Cytochemical localization of terminal N-acetyl-D-galactosamine residues in cellular compartments of intestinal goblet cells: implications for the topology of O-glycosylation

The O-linked oligosaccharides of mucin-type glycoproteins contain N- acetyl-D-galactosamine (GalNAc) that is not found in N-linked glycoproteins. Because Helix pomatia lectin interacts with terminal GalNAc, we used this lectin, bound to particles of colloidal gold, to localize such sugar residues in subcellular compartments of intestinal goblet cells. When thin sections of low temperature Lowicryl K4M embedded duodenum or colon were incubated with Helix pomatia lectin- gold complexes, no labeling could be detected over the cisternal space of the nuclear envelope and the rough endoplasmic reticulum. A uniform labeling was observed over the first and several subsequent cis Golgi cisternae and over the last (duodenal goblet cells) or the two last (colonic goblet cells) trans Golgi cisternae as well as forming and mature mucin droplets. However, essentially no labeling was detected over several cisternae in the central (medial) region of the Golgi apparatus. The results strongly suggest that core O-glycosylation takes place in cis Golgi cisternae but not in the rough endoplasmic reticulum. The heterogenous labeling for GalNAc residues in the Golgi apparatus is taken as evidence that termination of certain O- oligosaccharide chains by GalNAc occurs in trans Golgi cisternae.     

Heterogeneity of glycoconjugates in the secretory cells of the chinchilla middle ear and eustachian tubal epithelia: a lectin-gold cytochemical study

The present study was conducted to characterize and localize the glycoconjugates in the tubotympanum (auditory or eustachian tube and middle ear cavity) of chinchilla on an ultrastructural level, using lectin-gold complexes with six different lectins: BPA, ConA, RCA-1, WGA, LFA, and SNA. A comparison of the affinity of these lectins demonstrated the heterogeneity of secretory cells. The glandular serous cells and epithelial dark granulated cells produced "serum"-type glycoprotein. The glandular mucous cells and goblet cells produced dominantly "mucin"-type glycoprotein in the light granules, but "serum"-type glycoprotein in the dark cores. The labeling of LFA and SNA showed that sialic acids existed mainly in the mucinous granules of secretory cells and ciliated epithelium glycocalyx, and in the mucous blanket. The results also suggested that the dominant linkage of sialic acids of mucin is a Neu5Ac(alpha 2-6)Gal/GalNAc sequence. Furthermore, the data obtained from ConA and BPA suggested that initial O-glycosylation of mucin took place in the cis side of the Golgi apparatus and that initial N-glycosylation of the serum occurred in the rough endoplasmic reticulum.     

Altered GalNAc-alpha-2,6-sialylation compartments for mucin-associated sialyl-Tn antigen in colorectal adenoma and adenocarcinoma.

Sialyl-Tn (STn), a mucin-associated disaccharide antigen carried by apomucins such as MUC2, plays an important role in tumor biology. However, little is known about the subcellular localization and compartments involved in STn synthesis. In this study we used immunoelectron microscopy to localize STn and MUC2 apomucin in human colorectal tissues. MUC2 apomucin was localized predominantly in the rough endoplasmic reticulum (RER) in normal colorectal mucosa (n=6), colorectal adenoma (n=8), and colorectal adenocarcinoma (n=10). STn, recognized by monoclonal antibody TKH2, was not readily detectable in normal colorectal mucosa but becomes manifest in both trans-Golgi apparatus and mucin droplets in colorectal adenoma. In colorectal adenocarcinoma, STn was localized not only in late but also in early Golgi compartments, and particularly in some RER lumens. Furthermore, electron microscopic in situ hybridization revealed that gold particles representing MUC2 mRNA are primarily localized over the RER. Our findings indicate that in colorectal adenoma STn sialylation takes place in the trans-Golgi apparatus, whereas in colorectal cancer STn sialylation occurs in all the Golgi compartments and in the RER.     

Subcompartment sugar residues of gastric surface mucous cells studied with labeled lectins.

