JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 17:35-50 (1991)

Cytochemical Characteristics of the Golgi Apparatus MARGIT PAVELKA

AND

ADOLF ELLINGER

Institute of Micromorphology and Electron Microscopy, University

KEY WORDS

of

Vienna, A-1090 Vzenna, Austria

Lectinocytochemistry, Golgi subcompartments, Lectins, Glycosylation

ABSTRACT

Lectinocytochemistry provides a useful tool for localizing subcompartments of the complex reticular apparatus of Golgi. The technique is based on interactions of lectins with glycoconjugates present in the limiting membranes and luminal spaces of Golgi elements. Application of a series of lectins of different sugar specificities permits a differentiation between Golgi subcompartments containing glycoconjugates with different oligosaccharide side chains. These may be a) different glycoconjugates or b) glycoconjugates a t different stages during synthesis or repair of their glycans. The lectinocytochemical studies with mannose-, glucose-, N-acetyl-glucosamine-, N-acetylgalactosamine-, galactose-, fucose-, and sialic acid-recognizing lectins revealed predominating patterns that labeled distinct, i.e., cis, medial, trans, and transmost, regions of the Golgi apparatus. A further refinement could be achieved by differential lectin-inhibition that enables a dissection of lectin binding reactions on the basis of their binding affinities. High-affinity binding reactions showed that subcompartments are not necessarily confined to one single Golgi subregion and may change their position from one to another subregion. Some of the patterns observed may be interpreted in relation to certain steps during synthesis and modifications of glycans.

INTRODUCTION

Roth et al., 1984,1988; Sato and Spicer, 1982; Tartakoff and Vassalli, 1983; Taatjes et al., 1988; Velasco et al., It is well established that the Golgi apparatus (Golgi, 1988; Wasano e t al., 1988), and radioautographical 1898) is a continuous intracellular membrane system demonstration of sugar incorporation into glycans (e.g., composed of several functionally different subcompart- Bennett, 1984; Haddad and Bennett, 1987). Some enments (for review see Berger, 1984; Farquhar, 1985; zyme-cytochemical examples showing several phosFarquhar and Palade, 1981; Morre and Ovtracht, 1977; phatase activities in Golgi apparatus elements appear Pavelka, 1987; Rothman, 1985; Tartakoff, 1987). Our in Figure 1. By biochemical a s well a s cytochemical main interest focuses on questions a s to where in the methods, some of the Golgi-associated processes, e.g., complex apparatus functional subcompartments are lo- several steps during the synthesis and modifications of calized and as to how they are organized and cooperate glycans, have been found related to either cis, medial, trans, or transmost Golgi regions (Dunphy et al., 1985; to build up the overall Golgi entity. Morphologically, subunits of the Golgi apparatus ap- Goldberg and Kornfeld, 1983; Griffiths e t al., 1982; Lupear as stacks of flat cisternae, interconnected by tu- cocq et al., 1987; Morre et al., 1983,1984; Roth, 1984; bular-reticular and saccular elements. At each stack, Roth and Berger, 1982; Roth et al., 1984; Strous et al., cis, medial, and trans cisternae, as well as transmost 1983). However, multiple variations came to light (e.g., elements (trans Golgi reticulum, trans tubular net- Brown and Farquhar, 1987; Ellinger and Pavelka, work) have been defined morphologically and biochem- 1988; Oliver and Hand, 1983; Pavelka and Ellinger, ically. Morphological studies, successful in labeling 1987; Roth et al., 19861, thus making it difficult to distinct Golgi apparatus regions, include metal im- coordinate functional Golgi subcompartments with pregnation (Friend and Murray, 1965), enzymecy- morphological Golgi subregions. The present paper concentrates on affinity-cytotochemical and immunocytochemical localization of Golgi-associated enzymes (e.g., Dunphy et al., 1985; chemical studies, which make use of the sugar-binding Ellinger and Pavelka, 1982; Hand and Oliver, 1984; capacity of lectins (Goldstein and Poretz, 1986; Sharon Matsuo et al., 1988; Matyas and Morre, 1983; Novikoff and Lis, 1972); with this method subcompartments of and Novikoff, 1977; Paavola, 1978; Pavelka and El- the Golgi apparatus are differentiated on the basis of linger, 1983; Rambourg et al., 1988; Roth and Berger, interactions of lectins with carbohydrates present in 1982; Roth et al., 1985; Strous et al., 19831, immuno- the limiting Golgi membranes or in the luminal spaces. cytochemical localization of various molecules travers- The list of lectins which have been used for charactering the Golgi stacks (e.g., Bergmann and Singer, 1983; izing Golgi apparatus elements covers the entire specGeuze et al., 1985; Green et al., 1981; Griffiths et al., 1989; Posthuma et al., 1984; Saraste and Kuismanen, 1984; Slot and Geuze, 1983; Yokota and Fahimi, 19811, lectinocytochemical localization of glycoconjugates Received March 20, 1989; accepted in revised form May 25, 1989. (e.g.,Ellinger and Pavelka, 1988; Hedman et al., 1986; Address reprint requests to Univ.-Doz. Dr.Margit Pavelka, Institute of' MicroLucocq et al., 1987; Malchiodi et al., 1986; Pavelka and morphology, and Electron Microscopy Schwarzspanierstrape 17, A-1090 Vienna, Austria. Ellinger, 1985,1989; Quatacker, 1989; Roth, 1984;

