Migration of glycoprotein from the Golgi apparatus to the surface of various cell types as shown by radioautography after labelled fucose injection into rats

A single intravenous injection of L-[³H]fucose, a specific glycoprotein precursor, was given to young 35–45 g rats which were sacrificed at times varying between 2 min and 30 h later. Radioautography of over 50 cell types, including renewing and nonrenewing cells, was carried out for light and electron microscope study. At early time intervals (2–10 min after injection), light microscope radioautography showed a reaction over nearly all cells investigated in the form of a discrete clump of silver grains over the Golgi region. This reaction varied in intensity and duration from cell type to cell type. Electron microscope radioautographs of duodenal villus columnar cells and kidney proximal and distal tubule cells at early time intervals revealed that the silver grains were restricted to Golgi saccules. These observations are interpreted to mean that glycoproteins undergoing synthesis incorporate fucose in the saccules of the Golgi apparatus. Since fucose occurs as a terminal residue in the carbohydrate side chains of glycoproteins, the Golgi saccules would be the site of completion of synthesis of these side chains. At later time intervals, light and electron microscope radioautography demonstrated a decrease in the reaction intensity of the Golgi region, while reactions appeared over other parts of the cells: lysosomes, secretory material, and plasma membrane. The intensity of the reactions observed over the plasma membrane varied considerably in various cell types; furthermore the reactions were restricted to the apical surface in some types, but extended to the whole surface in others. Since the plasma membrane is covered by a "cell coat" composed of the carbohydrate-rich portions of membrane glycoproteins, it is concluded that newly formed glycoproteins, after acquiring fucose in the Golgi apparatus, migrate to the cell surface to contribute to the cell coat. This contribution implies turnover of cell coat glycoproteins, at least in nonrenewing cell types, such as those of kidney tubules. In the young cells of renewing populations, e.g. those of gastro-intestinal epithelia, the new glycoproteins seem to contribute to the growth as well as the turnover of the cell coat. The differences in reactivity among different cell types and cell surfaces imply considerable differences in the turnover rates of the cell coats.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

Distribution of secretory component in hepatocytes and its mode of transfer into bile

Immunoglobin A in bile and other external secretions is mostly bound to a glycoprotein known as secretory component. This glycoprotein is not synthesized by the same cells as immunoglobulin A and is not found in blood. We now report the mechanism by which secretory component reaches the bile and describe its function in immunoglobulin A transport across the hepatocyte. Fractionation of rat liver homogenates by zonal centrifugation was followed by measurement of the amounts of secretory component in the various fractions by rocket immunoelectrophoresis. Secretory component was found in two fractions. One of these was identified as containing Golgi vesicles from its isopycnic density and appearance in the electron microscope; the other contained principally fragments of the plasma membrane of the sinusoidal face of the hepatocyte, as shown by its particle size and content of marker enzymes. Only the latter fraction bound ¹²⁵I-labelled immunoglobulin A added in vitro. At 5min after intravenous injection of [¹⁴C]fucose, the secretory component in the Golgi fraction was labelled, but not that in the plasma membrane. The secretory component in the sinusoidal plasma membrane did, however, become labelled before the first labelled secretory component appeared in bile, about 30min after injection. We suggest that fucose is added to the newly synthesized secretory component in the Golgi apparatus. The secretory component then passes, with the other newly secreted glycoproteins, to the sinusoidal plasma membrane. There it remains bound but exposed to the blood and able to bind any polymeric immunoglobulin A present in serum. The secretory component then moves across the hepatocyte to the bile-canalicular face in association with the endocytic-shuttle vesicles which carry immunoglobulin A. Hence there is a lag before newly synthesized secretory component appears in bile.     

