Trans Cisternae Research Articles

Lectin-gold cytochemistry of mucin oligosaccharide biosynthesis in Golgi apparatus of airway secretory cells of the hamster.

To elucidate the mechanism for the biosynthesis of O-linked mucin oligosaccharides, airway secretory cells of the hamster trachea were embedded in Lowicryl K4M resin, and sections were examined by lectin-gold cytochemistry with special attention focused on the Golgi apparatus. The interrelations between the Golgi cisternae stained with five different lectins were determined by double-staining procedures using various combinations of lectins conjugated with 14-nm and 8-nm colloidal gold. Several cis cisternae were stained only with HPA (Helix pomatia agglutinin specific for terminal alpha-N-acetylgalactosamine). The next medial cisternae were not stained with HPA, but reacted positively with two lectins, GSII (Griffonia simplicifolia agglutinin II specific for terminal alpha- or beta-N-acetylglucosamine) and RCAI (Ricinus communis agglutinin I specific for beta-galactose). The trans cisternae as well as condensing and mature secretory granules were labeled with four lectins, UEAI (Ulex europaeus agglutinin I specific for terminal alpha-L-fucose) and LFA (Limax flavus agglutinin specific for terminal N-acetyl or N-glycolyl neuraminic acid) in addition to HPA and RCAI. The same number of trans cisternae were positive to HPA and UEAI, whereas LFA bound to a few transmost cisternae but fewer than were stained with HPA or UEAI. The observed sequential appearance of different sugar residues in different levels of Golgi cisternae (from cis to trans cisternae) coincides quite well with the sugar sequence of airway mucin oligosaccharide (from reducing to nonreducing ends) proposed by biochemical analysis. It is suggested that airway mucin oligosaccharides elongate during a vectorial movement through the Golgi stack from cis toward trans and that the stack consists of at least three functionally distinct segments, cis, medial, and trans; in these three segments there take place, respectively, the initial O-glycosylation of mucin core peptide, the formation of a core region of oligosaccharide chain, and the completion of chain growth by addition of terminal sugar moieties.

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: histochemical, ultrastructural cytochemical, and biochemical characterization.

The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained the trans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgi trans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.

Ultrastructural demonstration of lectin binding sites in the Golgi apparatus of rat epiphyseal chondrocytes.

Binding sites for wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ricinus communis I agglutinin (RCA I) and Limax flavus agglutinin (LFA) have been ultrastructurally detected in rat epiphyseal chondrocytes by a post-embedding cytochemical technique using colloidal gold as marker. The four lectins labelled exclusively the Golgi apparatus of chondrocytes embedded in Lowicryl K4M resin by two different methods. WGA binding sites were localized in medial and trans cisternae as well as in immature secretory vesicles, whereas those for DBA were seen concentrated in cis and medial cisternae. Labelling with both RCA I and LFA lectins was distributed throughout all the cisternae of the Golgi stack, and the latter also in vesicles and tubules at the trans face. Neuraminidase pretreatment of the sections abolished LFA staining, decreased reaction with WGA and increased that with RCA I, while it did not affect DBA staining. After chondroitinase ABC treatment only the RCA I reaction was modified, revealing new binding sites in the trans Golgi face, secretory granules and extracellular matrix. These results indicate that the distribution of subcompartments in the Golgi apparatus of chondrocytes is different from that in cells secreting glycoproteins as major products.

An outline of formal logic and its applications in medicine--II.