C 1991 WILEY-LISS, INC

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M. PAVELKA AND A. ELLINGER

Fig. 1

CYTOCHEMISTRY OF GOLGI APPARATUS

trum, i.e., the groups of mannoseiglucose (man/ glcl-binding lectins, N-acetyl-glucosamine (g1cNAc)-, galactose and N-acetyl-galactosamine (ga1igalNAc)binding, as well a s fucose (fuc)- and sialic acid-recognizing lectins. Owing to the prominent role of the Golgi apparatus in sugar chain synthesis and modification (for review see Farquhar and Palade, 1981; Hirschberg and Snider, 1987; Hubbard and Ivatt, 1981; Kornfeld and Kornfeld, 1985; Roth, 19871, lectinocytochemical analyses of Golgi elements are of particular interest. Results obtained by examinations of various cell types showed predominating lectin binding patterns with a distinct labeling of either cis, medial, trans, or transmost Golgi apparatus regions. Some of these patterns may be interpreted in relation to certain steps during the synthesis and modifications of glycans. However, localizations of lectinolabeled Golgi subcompartments proved to be complex, and significant variations became apparent during studies of differently specialized cells (Pavelka and Ellinger, 1986; Roth et al., 1986) and analyses of cell differentiation (Pavelka and Ellinger, 1987). In the present paper, we show dominant lectinocytochemical reaction patterns a s well a s variations; furthermore, we report on the localization of Golgi apparatus subcompartments a s visualized by means of a new differential lectin-inhibition technique that permits a differentiation between low- and high-affinity binding reactions.

MATERIALS AND METHODS Albino rats (Him: OFA SD SPF), inclusive of mated animals, were obtained from the “Forschungsinstitut fur Versuchstierzucht” of the University of Vienna. Animals were fed a standard diet (Altromin 1314,1324) and had free access to water; they were fasted overnight prior to those experiments, which were concentrated on intestinal or pancreatic tissue. Generally, tissues were excised under anesthesia with pentobarbital. For studies of embryonic tissues, a t the appropriate gestational ages, caesarian section was performed under pentobarbital anesthesia; the tissues were fixed in situ and excised with the aid of a preparation microscope. Rat fibroblasts ( W l ) were kindly provided by Dr. ~~~

Fig. 1. Morphology and enzyme cytochemical localization of phosphatase activities in the Golgi apparatus of serous secretory cells of rat tracheal epithelium. A: Morphology. Golgi stack exhibiting a polar organization with wide luminal spaces of the cisternae a t one (cis, arrows) side and narrow cisternae at the other (trans) side. A “rigid lamella” is located in the transmost position of this stack iarrowheads). x 45,000. B: Thiamine pyrophosphatase. Reactions for thiamine pyrophosphatase are contained in trans cisternae of this stack (curved arrows),whereas the transmost cisternae and secretory granules are devoid of label (arrowheads). x40,OOO. C: Inosine diphosphatase. Inosine diphosphatase activities are apparent in cisternae of the trans Golgi side; cis and transmost Golgi elements (arrowheads) as well as secretion granules are unreactive. x 40,000. D: Acid phosphatase. Acid phosphatase reaction products are concentrated in the transmost Golgi cisterna (arrowhead), in tubular and vesicular elements and in secretion granules; furthermore, lysosomes are intensely stained. x 45,000. E: Trimetaphosphatase. Reactions for trimetaphosphatase are confined to lysosomes, whereas Golgi cisternae are devoid of label. x 45,000.

37

Monika Vetterlein from the Institute of Tumor Biology of the University of Vienna.