We examined the intracellular localization of sugar residues of the rat gastric surface mucous cells in relation to the functional polarity of the cell organellae using preembedding method with several lectins. In the surface mucous cells, the nuclear envelope and rough endoplasmic reticulum (rER) and cis cisternae of the Golgi stacks were intensely stained with Maclura pomifera (MPA), which is specific to alpha-Gal and GalNAc residues. In the Golgi apparatus, one or two cis side cisternae were stained with MPA and Dolichos biflorus (DBA) which is specific to terminal alpha-N-acetylgalactosamine residues, while the intermediate lamellae were intensely labeled with Arachis hypogaea (PNA) which is specific to Gal beta 1,3 GalNAc. Cisternae of the trans Golgi region were also stained with MPA, Ricinus communis I (RCA I) which is specific to beta-Gal and Limax flavus (LFA) which is specific to alpha-NeuAc. Immature mucous granules which are contiguous with the trans Golgi lamellae were weakly stained with RCA I, while LFA stained both immature and mature granules. The differences between each lectin's reactivity in the rough endoplasmic reticulum, in each compartment of the Golgi lamellae and in the secretory granules suggest that there are compositional and structural differences between the glycoconjugates in the respective cell organellae, reflecting the various processes of glycosylation in the gastric surface mucous cells.     

Ultrastructural localization of mannoside residues on tissue sections: comparative evaluation of the enzyme-gold and the lectin-gold approaches

Mannoside residues were revealed at the ultrastructural level in different cellular and extracellular compartments by means of the enzyme-gold and the lectin-gold approaches. For the enzyme-gold technique, an alpha-mannosidase-gold complex was prepared and conditions for the preparation of this complex as well as for its application were determined. Labeling was found over the rough endoplasmic reticulum mainly at the level of the membranes, the lumen of the cisternae being devoid of labeling. In the nucleus, the dense chromatin and the edge of the fibrillar threads in the nucleolus were intensely labeled. Few gold particles were present over the Golgi apparatus and mitochondria. The secretory granules in pancreatic cells, the peroxisomes in liver and the mucin in duodenal goblet cells were devoid of labeling. In the extracellular space, the basal lamina was labeled. Over the glomerular basal lamina, the labeling was mainly towards the epithelial side, in close contact with the podocytes. The results with the concanavalin A horseradish peroxidase (Con A-HRP)-gold technique were similar to those found with the enzyme-gold approach. Some differences were, however, detected at the level of the rough endoplasmic reticulum and the nucleus. In the endoplasmic reticulum, Con A-HRP-gold labeling was present over both the membranes and the lumen of the cisternae. In the nucleus, the labeling was mainly over the dispersed chromatin. These differences may be due to the binding of Con A not only to mannoside but also to other sugar residues as well as to the affinity of HRP-gold for some nucleoplasmic components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin cytochemistry in the gastrointestinal tract with special reference to glycosylation in the Golgi apparatus of Brunner's gland cells.

Two hydrophilic, low temperature-embedding resins, Lowicryl K4M and LR White, were compared in lectin cytochemistry. Post-embedding staining of colloidal gold-labeled Griffonia symplicifolia agglutinin II (GSA-II) resulted in staining of the Golgi apparatus and mucous granules of mucous neck cells in the gastric fundic gland, pylorocytes, and Brunner's gland cells embedded in either resin, although it was much easier to make ultra-thin sections with LR White-embedded material than with the other. Post-fixation with uranyl acetate followed by LR White embedding improved general ultrastructure so that lectin binding sites were identified precisely. All examined lectins, soybean agglutinin (SBA), Maclura pomifera agglutinin (MPA), GSA-II, and Ulex europaeus agglutinin I (UEA-I), stained mucous granules and the Golgi apparatus, in which the staining pattern was characteristic of each lectin: cis cisternae were labeled with SBA and MPA, intermediate cisternae with GSA-II, and trans cisternae and mucous granules with SBA, GSA-II, UEA-I, and lightly with MPA. No labeling was observed in the rough endoplasmic reticulum with any lectin. These findings suggest that the Golgi apparatus is the site of O-linked glycosylation and can be divided into at least three distinct compartments with regard to the glycosylation.     