Helix pomatia agglutinin binds specifically to the Golgi apparatus in cultured human fibroblasts and reveals two Golgi apparatus-specific glycoproteins

Summary Fluorochrome-coupled Helix pomatia agglutinin (HPA), but not other lectin-conjugates with the same nominal specificity, bound specifically to the Golgi apparatus in cultured human fibroblasts, revealing a cytoplasmic juxtanuclear reticular structure. Unlike other Golgi-binding lectins the HPA-conjugates did not bind to the cell surface membrane or pericellular matrix. Experiments with ³⁵S-methionine-labeled cells showed that HPA recognized two glycoproteins of M_r 170000 and 400000 among the secreted products of fibroblasts and two major cellular glycoproteins of M_r 40000 and M_r 180000 in Triton X-100 extracts of the cells. The two cellular HPA-binding polypeptides were also found in cells depleted of secretory products and in cells pulselabeled shortly with ³⁵S-methionine and then chased with methionine containing medium up to 12 h. These findings suggest that the two cellular glycoproteins recognized by HPA are retained in the Golgi apparatus and are therefore not precursors of secretory proteins. The results suggest that there are two endogenous, Golgi apparatus-specific glycoproteins in cultured human fibroblasts with terminal non-reducing O-glycosidic N-acetyl galactosaminyl residues.     

Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 μg/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 μg/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of approximately 10 min. These results suggest a similar intracellular pathway in the maturation of both plasma membrane and secretory glycoproteins.     

Distribution of xylosylation and fucosylation in the plant Golgi apparatus

Antibodies have been immunopurified which are specific for carbohydrate epitopes containing the β1→2 xylose or α1→3 fucose residues found on complex N-linked glycans in plants. The antibody specificity was determined by taking advantage of an Arabidopsis thaliana N-glycosylation mutant which lacks N-acetyl-glucosaminyltransferase I and is unable to synthesize complex glycans. These antibodies were used to immunolocalize xylose- and fucose-containing glycoproteins in suspension-cultured sycamore cells (Acer pseudoplatanus). By mapping the enzymatic reaction products within the Golgi apparatus, the fucosyl- and xylosyltransferase subcellular localization was made possible using immunocytochemistry on thin sections of high-pressure frozen and freeze-substituted sycamore cells. This procedure allows a much better preservation of organelles, and particularly of the Golgi stack morphology, than that obtained with conventionally fixed samples. Glycoproteins containing β→2 xylose and α1→3 fucose residues were immunodetected in the cell wall, the vacuole, and the Golgi cisternae. The extent of immunolabeling over the different cisternae of 50 Golgi stacks was quantified after treatment with anti-xylose or anti-fucose antibodies. Labeling for xylose-containing glycoproteins was predominent in the medial cisternae, while fucose-containing glycoproteins were mainly detected in the trans compartment. Therefore, in plants, complex N-linked glycan xylosylation probably occurs mostly at the medial Golgi level and α1→3 fucose is mainly incorporated in the trans cisternae. Finally, fucose- and xylose-containing glycoproteins were also immunolocalized, albeit to a lesser extent, in earlier Golgi compartments. This indicates that the glycosylation events are a continuous process with some maxima in given compartments, rather than a succession of discrete and compartment-dependent steps.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

Morphological and cytochemical studies of the effects of ecdysteroids in a lepidopteran cell line (IAL-PID2)

Summary The Indian meal-moth cell line, IAL-PID2, established from larval wing discs was examined from the 250th to the 300th passages. The cultured cells retain various structural and functional qualities of epidermal cells. Under hormone-free conditions PID2 cells grow as a monolayer of round or spindle-shaped cells. They appear as weakly active epidermal cells. The endoplasmic reticulum and Golgi apparatus are poorly developed and secretory activity is reduced. Culture conditions resulted in considerable cellular expansions, abundance of storage products (glycogen, lipids), and hypertrophy of the lysosomal system. The PID2 cell line retains the ability to respond to ecdysteroids; 20-hydroxyecdysone treatment (2×10^-6 M) triggered morphogenetic and secretory processes. Cells formed pseudoepithelial aggregates interconnected and linked by desmosome-like structures. The hormone-stimulated cells are involved in the biosynthesis of N-acetyl-D-glucosamine-rich glycoproteins. The glycosylation sites were located, by use of WGA-gold particles, on cellular expansions and all along the plasma membrane. The possible significance of these glycoproteins is discussed.     