Tissue processing Following a brief rinse in buffer (0.1 M sodium cacodylate), tissues and cells were fixed in a mixture of 4% paraformaldehyde (freshly prepared) and 0.5% glutaraldehyde (electron microscope grade, Merck, Darmstadt, FRG) in 0.1 M sodium cacodylate, pH 7.2. Small pieces of tissue were immersed in the fixative for 30 min at 4°C and subsequently rinsed in buffer containing 10% dimethyl sulfoxide (DMSO). Free aldehyde groups were blocked by treatment with 0.05 M ammonium chloride for 2 h a t room temperature. Lectinocytochemistry The lectins of Helix pomatia (HPA), Griffonia simplicifolia I (GS I A, and B,), Ulex europaeus I (UEA I), Triticum uulgare-biotin (WGA-biotin), Pisum sativum (PSA), Lens culinaris (LCA), Erythrina cristagalli (ECA), as well a s concanavalin A (ConA) were purchased from Sigma Chem. Co. (St. Louis, MO); the Limax fLauus agglutinin (LFA) was purchased from Calbiochem, Behring Diagnostics (La Jolla, CAI; lectin-horseradish peroxidase (HRP)-conjugates and goldconjugated Ricinus communis I agglutinin (RCA I ) were from E-Y Laboratories (San Mateo, CAI. Preembedment procedures. After a 2-3-day rinse in 10% DMSO-containing cacodylate buffer, the tissues were frozen to -30°C in the presence of isopentane. Then 10 pm thick cryosections were prepared and thawed in phosphate-buffered saline (PBS). Cryosections as well as fibroblasts grown on coverslips were incubated in PBS-solutions of the lectin-HRP conjugates a t concentrations ranging from 30 to 100 pgiml for 4 h a t room temperature. In the case of ConA, a 2-step procedure (Bernhard and Avrameas, 1971) was employed: the ConA incubation was followed by treatment with HRP (grade VI, Sigma Chem. Co., St. Louis, MO; 200 pgiml) for 4 h a t room temperature. All incubation media, in addition, contained 0.1 mgiml saponin and 1 mgiml bovine serum albumin. The cryosections were continuously agitated during the incubations. Some of the specimens were refixed in 1%glutaraldehyde for 1 h a t 4°C. Diamino benzidine incubations (DAB, Serva Heidelberg, FRG) were used for visualizing the binding reactions; the incubation media contained 0.5 mgiml DAB and 20 p l / m l l % H,O, in 0.05 M Tris-HC1 buffer, pH 7.6; incubations were performed for 15 min at room temperature. Following several rinses in double-distilled water, the specimens were postfixed in 1%osmium ferrocyanide for 30 min, subsequently treated with 1% veronal acetate-buffered OsO, for 8 h a t 4”C, dehydrated, and embedded in Epon. Thin sections were examined unstained in a Philips 400 electron microscope. Depending upon the type of tissue studied and upon the lectin used, a various number of cryosections, ranging from 50 to 8596, exhibited the reactions throughout their entire 10 pm thickness; only these cryosections and here, only the central regions, were used for evaluation purposes.

M. PAVELKA A N D A. ELLINGER

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TABLE 1 Lectins that were used for analyzmg G o l g ~elements’ Canavalia ensiforinis Lens culiraaris Pisum sutivum Trztic u m uu lga re Helix pomatia Vicia uillosa Griffonia simplicifolia LA, Maclura pornifera Ricinus communzs I Erythrina cristagalli Griffonia simplicifolia I-B, Ulex ruropeus I Limax flavus ~~

ConA LCA’ PSA WGA HPA VVA GSI-A, MPA RCAI ECA GSI-R, UEA I LFA

aMan>aGlc>GlcNAc aMan>aGlc>GlcNAc ( + F u c ) aManYuGlc = GlcNAc ( + Fuc) GlcNAc>NANA aGalNAc>aGlcNAc>bGal aGalNAcsnGa1 aGalNAc>aGal aGalNAc>ruGal PGal>aGal>>GalNAc Gal~1,4GlcNAc>cuGalNAc aGal>>uGalNAc CYL-Fuc NANA

~

’ M a n , mannose: Glc, glucose; GlcNAc. N-acetyl-glucosamine; GalNAc, N-acetylqalactosamine: Gal, galactose, Fuc. fucnse; NANA. sialic acid. -A = agglutinin.

For cytochemical control, the specimens were incubated in PBS solutions of peroxidase (200 pgiml) for 4 h a t room temperature and subsequently subjected to the DAB protocol as described above, thus testing whether peroxidase binding sites, possibly extant in the tissues, contributed to the reactions. In order to exclude staining due to endogenous peroxidase, in other controls only the DAB reaction was performed. Postembedment procedures. For postembedment lectin staining, tissues were embedded in LR White (London Resin Company Ltd, Woking, Surrey, England; Ellinger and Pavelka, 1985; Newman et al., 1983). Preparation of lectin-gold conjugates. Colloidal gold particles with mean particle diameter of 14 nm were prepared by reducing chloroauric acid with trisodium citrate (Frens, 1973). The gold sols were stabilized with the respective lectins to be tested o r with fetuin; in each case, the minimum amount of coating agent required for stabilization of the gold sols, was determined by the salt flocculation test (Horisberger and Rosset, 1977). Following centrifugation a t 60,OOOg for 45 min, the gold complexes were resuspended in PBS. Fetuingold was resuspended in Tris-buffered saline (TBS, pH 7.2; 0.1 M Tris-HC1-0.15 M NaCI, 2 mM MnCI,, CaCI,, MgCl,, and CoCl,, and 0.2 mgiml polyethylene glycol, moI. wt. -20,000). Thin sections were mounted on gold or nickel grids and were preincubated in PBS or TBS (in the case of the 2-step LFA-fetuin incubations). Subsequently, the grids were immersed or floated on a drop of the lectin incubation media for 30 min a t room temperature; the final lectin-gold concentrations ranged from 8 to 20 pgiml. The WGA-biotin (100 pgiml) incubations were followed by treatment with strepavidin-gold (30 min; Amersham Int., UK); LFA (100 pgiml) incubations were followed by treatment with fetuin-gold (4 pglml; 30 min). Incubations were stopped by rinse in PBS or TBS (for LFAI; following additional rinses in distilled water, the sections were counterstained in 2% aqueous uranyl acetate (5 min) and 2% aqueous lead citrate (2 min). Control of specificities Controls of the sugar specificities were performed with the aid of competitive and non-competitive monosaccharides (man, glc, glcNAc, gal, galNAc, fuc,