Distribution of golgi apparatus-associated polyribosomes across the polarity axis of dictyosomes of wild carrot (Daucus carota L.)

Summary Endoplasmic reticulum-polyribosome-Golgi apparatus associations were a general feature of cells of suspension cultures of wild carrot (Daucus carota L.). Free polyribosomes occurred within the Golgi apparatus zone for all dictyosomes and with equal frequency at all levels within the stack including the most mature or trans face. When evaluated and quantified from electron micrographs, approximately 60% of the dictyosome profiles were characterized by a system of transition elements consisting of part smooth-part rough endoplasmic reticulum. These were encountered most frequently in the immediate vicinity of the immature, forming or cis face, usually toward the periphery of the stacked cisternae. Analysis of serial sections showed that those dictyosome profiles not exhibiting this characteristic did so primarily because of an unfavorable plane of sectioning. All dictyosomes examined in 5 or more serial sections revealed some type of close association with endoplasmic reticulum. Some of the associations were so close that direct connections between Golgi apparatus and endoplasmic reticulum tubules could not be excluded. Also present, especially at the forming or cis face, were small 600 nm transition vesicles with nap-like surface coats on nearly 90% of the dictyosomes examined. More than 50% exhibited spiny (clathrin-)coated vesicles at the mature or trans face.     

Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique

The protein A-gold immunocytochemical technique has been modified to allow labeling of cellular antigenic sites on osmium-fixed or postfixed tissues. Several strong oxidizing agents have been found able to restore protein antigenicity on osmicated tissue thin sections. According to the fine structural preservation and intensities of labeling, pretreatment with sodium metaperiodate gave optimal results. Pancreatic secretory proteins (and/or proproteins) as well as insulin (and/or proinsulin) were localized over perfectly preserved rough endoplasmic reticulum (rER), Golgi apparatus, and secretory granules of the corresponding pancreatic cells; carbamyl phosphate synthetase and catalase were revealed over liver mitochondria and peroxisomes, respectively. In addition to the higher resolution in the labeling obtained using osmium-fixed tissues, the present modification confers an additional advantage to the protein A-gold technique by allowing labeling on tissues processed for routine electron microscopy.     

Intracellular transport of albumin through the secretory apparatus of rat liver parenchymal cells. An immunocytochemical study

The fine structural localization of albumin in rat liver parenchymal cells was determined by an improved immunocytochemical method and serial sectioning. Albumin in the secretory apparatus of the parenchymal cells was present in segments of the rough endoplasmic reticulum, interrupted with negative segments, in transport vesicles, Golgi saccules, finely anastomosed tubules and vesicles on the trans side of the Golgi complex, and in secretion granules. Horizontally sectioned Golgi saccules contained lipoprotein particles on one side and albumin on the other side. After transport, the vesicles that contained albumin fused with the so-called rigid lamellae on the trans-side of the Golgi complex. Ultrathin serial sections revealed no true structural continuity between the endoplasmic reticulum and the cis-aspect of the Golgi complex. We concluded that secretory proteins are transported from the endoplasmic reticulum to the Golgi complex by transport vesicles that bud from the endoplasmic reticulum and fuse with the Golgi saccules. These vesicles fuse regularly with the Golgi saccules on the cis-side and occasionally with tubular elements on the trans-aspect that may belong to the so-called GERL.     

Localization of lipocaline-type prostaglandin D synthase in rat brain: immunoelectron microscopic study

The distribution of lipocaline-type prostaglandin D synthase (L-PGDS) in rat brain was investigated by immunoelectron microscopy using a protein A-gold technique. In perivascular cells adjacent to the basement membrane of arterioles in the pia-arachnoid and of blood vessels in the subpial cortex, gold labeling was confined to the lumen of the dilated rough endoplasmic reticulum, and not found in the few lysosomes present in the cytoplasm. The results suggest that the perivascular cells secrete L-PGDS and seem not to degrade lipophilic molecules carried by L-PGDS. Moreover, gold particles representing the antigenic sites of L-PGDS were found in the Golgi apparatus, rough endoplasmic reticulum, vesicles, and nuclear envelope of arachnoid trabecular cells, arachnoid barrier cells, and arachnoid pia mater cells. The labeling was less detectable in the same organelles of choroid plexus epithelial cells, compared with leptomeningeal cells. In meningeal macrophages and parenchymal microglia, L-PGDS was detected in lysosomes, multivesicular bodies, and endocytic vesicles. The production of L-PGDS in perivascular cells is important to the various functions of this enzyme in brain parenchyma.     