signal carried by the lysosomal enzyme and becomes responsible for carrying the protein to its correct destination. Both the bound lysosomal enzyme and the low density lipoprotein receptor then move in vesicles to the medial and then to the trans cisterna. Here they part company, each being separately packaged into budding vesicles. The lysosomal enzyme is carried to lysosomes, where the mannose-6-phosphate tag is hydrolysed. This releases it from the receptor, which recycles back to the eis Golgi cisterna for further rounds of lysosomal transport. It is not known how the low density lipoprotein receptor is directed to the cell surface. Many argue that this is the default pathway?that is, everything that leaves the endoplasmic reticulum is sent to the cell surface unless it is either removed or retained on the way. Once on the cell surface the low density lipoprotein receptor begins to cycle. To do that it must contain information that causes it to be trapped selectively in coated pits. The tag for this selective trapping resides in that part of the receptor exposed to the cytoplasm. Mutations in this part yield receptors which are expressed on the cell surface and bind low density lipoprotein, but they are not efficiently internalised. Cholesterol cannot be taken up, and children with this defect suffer from familial hypercholesterolaemia. The tag is extremely efficient, allowing a 5000-fold purification of the receptor in a single step. This ensures that only those proteins that are meant to go to the endosomes are delivered there. All others are excluded from coated pits. The mechanism which carries out this selection is still unknown. In summary we should remember that the three dimensional organisation of the cell demands two types of information: informa? tion to transfer the protein to that part of the cell where it is to function, and information which allows it to carry out its function. In the simplest case this entails merely keeping it in a particular compartment. In the more complicated case information is needed to move it specifically between compartments. Future work will uncover more of the signals which encode this information and determine the nature of the mechanisms which decode it.

The Golgi apparatus in the acinar cells of the developing embryonic pancreas: II. Localization of lectin-binding sites.

The reaction patterns of the Golgi apparatus following staining with the lectins concanavalin A (ConA), Ricinus communis I agglutinin (RCA I), and Helix pomatia lectin (HPA) were studied in the pancreas acinar cells of rat embryos in the course of cell differentiation from day 13 through day 20 of gestation. The binding reactions were localized by means of pre-embedment incubation of 10-microns-thick cryosections of pancreas tissue, prefixed in a mixture of 4% formaldehyde/0.5% glutaraldehyde, using horseradish peroxidase for electron microscope visualization. ConA, which preferentially binds to alpha-D-mannosyl residues, consistently stained the cisternae of the cis Golgi side. The majority of the stacks also showed ConA staining of medial cisternae. The reaction of the trans side was variable; in each stage of development, the cisternae of the trans Golgi side either were devoid of labeling or appeared intensely stained. The reactions obtained with RCA I, which recognizes terminal beta-D-galactosyl residues, changed in the course of cell differentiation; in the protodifferentiated and early differentiated states, the system of "rigid lamellae," located at the trans side of the Golgi stacks, was intensely labeled, but became unreactive after production of secretion granules had started, the reaction then being restricted to the stacked saccules. In regard to the Golgi stacks in each of the developmental stages, RCA I binding sites either were confined to the trans cisternae, or, in addition, were found distributed across elements of the medial and cis compartments.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Differential subcompartmentation of terminal glycosylation in the Golgi apparatus of intestinal absorptive and goblet cells.

Two terminal glycosyltransferases, a sialyltransferase and the blood group A alpha 1,3 N-acetylgalactosaminyltransferase, were found to exhibit differential subcompartmentation in the Golgi apparatus of intestinal goblet and absorptive cells. As expected from their role in terminal glycosylation, the two glycosyltransferases and their products, sialic acid residues and blood group A substance, were localized in the trans cisternae of the Golgi apparatus of goblet cells. In contrast, however, they were found throughout the Golgi apparatus stack of adjacent absorptive cells, with the exception of the fenestrated first cis cisterna. The results are in contrast to the general view that enzymes in the glycosylation pathway are arranged in a cis to trans gradient across the Golgi apparatus and that such polarized distributions may instead be cell type-specific.

Transport of horseradish peroxidase from the cell surface to the Golgi in insulin-secreting cells: preferential labelling of cisternae located in an intermediate position in the stack.

We have used serial sectioning to study the topology of Golgi cisternae in insulin-secreting cells during secretion-stimulated endocytotic uptake of exogenous horseradish peroxidase (HRP). HRP-labelled cisternae were followed on several series of consecutive sections. This revealed that labelled cisternae could always be traced to a position in the Golgi stack intermediate between the cis and the trans poles. This occurred in spite of the apparent cis or trans locations of HRP-containing cisternae on some sections. The latter images could be explained by the lack of the true cis or trans (clathrin-coated) cisternae at certain levels of the stack.

Phosphatase localization in the endomembrane system of the dinoflagellate Crypthecodinium cohnii.