sialic acid; E-Y Laboratories, San Mateo, CA; the purity of the sugars was checked by thin-layer chromatography and was found to be >95% in each case) and oligosaccharides (man3glcNAc, Biocharb Chem., Lund, Sveden; >95% purity by NMR); sugars were added to the incubation media 30 min prior to the incubations; by using graded series, the concentrations required for complete inhibition of the respective lectin binding reactions were determined. For differential inhibition of the lectin binding reactions, specimens were incubated in the presence of the respective competing sugars a t various concentrations. Graded concentration series were tested ranging from 0.001 M solutions up to those concentrations, which were necessary for a complete inhibition of the respective lectin reactions.

RESULTS Both preembedment methods using peroxidase for visualizing the lectin binding reactions and postembedment techniques making use of the colloidal gold system were employed. Lectins that were used for analyzing Golgi apparatus elements as well a s sugar specificities are listed in Table 1. Concanavalin A (ConA) ConA belongs to the group of maniglc-binding lectins (Debray et al., 1981; Goldstein and Poretz, 1986) and preferentially interacts with high mannose-type N-glycosidically linked oligosaccharide side chains of glycoproteins. In several cell types, ConA-binding sites were predominantly localized in cis and medial cisternae of the Golgi stacks, as well a s in endoplasmic reticulum cisternae, several Golgi-associated vesicles and lysosomes (Fig. 2a). The number of reactive cisternae was variable; possibly, reactive structures included all the stacked cisternae, such a s were shown in approximately 15% of the Golgi stacks in the small intestinal absorptive enterocytes (Pavelka and Ellinger, 1985) and in embryonic pancreatic acinar cells (Pavelka and Ellinger, 1987; Fig. 2c). Helix pomatia lectin and Griffonia simplicifolia I isolectin A, (HPA, GS I-A4) HPA and GS I-A, bind a t high affinities with galNAc-residues (Goldstein and Poretz, 1986). Both lectins induced intense labeling of cis Golgi cisternae; in intestinal goblet cells, in addition to the cis Golgi reactions, trans cisternae and transmost Golgi ele-

Fig. 2. Embryonic pancreatic acinar cells of the rat-day 16 of gestation. A ConA-HRP. ConA reaction is intense in the endoplasmic reticulum cisternae and in several vesicles. At the Golgi apparatus, only t h e cismost cisterna shows intense reaction (arrows), whereas the other cisternae a r e weakly stained or devoid of label. x 40,000. B: RCA I-HRP. At the upper section of this stack, RCA I reaction products a r e concentrated in cisternae of the trans Golgi side (arrows);at the lower section, cis and medial cisternae are labeled as well. x 50,000. C: ConA-HRP. ConA reaction products are distributed in cis, medial and trans cisternae of these stacks, whereas the transmost elements (arrowheads) are free of staining. One cis cisterna builds up the “backbone” of two neighbored stacks. x 40,000.

CYTOCHEMISTRY OF GOLGI APPARATUS

Fig. 2.

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M. PAVELKA AND A. ELLINGER

Fig. 3. Goblet cells of the rat colon. A HPA-gold. HPA reactions are concentrated in cis cisternae (arrows) as well as in trans and transmost cisternae and in secretion granules. x 40,000. B: HPA-gold 0.005 M GalNAc. Under this condition, the trans and transmost

HPA binding reactions as well as those in the secretion granules are abolished; HPA reactions are confined to cisternae of the cis Golgi side (arrows). x 40,000.

ments were labeled, whereas medial cisternae were weakly stained a t their rims or were free of reactions (Figs. 3a, 4b).

1986). In intestinal goblet and absorptive cells, WGA binding sites were predominantly localized in cis and medial cisternae of the Golgi stacks. In addition, inconstant staining of trans and transmost Golgi

-

Maclura pomifera and Vicia villosa lectins (MPA, VVA) MPA and VVA both belong to the group of galNAcbinding lectins (Goldstein and Poretz, 1986).The localization of MPA and VVA was studied in small intestinal absorptive cells and goblet cells; glycoconjugates recognized by these lectins were found concentrated in cis and medial cisternae of the Golgi stacks (Fig. 4a).