Recycling of proteins between the endoplasmic reticulum and Golgi complex.

Several lines of investigation have shown that protein transport from the endoplasmic reticulum to the Golgi is more complex than previously imagined. Dynamic sorting of both membrane and soluble proteins is now believed to occur on the cis side of the Golgi apparatus with some proteins returning to the endoplasmic reticulum while others travel onwards.     

A review of the study of protein secretion applying the protein A-gold immunocytochemical approach

Exocrine and endocrine types of secretion were investigated in various cells by applying the protein A-gold immunocytochemical approach. Several proteins secreted by rat pancreatic and parotid acinar cells, mouse ameloblasts, rat pancreatic B cells and lymph-node plasma cells, and frog hepatocytes were studied using specific antibodies. While light microscope immunohistochemistry has allowed for good topographical identification of positive cells in tissues, the protein A-gold approach used at the electron microscope level has demonstrated the presence of specific antigenic sites in particular cellular compartments. All secretory proteins studied were detected in the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules of the corresponding secreting cells. In addition, some of the proteins were also found in lysosome-like structures. When good ultrastructural preservation of the cellular organelles was achieved, the labeling was revealed with very high resolution and precise localization. In such cases, we found labeling over transitional elements of the endoplasmic reticulum and in smooth vesicles in the Golgi area. The Golgi apparatus was subdivided into three compartments according to differences in labeling: the cisternae on the cisside, those of the trans-side and the trans-most rigid one. Quantitative evaluations of the intensities of labeling have allowed for 1) demonstration of the high specificity of the different labelings; 2) revelation of the existence of a gradient of increasing intensity that follows precisely the progress of the proteins along their secretory pathway; and 3) identification of intracellular sites where increments of protein antigenicity occur. Furthermore, they have revealed the existence of alterations in protein processing that occurred under experimental and pathological conditions. Double-labeling approaches were performed to demonstrate two different antigenic sites on the same tissue section by applying protein A-gold complexes formed by gold particles of different sizes. Protein A-gold immunocytochemistry has also been combined with cytochemical and radioautographic techniques. This review thus demonstrates that high-resolution quantitative immunocytochemistry can contribute significantly to the investigation of the intracellular processing of secretory proteins. It also illustrates the potential and versatility of the protein A-gold technique, which in combination with other procedures constitutes a powerful method in cell biology.     

Ultrastructural localization of dentine phosphoprotein in rat tooth germs by immunogold staining

Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicle-associated nucleation.     

Asymmetrical microtubule capping structures in frog palate cilia

The three-dimensional ultrastructure of the Golgi apparatus in milk secreting epithelial cells of bovine mammary gland was explored. From computer-aided reconstructions of serial thin sections, it was determined that the Golgi apparatus was composed of a single set of stacked cisternae. The three-dimensional shape of the dictyosome varied from cell to cell, but the overall shape was that of a hollow cone, cylinder, or bowl. The cis and trans surfaces of the dictyosome were arranged in three-dimensional space such that the cis face was located on the outer surface of the hollow structure and the trans face on the inner surface. The cytoplasmic channel (secretory channel) that traversed the longitudinal axis of the hollow dictyosome contained secretory vesicles. Densely stacked cisternae of rough endoplasmic reticulum surrounded the dictyosome, and microvesicles appeared to fuse with, or bud from, cisternae of both organelles. These findings suggest that Golgi apparatus of the lactating epithelial cell is highly organized and that the Golgi apparatus and secretory channel are essentially an independent compartment within the cell.     