The distribution of four enzymes within the endomembrane system of the protist Crypthecodinium cohnii has been determined using cytochemical localizations with lead as a capture agent. Nucleoside diphosphatase (NDPase) activity, using inosine diphosphate (IDP) and thiamine pyrophosphate (TPP) as substrates, was observed in the Golgi apparatus, with a gradient of increasing reaction product noted in some cells from the cis to trans cisternae. Tubules and vesicles associated with the trans cisternae also contained reaction product. The endoplasmic reticulum exhibited a high activity of glucose-6-phosphatase [with glucose-6-phosphate (G-6-P) as substrate]. Traces of reaction product were also observed in the cis-most and trans-most cisternae of the dictyosomes. Activity of acid phosphatase (AcPase) was observed in Golgi cisternae as well as in associated cytoplasmic vesicles. Heaviest deposition was localized in medial and trans dictyosome cisternae. The cytoplasmic system of flattened vesicles subtending the surface membranes in these cells did not exhibit reactivity with any of the substrates used. The distribution of these enzymes in this algal cell appears similar to that observed in animal cells and suggests that these enzymes may represent markers for algal cell endomembrane compartments.

Transferrin receptors recycle to cis and middle as well as trans Golgi cisternae in Ig-secreting myeloma cells.

The recycling itinerary of plasma membrane transferrin receptors (TFR) was charted in IgG-secreting mouse myeloma cells (RPC 5.4) by tagging surface receptors with either bound anti-transferrin receptor antibodies (anti-TFR) or Fab fragments thereof and determining the intracellular destinations of the tagged receptors by immunocytochemistry. By immunofluorescence, TFR tagged with either probe were seen to be rapidly internalized and translocated from the cell surface to the juxtanuclear (Golgi) region. When localized by immunoperoxidase procedures at the electron microscopic level, the anti-TFR-labeled receptors were detected in all cisternae (cis, middle, and trans) of the Golgi stacks as well as in endosomes and trans Golgi reticular elements. There was no difference in the routing of TFR tagged with monovalent Fab and those tagged with divalent IgG. Tagged receptors were detected in Golgi stacks of approximately 50% of the cells analyzed. The position of the labeled cisternae within a given stack was found to be quite variable with cis and middle cisternae more often labeled at 5 min and trans cisternae at 30 min of antibody uptake. The finding that recycling plasmalemmal TFR can visit all or most Golgi subcompartments raises the likely possibility that any Golgi-associated posttranslational modification can occur during recycling as well as during the initial biosynthesis of plasmalemma receptors and other membrane proteins.

Special secretory cells in the soybean seed coat

The seed coat of soybean (Glycine max L. Merr.) is of physiological interest for synthesis and transport of amino acids and photosynthates during embryo development. A transmission and scanning electron microscopic study to elucidate the structure of the seed coat disclosed a specialized convex area (antipit) appressed to a concave pit in the center of the abaxial surface of the cotyledon. The antipit, which lies on the inner surface of the seed coat at a medial point in the anterior to posterior direction of the seed, contained specialized secretory cells bounded by loose multi-layered cell walls. These cells were rectangular in the developing seed, varied in length, and contributed directly to the convex morphology of the antipit seen on the ventral surface of the seed coat. At maturity these cells assumed the shape of a “cone”, extending from the aleurone layer in a perpendicular array. The aleurone and cone cells contained numerous Golgi apparatus, laminated rough endoplasmic reticulum, secretory vesicles, and amyloplasts. Secretory vesicles arose directly from tubules of fenestrated trans cisternae of the Golgi apparatus. Mitochondria were clustered with the amyloplasts; stacks of lamellar cisternae of rough endoplasmic reticulum were associated with the nucleus and Golgi apparatus. The cellular contents, the interconnections by plasmodesmata, and the close physical association with the cotyledon suggested that the aleurone and cone cells may be involved in symplastic transport of nutrients for use by the developing embryo.

Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry

This study was undertaken to measure the effect of maximal stimulation of sodium pump activity on the rate of energy metabolism in mouse cerebral astrocytes. The rate of uptake of 3H-2-deoxyglucose (3H-2-DG) was measured in astrocyte cultures sodium-loaded either by incubation in a K+-deficient solution or by use of the carboxylic sodium ionophore monensin. Sodium-loading by the first method caused 3H-2-DG uptake to increase by 80%, but the effect was brief (about 5 min) compared with the period of uptake measurement (20 min). In contrast, the presence of monensin (20 microM) caused a sustained 3.4-fold increase in the rate of 3H-2-DG uptake. The concentration-response relationship for monensin indicated a Kd of 1.5 microM and a maximum uptake enhancement of approximately fourfold. The monensin-stimulated uptake of 3H-2-DG was totally inhibited by incubation of the cultures in either K+-free or Na+-free solutions, or in the presence of ouabain (0.4 mM), indicating that the enhancement of uptake was the result of Na+ influx and sodium pump activation. These results raise the possibility that astroglia contribute significantly to regional variations in glucose consumption associated with functional activity in the brain. Ultrastructural analysis showed that sodium-loading in K+-free solution caused swelling confined to the trans face of Golgi stacks. However, monensin (5 microM) caused swelling of the entire Golgi stack, with progressively more severe swelling from cis to trans cisternae and formation of cytoplasmic vacuoles.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the epidermal growth factor (EGF) receptor within the endosome of EGF-stimulated epidermoid carcinoma (A431) cells.

We have followed the internalization pathway of both epidermal growth factor (EGF) and its receptor in human epidermoid carcinoma (A431) cells. Using EGF conjugated with horseradish peroxidase and anti-receptor monoclonal antibodies (TL5 and EGFR1) coupled either directly or indirectly to colloidal gold we have identified an extensive elaboration of endosomal compartments, consisting of a peripheral branching network of tubular cisternae connected to vacuolar elements that contain small vesicles and a pericentriolar compartment consisting of a tubular cisternal network connected to multivesicular bodies. Immunocytochemistry on frozen thin sections using receptor-specific antibody-gold revealed that at 4 degrees C in the presence of EGF, receptors were mainly on the plasma membrane and, to a lesser extent, within some elements of both the peripheral and pericentriolar endosomal compartments. Upon warming to 37 degrees C there was an EGF-dependent redistribution of most binding sites, first to the peripheral endosome compartment and then to the pericentriolar compartment and lysosomes. Upon warming only to 20 degrees C the ligand-receptor complex accumulated in the pericentriolar compartment. Acid phosphatase cytochemistry identifies hydrolytic activity only within secondary lysosomes and trans cisternae of the Golgi stacks. Together these observations suggest that the prelysosomal endosome compartment extends to the pericentriolar complex and that the transfer of EGF receptor complexes to the acid phosphatase-positive lysosome involves a discontinuous, temperature-dependent step.

Golgi and secreted galactosyltransferase.

Galactosyltransferase (GT) belongs to the glycosyltransferases. In several tissues and cell lines, the enzyme is localized by immunocytochemistry to the two to three trans cisternae of the Golgi complex and may thus be considered a specific membrane component of this type of endomembrane. As a consequence, it is the most common Golgi "marker" enzyme in cell fractionation studies. Study of its biosynthesis, membrane orientation, and turnover in several tissues and cultured cell lines has broadened our knowledge about Golgi function itself. The enzyme is oriented towards the lumen of the cisternal space. In this orientation, it catalyzes the transfer of galactose to glycoprotein-bound acetylglucosamine and, in the presence of alpha-lactalbumin, to glucose, as shown in the Golgi complex of mammary gland epithelial cells. The enzymatic properties of GT are well known. The metabolism of GT has been extensively studied in HeLa and human hepatoma cells. The enzyme is synthesized in the rough endoplasmic reticulum (RER) and provided with one N-linked oligosaccharide and palmitate residues. In the Golgi complex, terminal sugars are attached to the N-linked oligosaccharide and extensive O-glycosylation takes place. The half-life of the enzyme is about 20 hr, after which a soluble form appears in the culture medium. Release of GT into the medium is observed in all cell lines studied. This phenomenon is in accordance with the presence of soluble GT in body fluids such as serum, ascites, milk, and saliva. In patients suffering from ovarian and breast cancer, increased levels of GT enzyme activity have been reported. Whether extracellular GT is of biological significance is still a point of discussion.

Influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate by skin fibroblasts.

The influence of monensin on biosynthesis, processing and secretion of proteodermatan sulfate from human skin fibroblasts was studied with the aid of a specific immunological procedure. Double-labeling experiments with [3H]leucine and [35S]sulfate indicated that monensin caused a dose-dependent parallel decrease of sulfate incorporation into total and of secretion of 3H-labeled proteodermatan sulfate. Compared with the untreated control, a greater proportion of incorporated [35S]sulfate than of incorporated [3H]leucine became secreted. Other monensin effects were a moderate intracellular accumulation of glycosaminoglycan-free core protein, a reduced chain length and a greatly reduced epimerization of D-glucuronic to L-iduronic acid residues. In contrast to the formation of N-acetylgalactosamine 4-sulfate residues 6-sulfation was not affected. Conversion of high-mannose-type oligosaccharides to complex-type N-glycans which normally occurred concomitantly with glycosaminoglycan biosynthesis was inhibited. Withdrawal of monensin made possible an additional sulfation of intracellularly accumulated proteodermatan sulfate. The newly formed sulfate esters did not cluster at the non-reducing ends of the glycosaminoglycan chains. Cells preexposed to monensin and labeled with [3H]glucosamine either in the absence or continuous presence of the drug incorporated similar amounts of 3H radioactivity into proteodermatan sulfate. The results suggest that epimerization of D-glucuronic acid residues and 4-sulfation occur predominantly in the trans cisternae of the Golgi apparatus whereas chain polymerisation and 6-sulfation take place predominantly in the cis Golgi complex.

Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane.

The intracellular location at which the G protein of vesicular stomatitis virus accumulated when transport was blocked at 20 degrees C has been studied by biochemical, cytochemical, and immunocytochemical methods. Our results indicated that the viral G protein was blocked in that cisterna of the Golgi stack which stained for acid phosphatase. At 20 degrees C this trans cisterna became structurally altered by the accumulation of G protein. This alteration was characterized by extensive areas of membrane buds which were covered by a cytoplasmic coat. These coated structures were of two kinds--those that labeled with anti-clathrin antibodies and those that did not. The clathrin-coated pits consistently did not label with anti-G antibodies. Upon warming infected cells to 32 degrees C, G protein appeared on the surface within minutes. Concomitantly, the trans cisterna lost its characteristic structural organization. Double-labeling experiments were performed in which G protein localization was combined with staining for horseradish peroxidase, which had been taken up from the extracellular medium by endocytosis. The results suggest that the trans cisterna was distinct from the endosome compartment and that the latter was not an obligatory station in the route taken by G protein to the cell surface.

Localization of binding sites for concanavalin A, Ricinus communis I and Helix pomatia lectin in the Golgi apparatus of rat small intestinal absorptive cells.

Binding sites for concanvalin A (Con A), Ricinus communis I agglutinin (RCA I), and Helix pomatia lectin (HPA) were localized in the Golgi apparatus of rat small intestinal absorptive cells. A preembedment technique, a modification of the one originally used by Bernhard and Avrameas (Exp Cell Res 64:232, 1971), was employed, with horse-radish peroxidase being used for cytochemical visualization. Incubations were performed on 10 microns thick cryosections of duodenal segments that were fixed in a mixture of 4% formaldehyde and 0.5% glutaraldehyde; fixation was preceded by a 2-min rinse in 0.1 M sodium cacodylate and followed by storage in the same buffer for up to 7 days. Incubation with Con A, which binds preferably to alpha-D-mannose and alpha-D-glucose residues, caused intense reaction of the dilated cisternae of the cis Golgi side; staining was variable in intermediate and trans cisternae. RCA I, recognizing beta-D-galactose residues, could only be demonstrated in intermediate cisternae. Reaction for HPA, which indicates alpha-N-acetyl-D-galactosamine residues, stained intensely 1 to 2 cisternae of the cis Golgi side, as well as being localized in the peripheral regions of the cisternae of the intermediate compartment of the stacks. Deposits of reaction product covered the luminal surface of the cisternal membranes, but usually left the lumen itself, as well as lipid particles, devoid of reaction. The differences in Con A, RCA I, and HPA reactivity between cis, intermediate, and trans cisternae suggest compositional and structural differences of the carbohydrates in the respective compartments; they may reflect conversion processes that are known to occur in the oligosaccharide side chains of glycoconjugates at the Golgi complex level.