RADIOAUTOGRAPHIC STUDY OF IN VIVO AND IN VITRO INCORPORATION OF FUCOSE-3H INTO THYROGLOBULIN BY RAT THYROID FOLLICULAR CELLS

The incorporation of fucose-³H in rat thyroid follicles was studied by radioautography in the light and electron microscopes to determine the site of fucose incorporation into the carbohydrate side chains of thyroglobulin, and to follow the migration of thyroglobulin once it had been labeled with fucose-³H. Radioautographs were examined quantitatively in vivo at several times after injection of fucose-³H into rats, and in vitro following pulse-labeling of thyroid lobes in medium containing fucose-³H. At 3–5 min following fucose-³H administration in vivo, 85% of the silver grains were localized over the Golgi apparatus of thyroid follicular cells. By 20 min, silver grains appeared over apical vesicles, and by 1 hr over the colloid. At 4 hr, nearly all of the silver grains had migrated out of the cells into the colloid. Analysis of the changes in concentration of label with time showed that radioactivity over the Golgi apparatus increased for about 20 min and then decreased, while that over apical vesicles increased to reach a maximum at 35 min. Later, the concentration of label over the apical vesicles decreased, while that over the colloid increased. Similar results were obtained in vitro. It is concluded that fucose, which is located at the end of some of the carbohydrate side chains, is incorporated into thyroglobulin within the Golgi apparatus of thyroid follicular cells, thereby indicating that some of these side chains are completed there. Furthermore, the kinetic analysis demonstrates that apical vesicles are the secretion granules which transport thyroglobulin from the Golgi apparatus to the apex of the cell and release it into the colloid.     

Glycoprotein secretion in the mouse submandibular gland as revealed by radioautography after L-3H-fucose injection

Summary Glycoprotein secretion in the mouse submandibular gland was investigated by light microscope radioautography of semi-thin sections after the administration of L-³H-fucose. The incorporation of the precursor in the acini was negligible. ³H-fucose was taken up in the paranuclear region of the cells lining the intercalated, secretory, striated and excretory ducts. This labeling pattern was interpreted as addition of the precursor to glycoproteins within the Golgi apparatus. Incorporation in the intercalated duct was restricted to the cells with fine cytoplasmic granules. The glycoproteins synthesized by the intercalated and secretory ducts were transported to the saliva by the secretion granules. It is assumed that the glycoproteins synthesized in the striated and excretory ducts are plasma membrane glycoproteins which seem to renew continuously. Quantitation of the radioautographs supplied data concerning the incorporation of ³H-fucose into newly synthesized glycoproteins as well as the renewal of the labeled macromolecules in each duct.     

Synthesis and migration of 3H-fucose-labeled glycoproteins in the retinal pigment epithelium of albino rats, as visualized by radioautography

3H-fucose was injected into the vitreous body of the eye(s) of 250-gm rats, which were then killed by means of an intracardiac perfusion with glutaraldehyde after intervals of 10 min, 1 and 4 hr, and 1 and 7 days. The eyes were removed and further fixed, and pieces of retina were processed for light and electron microscope radioautography. Light microscope radioautography showed that the pigment epithelial cells actively incorporated 3H-fucose label. The intensity of reaction peaked at 4 hr after injection of the label and then slowly declined. Quantitative electron microscope radioautography revealed that, at 10 min after 3H-fucose injection, over 70% of the label was localized to the Golgi apparatus, indicating that fucose residues are added to newly synthesized glycoproteins principally at this site. With time the proportion of label associated with the Golgi apparatus decreased, but that assigned to the infolded basal plasma membrane, the apical microvilli, and various apical lysosomes increased. These results indicate that in retinal pigment epithelial cells newly synthesized glycoproteins continuously migrate from the Golgi apparatus to lysosomes and to various regions of the plasma membrane. In this case, the membrane glycoproteins may play specific roles in receptor functions of the basal plasma membrane or phagocytic activities at the apical surface. Very little label migrated to Bruch's membrane, indicating either a very slow turnover or a paucity of fucose-containing glycoproteins at this site.     

