The data provide evidence that apical plasma membrane is internalized and distributed to a number of intracellular compartments.     

Structure of the tegument and ectocommensal microorganisms of Gyliauchen nahaensis (Digenea: Gyliauchenidae), an inhabitant of herbivorous fish of the Great Barrier Reef, Australia

The ultrastructure of the tegument and tegument-associated microorganisms of the gyliauchenid digenean Gyliauchen nahaensis is described by transmission and scanning electron microscopy. The tegument is devoid of surface spines and is characterized by a moderately folded apical membrane, abundant vesicles, basal mitochondria, a folded basal plasma membrane, and a thick basal matrix. Microorganisms form a dense biofilm on the tegument of the posterodorsal surface and the excretory papilla. At least 7 microbial morphotypes were identified, including eubacteria, spirochaetes, and nanobacteria.     

Internalization of macromolecules from the medium in Suctoria

Takophrya infusionum like all other Suctoria lacks an oral cavity. Its feeding apparatus consists of tentacles, long narrow tubes through which the contents of the living prey are ingested. For normal growth, reproduction, and longevity of clones, Tokophrya needs supplements deriving from the medium in addition to living prey. Since Tokophrya lacks a mouth, these supplements can reach the cytoplasm only through the complex structure of the cortex, which is composed of a three- membraned pellicle and a dense epiplasm. In addition, external to the cortex, an extraneous coat covers the whole organism. Only the outer pellicular plasma membrane is continuous; the other two and the epiplasm are interrupted by the outer plasma membrane which invaginates at intervals forming the so-called pits. The invaginated plasma membrane dips down into the cytoplasm where it extends to form a saccule. Experiments with cationized ferritin and Thorotrast provide evidence that internalization of these macromolecules takes place through the pits by pinocytosis. The membrane of the saccules of the pits forms invaginations which pinch off giving rise to small, flattened vesicles containing the tracers. The tracers were never found free in the cytoplasm but exclusively in the flat vesicles. These vesicles are thus the vehicles transporting macromolecules from the medium to the cytoplasm. The saccules of the pits are the natural loci of pinocytosis and together with the flattened vesicles perform an important function in Suctoria, supplying the organisms with macromolecules from the medium.     

Adrenal cortex hormones and small intestine of adult rat: morphology, purity and enzymatic activities of isolated microvillous vesicles

An electron microscopic and enzymatic investigation was carried out to assess the purity as well as the morphologic integrity of small intestine microvillous membrane vesicles prepared from the following groups of adult rats: untreated (normals); adrenalectomized and pair-fed controls; treated for 8 days with corticosterone (1 and 10 mg X 100 g-1 body weight X day-1), and relative controls. Electron microscopy observation of ultrathin sections of ruthenium red stained vesicles from all groups showed that the microvillous membranes were structurally intact and virtually uncontaminated by material from other subcellular structures. The vesicles presented a fuzzy, intensely ruthenium red positive layer in their outer membranes. Apparently no alteration was induced in microvillous membrane by adrenalectomy, semistarvation or corticosterone treatment. The values of the enrichment factors for alkaline phosphatase and sucrase were similar for all the groups, indicating a high grade of purity of the preparations. Adrenalectomy and high dose of corticosterone decreased the vesicles content of alkaline phosphatase; sucrase was increased by semistarvation and adrenalectomy.     

Influence of cations at the plasma membrane in controlling polysaccharide secretion from sycamore suspension cells

1. Three soluble polysaccharides and a soluble protein containing hydroxyproline were secreted by sycamore suspension cultures. l-[1-(3)H]Fucose was incorporated solely into the fucose of fucoxyloglucan and l-[1-(14)C]arabinose mainly into the arabinose of arabino-galactan. [U-(14)C]Glucose was a general precursor for soluble protein and polysaccharides. 2. The steady-state rate of secretion of all the polymers was increased within seconds of adding various electrolytes and polyelectrolytes to the growth medium. The increased secretion was induced by cations at the outer surface of the plasma membrane. It was brought about by a stimulation of the normal mechanisms of cell-wall polysaccharide secretion. It was partly inhibited by anaerobiosis or sodium arsenate and was unaffected by temperature changes in the range 0-35 degrees C. 3. The precursor pool from which secretion was induced contained completely synthesized polysaccharides and was probably located in the Golgi-derived vesicles. The results indicated that the endoplasmic reticulum did not secrete polysaccharide directly to the cell exterior. 4. The various cations probably induced secretion by causing a depolarization of the negative electric potential of the cell surface, and this resulted in the fusion of vesicles with the plasma membrane. 5. Analogy with exocytosis and pinocytosis in various animal tissues suggested that the decreased surface potential brought about membrane fusion by causing an increase in plasma-membrane permeability to Ca(2+). 6. The results showed that the fusion of vesicles with the plasma membrane was rate-limiting and a potential control point. Auxin-stimulated cell-wall deposition could be a result of a stimulated influx of Ca(2+) causing vesicle fusion with the plasma membrane.     