Protein glycosylation in the endoplasmic reticulum and the Golgi apparatus and cell type-specificity of cell surface glycoconjugate expression: analysis by the protein A-gold and lectin-gold techniques

High resolution immunolabeling applying the protein A-gold technique and carbohydrate cytochemistry using lectin-gold labeling on Lowicryl K4M and thawed-frozen thin sections are most useful approaches for the detection of protein antigens and lectin binding sites in intracellular organelles and the plasma membrane. They provided the basis for modern electron microscopic studies on protein gylcosylation reactions and the identification of their subcellular localization as reviewed here. These studies have demonstrated organelle subcompartments and the cell type-specific compartmentation of endoplasmic reticulum and Golgi apparatus-associated glycosylation reactions. The other subject reviewed in this paper is cell surface glycoconjugates, as they are expressed in relation to specific cell types present in various organs and during cellular differentiation processes.     

Immunocytochemical localization of pepsinogen in rat stomach

The localization of pepsinogen in rat stomachs was investigated by a postembedding immunoferritin method. When the preparations embedded in Epon were used, the secretory granules of chief cells were stained heavily and the granules of mucous neck cells were stained moderately. The secretory granules of cells intermediate between mucous neck cells and chief cells showed a bizonal staining; the electron dense parts were stained heavily and the electron lucent parts were stained moderately. The secretory granules of pyloric gland cells, on the other hand, were labeled faintly. However, the secretory granules of surface mucous cells, foveolar mucous cells, endocrine cells, cardiac mucous cells and cardiac serous cells were not stained by the method. The protein A-gold method showed a similar staining pattern of pepsinogen to that of the immunoferritin method. When the samples embedded in Lowicryl K4M were used to enhance the stainability of pepsinogen, essentially the same staining pattern as that of the samples embedded in Epon was obtained. In addition, the Golgi apparatus and the rough surfaced endoplasmic reticulum were more easily stained.     

Immunocytochemical localization of beta 1,4 galactosyltransferase in epithelial cells from bovine tissues using monoclonal antibodies.

Post-embedding immunocytochemistry was employed to investigate the distribution of UDP-galactose:N-acetylglucosamine galactosyltransferase (beta 1,4-GT) in epithelial cells from various bovine organs. Several well characterized monoclonal antibodies previously demonstrated to recognize distinct polypeptide epitopes within the primary structure of beta 1,4-GT were applied to thin sections from tissues embedded in Lowicryl K4M, followed by the protein A-gold technique. Immunoreactivity was observed in the Golgi apparatus of epithelial cells from intestine, thymus and trachea. No immunoreactivity was observed in other intracellular structures, including rough endoplasmic reticulum, nuclear envelope and goblet cell mucus droplets. Within the Golgi apparatus, the staining was restricted to several cisternae in the trans region, with most portions of the trans-Golgi network appearing unlabelled. However, in thymic epithelial-reticular cells trans-Golgi network portions resembling classical GERL elements were stained by the antibodies. Thus, although immunoreactivity was subcompartmentalized within the Golgi apparatus in all epithelial cell types examined, the extent of staining within the trans-Golgi network was variable. Immunoreactivity was not detected at the plasma membrane (ecto-galactosyl-transferase), except in the case of a subpopulation of tracheal cells that resemble brush cells. These results suggest that in the epithelial cells examined, the subcompartmental distribution of beta 1,4-GT within the Golgi apparatus is maintained across different types of epithelial cell organization. Moreover, no evidence for a general epithelial cell ecto-galactosyltransferase could be discerned with these reagents.     