Wheat germ agglutinin (WGA) WGA binds with glcNAc a t high affinity and, furthermore, recognizes sialic acid (Goldstein and Poretz,

Fig. 4. A: MPA-HRP. Rat small intestinal absorptive cell. MPA reactions are intense in the cis (arrows)and medial cisternae of these stacks, whereas trans and transmost Golgi elements appear unstained. ~ 4 4 , 0 0 0B: GS I-A4-gold. Rat small intestinal goblet cell. Densely packed gold marker particles label cis (arrows) and trans/ transmost Golgi cisternae as well as secretion granules; medial cisternae are weakly stained. x 40,000. C: UEA I-gold. Rat small intestinal goblet cell. UEA I-colloidal gold particles are densely packed in the trans/transmost Golgi elements and in secretion granules. Cis (arrows)and medial cisternae appear free of reactions. x 40,000.

CYTOCHEMISTRY OF GOLGI APPARATUS

Fig. 4.

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M. PAVELKA AND A. ELLINGER

Fig. 5

CYTOCHEMISTRY OF GOLGI A P P A R A T U S

elements a s well as of secretory granules was noticed. On the other hand, in other cell types the WGA binding reactions were found to predominate in the transitransmost Golgi section (Tartakoff and Vassalli, 1983).

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the exception of the cismost one (Hedman et al., 1986; Roth et al., 1986); by contrast, in goblet cells, LFA reactions were found concentrated in the transitransmost Golgi stack regions (Roth et al., 1986).

Golgi apparatus subcompartments as visualized by differential lectin-inhibition technique One of the reasons for the complexity of lectinocytochemical patterns is probably a n overlapping of reactions. It has to be assumed that a given lectin interacts not only with one but with a number of different molecules present in the cells and tissues and binds with them a t different, either high or low, affinities. The patterns obtained following lectin incubations have to be seen as a sum of various binding reactions both high- and low-affinity ones. In order to unravel these binding patterns and to get more differentiated images of cell compartments, including Golgi subcompartments, new incubation protocols were worked out which permitted a delineation of high- and low-affinity binding reactions. With the aid of competitive sugars, Erythrina cristagalli lectin (ECA) added to the incubation media a t graded concentraECA is another N-acetyl-lactosamine recognizing tions, a differential inhibition of binding reactions lectin (Debray et al., 1986; Goldstein and Poretz, 1986). could be achieved. Since low-affinity binding reactions In several cell types, including small intestinal absorp- are already abolished a t low concentrations of compettive cells, pancreatic acinar cells and fibroblasts, the ing sugars, these protocols allow a selective demonstrareactions increased in intensity from the medial to tion of compartments and subcompartments in which transitransmost Golgi sections; a particular concentra- high-affinity binding sites, with respect to a given lection of ECA reactions was found in trans Golgi vesicles tin, are present. Differential Iectin-inhibition protocols and in the transmost tubular-reticular elements. proved valuable in the case of both preembedment-peroxidase and postembedment-gold labeling techniques. Griffoniasimplicifolia I isolectin B, (GS I-B4) This differential lectin-inhibition technique will be a-Gal residues are the binding sites for GS I-B, presented in the light of results obtained with the pea (Goldstein and Poretz, 1986).Studies with colonic gob- and the lentil lectins a s well as with HPA and GS I-A,. let cells showed a predominance of GS I-B, binding Pisum sativum and Lens culinaris lectins reactions in cis and medial Golgi cisternae. (PSA, LCA) Ulex europaeus I lectin (UEA I) Both PSA and LCA are man-, glc-, and glcNAcUEA I belongs to the group of fuc-binding lectins binding lectins (Debray et al., 1981; Goldstein and (Goldstein and Poretz, 1986). In small intestinal ab- Poretz, 1986; Kornfeld e t al., 1981); they possess exsorptive cells, UEA I induced weak reactions in medial tended binding sites and preferentially interact with and possibly in cis Golgi cisternae, whereas intense two- or more-antennary N-linked glycans. As opposed reaction products were concentrated in the transitrans- to ConA , PSA and LCA, for high-affinity binding to most Golgi apparatus regions. In goblet cells, the reac- glycopeptides, require a fucose residue attached to the tions were confined to the transitransmost Golgi re- asparagine-linked glcNAc; core-fucosylated N-linked oligosaccharide chains are to be considered the main gions (Fig. 4c; Ellinger and Pavelka, 1988). high-affinity binding sites for the pea and the lentil lectins. Limax flavus lectin (LFA) Binding reactions for both lectins were inhomogeLFA exhibits a narrow specificity for sialic acid neous in the endoplasmic reticulum cisternae (Figs. (Goldstein and Poretz, 1986). Variable Golgi apparatus 5a,b, 6a, 7a), the LCA staining generally being more reaction patterns were observed: in intestinal absorp- intense than the label induced by PSA. As to the Golgi tive cells, a s well a s in 3T3 cells, LFA label was ob- apparatus, in several cell types the reactions domiserved in the entire set of stacked Golgi cisternae with nated in cis and medial cisternae of the stacks (Figs. 5a,b, 7a). Trans cisternae and transmost Golgi elements, if reactive at all, showed weak label and often showed the reactions confined to distinct subregions of Fig. 5. Pancreatic acinar cells of the rat. A: LCA-HRP. Survey the cisternae. Frequently it was the utmost or the penmicrograph showing LCA reaction products in endoplasrnic reticulum ultimate cisterna a t the cis Golgi side that showed the cisternae as well as in the Golgi apparatus; here, label is apparent in most intense PSAiLCA label (Figs. 5a,b, 7a). This was one single cisterna, which is in the penultimate cis position (arrow!. noticed in various cell types, e.g., in mature intestinal ~ 3 1 , 0 0 0B: . LCA-HRP. LCA label is apparent in the endoplasmic reticulum; in the Golgi apparatus, LCA reactions are confined to the absorptive cells (Fig. 7a), in goblet and Paneth cells of the duodenum, in plasma cells and capillary endothepenultimate cis cisternae (arrows! of the stacks. X 50,000. Ricinus communis I lectin (RCA I) RCA I exhibits high affinity for p-gal residues (Debray et al., 1981; Goldstein and Poretz, 1986); it recognizes the N-acetyl-lactosamine sequence P-gal-(1-6)glcNAc occurring in terminal position at intermediates during the synthesis of N-linked glycans as well as being extant a t internalized asialoglycoproteins. Variable RCA I binding patterns of the Golgi apparatus were found (Pavelka and Ellinger, 1986,1987; Fig. 2b); in several cell types, RCA I binding glycoconjugates were shown confined to trans and transmost Golgi regions, whereas in other cell types, cis and medial cisternae showed intense reactions as well. In addition, various vesicles and lysosomes were reactive.