Clathrin-immunoreactive sites in the Golgi apparatus are concentrated at the trans pole in polypeptide hormone-secreting cells.

By analogy with receptor-mediated endocytosis, clathrin-coated membrane segments at locations other than at the plasma membrane have been implicated in intracellular membrane transport/sorting. The crucial role of the Golgi apparatus in these processes prompted us to study the distribution at this level of clathrin-immunoreactive sites in various polypeptide hormone-secreting cells. With a polyclonal antibody recognizing the polymerized form of clathrin (structural coats or cages), we found that the Golgi area had the highest degree of specific labeling of various cytoplasmic regions, including the subplasmalemmal web. Moreover, the Golgi labeling was not homogeneously distributed, most of the immunoreactive sites being associated with membrane segments at the trans pole. The labeled membranes comprised regions of trans cisternae with and without condensing secretory material, newly formed (maturing) secretory granules freshly released from trans cisternae, and typical coated vesicles. The cis Golgi cisternae, as well as the cytoplasmic region containing transfer (shuttle) vesicles and transitional cisternae of the rough endoplasmic reticulum, were only weakly labeled. Clathrin concentration, degree of assembly of triskelions into coats, duration of clathrin association with membranes, or presence of a coat protein different from clathrin could account for the low immunoreactivity observed at the cis pole as compared to the trans pole.

Vesicles and cisternae in the trans golgi apparatus of human fibroblasts are acidic compartments

Recently we demonstrated that low-pH compartments can be visualized with the electron microscope using a basic congener of dinitrophenol, 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP), which concentrates in acidic compartments and can be detected by immunocytochemistry with a monoclonal anti-dinitrophenol antibody. We now report that DAMP also accumulates in cisternae and vesicles associated with the trans face of the Golgi apparatus. DAMP rapidly leaves this compartment when cells are incubated with the ionophore monensin, which indicates that accumulation is due to the acidic pH in this compartment. Using indirect protein A-gold immunocytochemistry, we localized fibronectin, a major secretory protein in fibroblasts, to the trans Golgi vesicles that took up DAMP. Therefore, the trans cisternae of the Golgi apparatus and forming secretory vesicles have an acidic pH.

Effect of monensin on the Golgi apparatus of absorptive cells in the small intestine of the rat. Morphological and cytochemical studies.

SummaryThe effect of short-time treatment with the ionophore monensin, administered intraluminally at concentrations of 5 and 10 μM, was studied on the Golgi apparatus of absorptive cells in the small intestine of the rat. At 2–3 min after treatment most of the Golgi stacks exhibited dilated cisternae. At 4–5 min stacked cisternae were absent; they were replaced by groups of smooth-surfaced vacuoles. Dilatation and vacuolization occurred in the entire stacks without preferential effect on any particular Golgi subcompartment.Monensin did not influence the cytochemical Golgi reaction of thiamine pyrophosphatase and acid phosphatase. The characteristic staining pattern of these two enzymes in all Golgi cisternae of absorptive cells in the proximal small intestine, and the reactivity restricted to trans cisternae in distal segments of the small intestine, were unchanged after treatment with monensin. In the distal small intestine, the cytochemical pattern allowed the monensin-induced vacuoles to be attributed to the former cisor trans-Golgi face. Further, the cytochemical results demonstrate that vacuolization is not restricted to the stacked cisternae, but includes the trans-most cisterna. The latter, usually located at some distance from the Golgi stacks, has been defined as belonging to the GERL system in several types of cells. The clear response to monensin, an agent that selectively affects the Golgi apparatus, indicates common properties between trans-most and stacked Golgi cisternae.