Detection of plasma cell immunoglobulins in tissue sections optimally fixed for ultrastructural immunocytochemistry

We describe the ultrastructural localization of plasma cell immunoglobulins in vibratome sections of popliteal lymph nodes. Fixation with glutaraldehyde-paraformaldehyde gave better tissue and antigen preservation than paraformaldehyde or periodic acid lysine-paraformaldehyde; biotinylated Fab fragments of sheep anti-mouse IgG-streptavidin-biotinylated horseradish peroxidase (HRP) or Fab-HRP conjugates gave similar results. With both immunoreagents, excellent tissue preservation and antigen detection was observed in the first layer of cells sectioned with the vibratome. Conjugates of anti-mouse IgG with HRP did not show any staining. Peroxidase stain was observed in the nuclear envelope, cisternae of the rough endoplasmic reticulum, and the Golgi apparatus complex. In the Golgi apparatus, staining was seen consistently in cisternae of the cis face and in adjacent vesicles; the trans cisternae showed weak or no stain, and adjacent vesicles, "coated" vesicles, and granules were not stained. This study shows that high quality of tissue preservation and antigen detection, by both light and ultrastructural immunocytochemistry, is feasible in tissue fixed with glutaraldehyde-paraformaldehyde followed by vibratome sectioning and immunostaining with Fab-biotin-streptavidin-biotin-HRP, or Fab-HRP.     

The ultrastructure of mammary explants from virgin ovariectomized goats during hormone-induced lactogenesis

The effect of insulin (I), cortisol (F) and prolactin (P) on the ultrastructural morphology of epithelial cells of cultured mammary explants from virgin ovariectomized (OV-X) goats were studied. The epithelial cells showed little structural organization and were devoid of fat droplets and secretory protein granules at zero time of culture. The cytoplasm contained few profiles of smooth and rough endoplasmic reticulum and the Golgi apparatus was rudimentary. After being cultured in Waymouth's medium without added hormones the epithelial cells were indistinguishable from epithelial cells of uncultured explants. The addition of I induced changes mainly in the appearance of nucleoli. The nucleoli were enlarged and fibrillogranular areas with light spaces were observed. The most obvious cytological changes of epithelial cells of explants cultured in the presence of I and F are translocation of the nucleus into the basal cytoplasm, increase of rough endoplasmic reticulum, an increase in the size of the Golgi apparatus, presence of one or two lipid droplets and in some cells vacuoles with protein granules were present. Mitochondria were more abundant. The epithelial cells of explants cultured in the presence of I, F and P were characterized by the polarization of organelles within the cytoplasm and by the formation and release of protein granules and small and large fat droplets. The cell nucleus was in the basal cytoplasm, the Golgi apparatus was supranuclear. The rough endoplasmic reticulum was extensively developed and formed large sacs. Golgi vacuoles contained protein granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-free transfer of membrane lipids. Evidence for lipid processing

A latent phospholipase A is concentrated in cis elements of rat liver Golgi apparatus, the presumed sites of fusion of the 50-70-nm transition vesicles formed from endoplasmic reticulum. As a result, conversion of transferred phospholipids to their corresponding lysoforms may provide an index of post transfer lipid processing in a corresponding reconstituted membrane transfer system. To label the phosphatidylcholine of transitional endoplasmic reticulum in vitro, [14C]CDP-choline and endogenous cytidyltransferases were used. In the reconstituted transfer system, the radiolabeled phosphatidylcholine was transferred via transition vesicles to Golgi apparatus immobilized on nitrocellulose strips in a time- and temperature-dependent process. Transfer was promoted by ATP and the ATP-dependent transfer was specific for cis Golgi apparatus elements as acceptor. Trans Golgi apparatus elements were ineffective as acceptors. Median Golgi apparatus elements were intermediate. A portion of the transferred phosphatidylcholine was converted subsequently to lysophosphatidylcholine also in a time- and ATP-dependent manner. The phospholipase A activity of the Golgi apparatus was more than 90% latent (active site located on the lumens of the Golgi apparatus membranes). Therefore, the lipid-containing vesicles derived from endoplasmic reticulum must have combined with cis Golgi apparatus membranes as the basis for Golgi apparatus-dependent phospholipase A processing of endoplasmic reticulum-derived phosphatidylcholine. Since the lipids were processed by phospholipase A in approximately the same proportion as occurs in situ, the findings offer evidence both for the specificity of the ATP-dependent component of cell-free lipid transfer from endoplasmic reticulum to Golgi apparatus and its fidelity to lipid transfer observed in vivo.