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Fig. 6

CYTOCHEMISTRY OF GOLGI APPARATUS

lial cells, and was particularly conspicuous in the case of the LCA label in the secretory pancreatic acinar cells (Fig. 5a,b); here, the LCA Golgi apparatus reactions were often exclusively localized in the penultimate cis cisterna. Deviating from these patterns, in the immature crypt cells of the small intestine, PSA and LCA binding reactions were frequently detected in all of the stacked Golgi cisternae (Fig. 6a). Similarly, in fibroblasts, intense PSA and LCA label was found in all cisternae throughout the stacks, with the exception of the cismost cisterna, which was weakly stained or devoid of reactions. In all cell types studied, Golgi-associated vesicles of various sizes and, in part, lysosomes were reactive. Addition to the incubation media of competing monosaccharides (man, glc, glcNAc) or oligosaccharides (man3glcNAc) influenced the reactions depending on the concentrations used. Low concentrations, e.g., 0.015 M man3glcNAc, only reduced or abolished the endoplasmic reticulum reactions, but did not influence staining of any other cell compartment (Fig. 6b). Higher concentrations of competing sugars, inhibited not only the endoplasmic reticulum reactions, but also reactions in lysosomes and some of the Golgi reactions (Fig. 7b,c, 8a). The highest sugar concentrations were required for the complete abolition of the reactions in Golgi cisternae and in some of the Golgi-associated vesicles. These results show that lectin binding reactions can be subdivided and that in the case of the PSA and LCA reactions, the reactive glycoconjugates in the endoplasmic reticulum cisternae bind a t lower affinities than those localized in the Golgi apparatus. Our findings demonstrate that various PSA- and LCA-reactive glycoconjugates differing in their binding affinities are present in the Golgi cisternae. Use of competing sugars a t concentrations just beneath those required for complete inhibition of the binding reactions permits a selective labeling of those Golgi subcompartments that contain high-affinity PSAiLCA binding glycoconjugates (Figs. 7b,c, 8a,b). Depending on cell type and stage of differentiation, these PSAiLCA high-affinity binding Golgi subcompartments comprise one single cisterna (Fig. 7b,c), or several cisternae (Fig. 8b), or parts of cisternae. In several cell types, such a s in the mature intestinal absorptive cells (Fig. 7c), goblet (Fig. 7b), Paneth and pancreatic acinar cells, the high-affinity PSAiLCA compartment was often one single cisterna in the penultimate cis position of the Golgi

Fig. 6. Rat small intestinal epithelial cells from the upper crypt region. A: LCA-HRP. Survey micrograph demonstrating intense LCA label in the endoplasmic reticulum cisternae; the Golgi stacks exhibit LCA reactions in cisternae of each, i.e., cis, medial, trans, and transmost, subsection. x 24,000. B: LCA-HRP i-0.015 M ManSGlcNAc. The LCA reactions are significantly reduced in the endoplasmic reticulum cisternae as compared to the normal LCA reactions, but are unaltered in Golgi elements; here, LCA-reactive cisternae are apparent in cis, medial, and transitransmost subregions. Unlabeled cisternae are interposed in between others which show intense LCA reactions. At the right-hand stack, one reactive cisterna (arrows)changes from the cismost to a medial position. x 40,000.