Effect of brefeldin A on the structure of the Golgi apparatus and on the synthesis and secretion of proteins and polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells.

Brefeldin A (BFA), a specific inhibitor of Golgi-mediated secretion in animal cells, has been used to study the organization of the secretory pathway and the function of the Golgi apparatus in plant cells. To this end, we have employed a combination of electron microscopical, immunocytochemical, and biochemical techniques to investigate the effects of this drug on the architecture of the Golgi apparatus as well as on the secretion of proteins and complex cell wall polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. We have used 2.5 and 7.5 micrograms/mL of BFA, which is comparable to the 1 to 10 micrograms/mL used in experiments with animal cells. Electron micrographs of high-pressure frozen and freeze-substituted cells show that although BFA causes swelling of the endoplasmic reticulum cisternae, unlike in animal cells, it does not induce the disassembly of sycamore maple Golgi stacks. Instead, BFA induces the formation of large clusters of Golgi stacks, an increase in the number of trans-like Golgi cisternae, and the accumulation in the cytoplasm of very dense vesicles that appear to be derived from trans Golgi cisternae. These vesicles contain large amounts of xyloglucan (XG), the major hemicellulosic cell wall polysaccharide, as shown by immunocytochemical labeling with anti-XG antibodies. All of these structural changes disappear within 120 min after removal of the drug. In vivo labeling experiments using [3H]leucine demonstrate that protein secretion into the culture medium, but not protein synthesis, is inhibited by approximately 80% in the presence of BFA. In contrast, the incorporation of [3H]fucose into N-linked glycoproteins, which occurs in trans-Golgi cisternae, appears to be affected to a greater extent than the incorporation of [3H]xylose, which has been localized to medial Golgi cisternae. BFA also affects secretion of complex polysaccharides as evidenced by the approximate 50% drop in incorporation of [3H]xylose and [3H]fucose into cell wall hemicelluloses. Taken together, these findings suggest that at concentrations of 2.5 to 7.5 mu g/mL BFA causes the following major changes in the secretory pathway of sycamore maple cells: (a) it inhibits the transport of secretory proteins to the cell surface by about 80% and of hemicelluloses by about 50%; (b) it changes the patterns of glycosylation of N-linked glycoproteins and hemicelluloses; (c) it reduces traffic between trans Golgi cisternae and secretory vesicles; (d) it produces a major block in the transport of XG-containing, dense secretory vesicles to the cell surface; and (e) it induces the formation of large aggregates of Golgi apparatus of plant and animal cels share many functional and structural characteristics, the plant Golgi apparatus possesses properties that make its response to BFA unique.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucose injection

In the first paper of this series (Bennett et al., 1984), light-microscope radioautographic studies showed that colchicine or vinblastine inhibited intracellular migration of glycoproteins out of the Golgi region in a variety of cell types. In the present work, the effects of these drugs on migration of membrane glycoproteins have been examined at the ultrastructural level in duodenal villous columnar cells and hepatocytes. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In duodenal villous columnar cells, 3H-fucose labeling of the apical plasma membrane was reduced by 51% after colchicine and by 67% after vinblastine treatment; but there was little change in labeling of the lateral plasma membrane. Labeling of the Golgi apparatus increased. This suggests that labeled glycoproteins destined for the apical plasma membrane were inhibited from leaving the Golgi region, while migration to the lateral plasma membrane was not impaired. In hepatocytes, labeling of the sinusoidal plasma membrane was reduced by 83% after colchicine and by 85% after vinblastine treatment. Labeling of the lateral plasma membrane also decreased, although not so dramatically. Labeling of the Golgi apparatus and neighboring secretory vesicles increased. This indicates that the drugs inhibited migration of membrane glycoproteins from the Golgi region to the various portions of the plasma membrane. Accumulation of secretory vesicles at the sinusoidal front suggests that exocytosis may also have been partially inhibited. In both cell types, microtubules almost completely disappeared after drug treatment. Microtubules may, therefore, be necessary for intracellular transport of membrane glycoproteins, although the possibility of a direct action of these drugs on Golgi or plasma membranes must also be considered.     