INTESTINAL TRANSPORT OF ANTIBODIES IN THE NEWBORN RAT

Evidence has been reported that the proximal small intestine of the neonatal rat selectively transports antibodies into the circulation. This study describes the morphology of the absorptive epithelial cells in this region of the intestine and their transport of several immunoglobulin tracers: ferritin-conjugated immunoglobulins (IgG-Ft) and antiperoxidase antibodies. Cells exposed to rat IgG-Ft bound the tracer on the membrane of tubular invaginations of the apical cell surface. Tubular and coated vesicles within the cell also contained the tracer, as did the intercellular spaces. Uptake of tracer was highly selective and occurred only with rat or cow IgG-Ft; when cells were exposed to chicken IgG-Ft, ferritin-conjugated bovine serum albumin, or free ferritin, tracer did not enter the cell or appear in the intercellular spaces. Experiments with rat and chicken antiperoxidase showed a similar selective uptake and transport of only the homologous antibody. When cells from the distal small intestine were exposed to the tracers, all tracers were absorbed nonselectively but none were released from the cells. Cells from the proximal small intestine of the 22-day-old rat failed to absorb even rat IgG-Ft. A model is presented for selective antibody transport in proximal cells of the neonatal rat in which antibodies are selectively absorbed at the apical cell surface by pinocytosis within tubular vesicles. The antibodies are then transferred to the intercellular space within coated vesicles. Distal cells function only to digest proteins nonselectively.     

The Neurospora crassa exocyst complex tethers Spitzenk?rper vesicles to the apical plasma membrane during polarized growth

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.     

Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm

Hockley D. J. and McLaren D. J. 1973. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. International Journal for Parasitology3: 13–25. The tegumental outer membrane of the cercaria is trilaminate: the adult worm, however, has a seven-layered membrane. Formation of the heptalaminate membrane commences immediately after the cercaria has penetrated the vertebrate host: multilaminate membrane-bounded vacuoles are passed from subtegumental cells into the tegument where they enlarge, join to the outer membrane and open to the exterior. The heptalaminate limiting membrane of the vacuole thus becomes the outer membrane of the tegument. At the same time the original trilaminate tegumental membrane is formed into microvilli which are cast off and thus the cercarial outer membrane is lost. Schistosomula usually have a heptalaminate outer membrane within three hours of penetration. After this time the large vacuoles are replaced by smaller membraneous bodies which presumably contribute to the outer membrane during growth of the schistosomulum. The membraneous bodies are also present in the tegument of the adult worms and there is some evidence that the outer membrane is continually renewed.     

Fine structure of male genital tracts of some Acrididae and Tettigoniidae (Insecta: Orthoptera)

A morphological, histological and ultrastructural study was carried out on the spermiducts and seminal vesicles of some species of Acrididae and Tettigoniidae. In all the species examined, the spermiducts and seminal vesicles have a monolayered secretory epithelium. Only the species of Acrididae have the sac with a flattened epithelium. Furthermore, in the most distal tubule region of the seminal vesicles of Eyprepocnemis plorans plorans, a rather characteristic secretory mechanism was found: the cytoplasm of the epithelial cells contained a large vesicle delimited by tightly packed microvilli. Numerous small vesicles open into this large vesicle which gradually dilates to merge with the apical plasma membrane releasing its contents into the lumen. Spermiophagic activity was found in all the species investigated. In the Tettigoniidae, this activity was found only in some epithelial cells of the seminal vesicle wall; in the species of the Acrididae the spermiophagic activity was carried out in the spermiduct lumen by an epithelial‐type cellular group. Spermiophagic activity is discussed as well as its role in the reproduction of these insects.     

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.     

A quantitative ultramorphological approach for systematic assessment of sperm head regions: an example in rams

Examination of the type and frequency of damage to the head of spermatozoa using electron microscopy can be used to evaluate the quality of differently treated sperm. This report describes a systematic approach based on 29 morphological categories of sperm heads assessed from discrete regions in raw, chilled and frozen-thawed spermatozoa. Injury occurred principally at the plasma membrane and could be present or absent in all regions. In the anterior segment, when the plasma membrane is present, it can be intact, dilated, very dilated, disrupted, or contain vesicles characteristic of acrosomal reaction-like capacitation changes. When the plasma membrane is absent, the acrosome may be intact, exhibit a complete loss of contents, or retain some contents of the apical ridge and present a very dilated outer acrosomal membrane. The plasma membrane in the equatorial segment and the boundary between regions can be intact, dilated, very dilated or disrupted. The post-acrosomal plasma membrane is classified as intact, dilated or very dilated, whereas the dense lamina is intact, dilated or fragmented. The morphology of the heads most frequently observed in chilled spermatozoa consists of anterior and equatorial segments with a dilated, or dilated and disrupted plasma membrane; a boundary between regions with an intact and dilated plasma membrane; and a post-acrosomal region with an intact plasma membrane and dense lamina, both dilated. In frozen-thawed spermatozoa, the morphology of the heads is more frequently characterised by no plasma membrane and an acrosome showing complete or some loss of contents in the apical ridge and very dilated outer acrosomal membrane, presenting mostly dilated and fragmented dense lamina in the post-acrosomal region. These findings are consistent with the conclusion that the freezing process produces an increase in the degree of damage to the cells when they are subjected to increasing degrees of cold shock. There are still difficulties in developing a good diluent and process for preserving the plasma membrane in ram spermatozoa. This systematisation, using different categories, allows characterisation of multiple transmission electron microscopy images. Thus, the different changes observed due to cryopreservation may be correlated.     

Insecticidal Activity Associated with the Outer Membrane Vesicles of Xenorhabdus nematophilus

Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.     

Subcellular fractionation of pig coronary artery smooth muscle

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.