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stacks. It became apparent t h a t the high-affinity reactive compartment may change its position (Fig. 6b), e.g., from the utmost cis to the penultimate cis position as well as to medial and trans positions of the stacks. In the case of LCA, the high-affinity binding reactions were mostly apparent along the entire length of the cisternae; by contrast, high-affinity PSA reactions were frequently confined to distinct Golgi cisternae subregions. In small intestinal crypt cells, a s well as in fibroblasts, the high-affinity PSAiLCA-binding compartment mostly included more than one Golgi cisterna. The reactive cisternae were distributed across cis, medial, as well as transitransmost Golgi regions (Fig. 8b); here unreactive saccules were often interposed in between high-affinity reactive cisternae. Again, positional changes of high-affinity binding compartments (Fig. 6b), e.g., from cis to medial positions or from medial to trans positions, were observed. HPA and GS I-A, Golgi apparatus patterns of goblet cells provide another example of differential lectin-inhibition. The reactions a t the transitransmost Golgi elements and at the rims of the medial cisternae were abolished by competitive monosaccharides a t low concentrations (0.003 M), whereas significantly higher sugar concentration (0.015 M) were required for the inhibition of the cis-located reactions (Fig. 3a,b).

DISCUSSION Lectinocytochemical dissection of the Golgi apparatus includes a differentiation between subcompartments which contain either a) different glycoconjugates or b) glycoconjugates a t different stages during synthesis of their glycans or during glycan repair, in the case of newly synthesized or recycling molecules, respectively. The method does not allow a differentiation between endogenous Golgi apparatus molecules on the one hand, and newly synthesized and recycling molecules traversing the Golgi stacks, on the other. Some of the lectinocytochemical patterns observedmay be interpreted in relation to glycan synthesis and glycan modifications occurring in the Golgi apparatus: The predominance of binding sites for man-binding lectins in cis Golgi cisternae and, on the other hand, the preferential localization of reactions for gal-, fuc-, and sialic acid-recognizing lectins in the transitransmost sections of the Golgi stacks, may signal the conversion of high-mannose N-glycosidically linked oligosaccharide chains into complex-type glycans. Such patterns indicate that subsequent events during the synthesis of N-linked glycans are predominantly oriented from the cis to the trans side of the Golgi stacks; this is in accordance with the predominating immunocytochemical localizations of some of the enzymes involved in the processing of N-linked glycans in some cell types, i.e., Golgi glcNAc-transferase I in medial Golgi cisternae (Dunphy et al., 19851, and galactosyl- and sialyltransferase in transitransmost Golgi elements (Roth and Berger, 1982; Roth et al., 1985; Strous et al., 1983). Likewise, the cis-predominating localizations of binding sites for galNAc-recognizing lectins may correspond to intermediates occurring after insertion of the serithr-linked galNAc residue during synthesis of

46

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Fig. 7

Fig. 8. A LCA-HRP + 0.3 M GlcNAc. Small intestinal absorptive cell of the rat. Reactions for LCA are absent from the endoplasmic reticulum. At the Golgi apparatus, LCA label is confined to cisternae at the cis side of the stacks (arrow). x 40,000. B: PSA-HRP 1 0.1 M Man. Fibroblast. Endoplasmic reticulum cisternae are free of PSA

reactions. The Golgi PSA reactions include cis, medial, as well as trans and transmost cisternae; unlabeled cisternae are interposed in between others that contain densely packed reaction products. x 62,000.

Fig. 7. A: PSA-HRP. Small intestinal absorptive cell of the rat. Reactions are weak in the endoplasmic reticulum. At the Golgi apparatus, PSA label is concentrated in cis (arrows)and medial cisternae, whereas trans and transmost elements are weakly stained or free of staining. x 40,000. B: LCA-HRP + 0.3 M Man. Goblet cell of the rat small intestine. Endoplasmic reticulum cisternae are free of reac-

tions. At the Golgi stacks, LCA reactions are limited to one single cisterna that is in the penultimate cis position (arrows). x 50.000. C: LCA-HRP + 0.3 M Glc. Small intestinal absorptive cell of the rat. LCA label is confined to one Golgi cisterna (arrow),which is located in the penultimate position a t one side of this stack. x 50,000.