Radioautographic analysis of the secretory pathway for glycoproteins in principal cells of the mouse epididymis exposed to [3H] fucose

The secretory process for glycoproteins in principal cells of the mouse caput epididymis was studied by electron microscope radioautography at intervals after exposure to [3H] fucose in vitro. The large Golgi apparatus showed very heavy labeling at the initial interval, followed by a steady decline in percent of grains and relative grain concentrations. Conversely, the epididymal lumen and the apical cell surface began low and increased in radioactivity at the 30-min interval. The extensive sparsely granulated endoplasmic reticulum showed modest increases in percent of grains and relative grain concentrations 30 min after administration of the percursor. Subdivision of the sparsely granulated reticulum into "intermediate" profiles (some ribosomes attached to the membranes) and "smooth" profiles (lacking ribosomes) showed that this increase was due to silver grains assigned to the smooth portions. After the initial interval, high relative grain concentrations were calculated for vesicles. The results indicate that glycosylation of epididymal secretory glycoproteins occurs in the Golgi apparatus, which is, therefore, not bypassed as its morphological features had suggested. The kinetics of the secretory process in the principal cells includes 15 to 30 min for synthesis of the polypeptide parts of secretory products and addition of sugars in the Golgi apparatus, and a similar time for subsequent release from the Golgi apparatus, transport to the apical end of the cell and discharge to the lumen. Ribosome-studded (intermediate) portions of the sparsely granulated endoplasmic reticulum are probably involved in synthesis of polypeptide parts of secretory products, while vesicles or smooth portions of the sparsely granulated reticulum may play a role in intracellular transport of glycoproteins.     

Ethanol-induced alterations of plasma membrane assembly in the liver

The effects of acute ethanol administration on the assembly of glycoproteins into the hepatic plasma membrane were studied in the rat. When [14C]fucose and N-acetyl[3H]mannosamine, a sialic acid precursor, were injected following an acute dose of ethanol, the incorporation of these precursors into the total pool of membrane glycoproteins was minimally affected. This finding indicated that ethanol treatment did not appreciably alter the glycosylation of proteins in the Golgi apparatus. However, the assembly of labeled fucoproteins and sialoproteins into the plasma membrane was markedly inhibited in the ethanol-treated animals. This inhibition of plasmalemmal glycoprotein assembly was accompanied by a corresponding accumulation of labeled glycoproteins in the cytosolic fraction of the hepatocyte. The content of labeled glycoproteins in the Golgi complex was not significantly altered by ethanol treatment. These results indicate that ethanol administration impairs the late stages of hepatic plasma membrane assembly and further suggest that ethanol administration interferes with the flow of membrane components from the Golgi apparatus to the surface membrane.     

Ultracytochemistry of glycoproteins in the eccrine nasolabial glands of the North American raccoon (Procyon lotor)

The distribution and quality of glycoproteins was studied by means of electron microscopic cytochemical methods, particularly lectin cytochemistry, in the secretory cells of the eccrine nasolabial glands of the North American raccoon (Procyon lotor). In the dark and clear glandular cells, complex glycoconjugates were demonstrable, predominantly, in secretory granules, the cisternae of the Golgi apparatus, the surface coat of the plasma membrane, and as glycogen particles. Secretory granules found in the dark cells contained a variety of saccharide residues, such as α-d-mannose, β-d-galactose, β-N-acetyl-d-glucosamine and sialic acid. Several sugars were also detectable in the surface coat of the plasma membrane and the Golgi apparatus.The results obtained may be helpful to understand the specific functions of the glandular secretions of the raccoon nasolabial glands. These could be, particularly, binding of water on the snout surface and protection against microbial hazards, to maintain the structural and functional integrity of the relatively thin snout epidermis in carnivores.     

Isolation of plasma membrane,Golgi apparatus,and endoplasmic reticulum fractions from single homogenates of mouse liver

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2–3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K⁺-stimulated p-nitrophenyl phosphatase and (Na⁺ K⁺) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.     

Incorporation of L-(3H)-fucose into glycoproteins of the adrenal gland of mice. Light microscope radioautographic study on semi-thin sections

Summary To study the biosynthesis and intracellular migration of glycoproteins in the adrenal gland, adult mice were injected intravenously with L-(³H) fucose and killed from 10 min to 14 days after injection. Semi-thin sections of the adrenal glands were then processed for radioautography. Incorporation of labeled fucose occurred in the steroid-secreting cells of the three zones of the cortex as well as in the adrenalin (A) and noradrenalin (NA) cells of the medulla. At short intervals after injection, the main site of incorporation was the paranuclear region of the cells, suggesting uptake by the Golgi apparatus. Subsequently, labeled glycoproteins migrated from the paranuclear region to other cell sites. The labeling pattern observed in the adrenocortical parenchyme strongly suggests that the glycoproteins are transferred to lysosomes, lipofuscin granules and the cell coat (glycocalyx). Counts of silver grains clearly indicate that these glycoproteins undergo renewal. The qualitative and quantitative analysis of the radioautographs also suggest that glycoproteins, acting as intracellular carriers of steroids, may be released to the extracellular environment together with the hormones. Most of the glycoproteins synthesized by the A and NA cells of the adrenal medulla seem to be transferred to secretion granules in which they may play some role in the cytophysiology of these structures. It is likely that glycoproteins are released from the cells during exocytosis of secretory granules.     

Fucosylation of glycoproteins in rat spermatocytes and spermatids

Glycoprotein synthesis in pachytene spermatocytes and round spermatids, isolated from rat testes, was studied by analysis of the incorporation of (³H)-fucose. The isolated germ cells were capable of incorporating (³H)-fucose into cell-bound, acid-precipitable components for an incubation period of at least 23 hours (at 32°C). In young spermatids, engaged in the formation of the acrosome, (³H)-fucose was incorporated into more than 16 different glycoproteins within the molecular weight range of 20.000–100,000. A qualitatively similar set of glycoproteins was found to be labeled in spermatocytes. Radioautography showed that after 4 hr most of the incorporated radioactivity was present at one pole in the perinuclear zone of spermatocytes and spermatids, which could reflect incorporation of fucose in the Golgi apparatus. The newly fucosylated glycoproteins were associated with a particulate subcellular fraction (membrane fraction). Trypsin treatment of whole cells after 25 hours of incubation with (³H)-fucose, however, did not cause significant lysis of tritiated glycoproteins. From the results it was concluded that the majority of the newly fucosylated glycoproteins in spermatocytes and spermatids remained associated with an intracellular membrane system, presumably the Golgi apparatus and the vesicles that arise from this structure, to be deposited subsequently in proacrosomic granules and the acrosome. The results also suggest that initiation of the synthesis of spermatidal glycoproteins occurs during the prophase of meiosis in spermatocytes.     

Carbohydrates in the epidermal mucous cells of a fresh-water fish Mastacembelus pancalus (mastacembelidae,Pisces) as studied by electron-microscopic cytochemical methods

Carbohydrates in the mucous cells of the epidermis of the fish Mastacembelus pancalus were studied by means of electron-microscopic cytochemical methods using physical development procedures. Three types of mucous cells (types A-C) were differentiated on the basis of the reactivities of the secretory products elaborated by them. The carbohydrate contents of mucous globules predominantly comprised sulfate esters and traces of oxidizable vicinal diols in type-A cells, oxidizable vicinal diols in type-B cells, and moderate amounts of both sulfate esters and oxidizable vicinal diols in type-C cells. Glycogen particles were also found to occur in the cytoplasm of these cells, and glycoproteins containing oxidizable vicinal diols were visualized in Golgi cisternae, rough endoplasmic reticulum, nuclear envelopes, and plasma membranes. In the type-A and type-B cells situated in the superficial layers of the epidermis, extensive cisternae of the Golgi apparatus and copious rough endoplasmic reticulum suggested the active syntheses of secretory contents, in contrast to the type-C mucous cells, which displayed poor development of these organelles, in the deeper layers.