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0-linked saccharide chains (Roth, 1984; Elhammer and Kornfeld, 1984); reduced accessibility of the core galNAc residues after insertion of other sugars may be the reason for the reduction or absence of label in medial cisternae; furthermore, galNAc residues in terminal position, such as present a t glycoconjugates of blood group A-positive individuals, may be the binding sites responsible for the transitransmost reactions obtained with galNAc-binding lectins (Roth, 1984; Roth et al., 1988). Hence, the reactions related to 0-glycosidically linked saccharide chains may be interpreted in the light of a cis-to-trans orientation of subsequent events during their synthesis. However, owing to the variations observed with the galNAc- and man-, as well as gal-, fuc-, and sialic acidbinding lectins, a general model cannot be deduced. None of the lectins so far tested can be used a s a marker for a certain, cis, medial, trans, or transmost, Golgi apparatus subsection. Differential lectin-inhibition experiments proved to be an approach for refined localization of Golgi subcompartments. This technique helped to unravel lectin reactions based on binding reactions of various affinities. By using competing sugars a t concentrations just beneath those necessary for complete inhibition of all staining, this technique made it possible to label selectively compartments containing high-affinity binding glycoconjugates with respect to a given lectin. With this technique, it was demonstrated that, in the case of the galNAc-reactive lectins, such a s HPA and GS I-A4, the transkransmost Golgi reactions are based on interactions with glycans binding a t lower affinities than those responsible for the cis Golgi reactions; it is exclusively in the cis Golgi cisternae that high-affinity-HPA and GS I-A4 binding glycoconjugates reside in the goblet cells. With respect to the pea and lentil lectins it was shown that the glycans binding a t highest affinities, in several cell types, are concentrated in one single cisterna or are even limited to a subregion of this cisterna. According to the PSA and LCA-binding characteristics, the high-affinity binding reactions may correspond to core-fucosylated intermediate sugar chains occurring a t distinct stages during the synthesis of N-glycosidically linked glycans, prior to or after the action of Golgi mannosidase I1 (Kornfeld and Kornfeld, 1985; Longmore and Schachter, 1982). In mature absorptive enterocytes, a s well as in goblet and Paneth cells and in the pancreatic acinar cells, these high-affinity binding glycoconjugates were dominant or appeared exclusively in one cisterna that was localized most frequently in the penultimate cis position of the Golgi stacks, thus suggesting a significant role of this cisterna during modifications of N-linked glycans. On the other hand, it was shown by differential lectininhibition studies of intestinal crypt cells and fibroblasts that high-affinity PSA and LCA-binding glycoconjugates may be present in cisternae of all Golgi regions and positional changes may occur. Summarizing, lectinocytochemistry refined by differential inhibition of the binding reactions permits a dissection of the Golgi apparatus in subcompartments. The binding patterns obtained with a list of lectins of

different sugar specificities showed functional subcompartments in correspondence with cis, medial, trans, and transmost Golgi cisternae. High-affinity binding reactions demonstrated that subcompartments are not necessarily limited to one single subregion of the Golgi apparatus and may change their position from one to another subregion.

ACKNOWLEDGMENTS The authors gratefully acknowledge the excellent technical assistance of Mrs. J u t t a Selbmann, Mrs. Elfriede Scherzer, Mrs. Gertrude Hartl, Mr. Helmut Oslansky, and Mr. Richard Reichhart. This work was supported by “Verlassenschaft Maria Buss.” REFERENCES Bennett, G. (1984)Role of the Golgi complex i n t h e secretory process. In: Cell BIology of the Secretory Process. M. Canin, ed. Karger, Basel. DU. 102-147. Berger, ‘E.G. (1984)The functional organization of the Golgi apparatus. INSERM, 126:lll-122. Bergmann, J.E.,and Singer, S.J. (1983)Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein ( G )of vesicular stomatitis virus in infected Chinese hamster ovary cells. J. Cell Biol., 97:1777-1787. Bernhard, W., and Avrameas, S. (1971)Ultrastructural visualization of cellular carbohydrate components by means of concanavalin A. Exp. Cell Res.. 64:232-236. Brown, W.J., and Farquhar, M.G. 11987) The distribution of 215kilodalton mannose 6-phosphate receptors within cis (heavy) and trans (light) Golgi subfractions varies in different cell types. Proc. Natl. Acad. Sci. USA, 84:9001-9005. Dehray, H., Decout, D., Strecker, G., Spik, G., and Montreuil, J. 119811 Specificity of twelve lectins towards oligosaccharides and glycopeptides related to N-glycosylproteins, Eur. J. Biochem., 117:41-55. Dehray, H., Montreuil, J., Lis, H., and Sharon, N. (19861Affinity o f four immobilized Erythrina lectins toward various N-linked glycopeptides and related oligosaccharides. Carbohydr. Res., 15L:359370. Dunphy, W.G.. Brands, R., and Rothman, J.E. (1985, Attachment of terminal N-acetyl-glucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack. Cell. 40:463472. Elhammer, A.,and Kornfeld, S. (1984)Two enzymes involved in the synthesis of 0-linked oligosaccharides are localized on membranes of different densities in mouse lymphoma BW5147 cells. J. Cell Biol., 98:327-331. Ellinger, A., and Pavelka M. (1982)The Golgi apparatus of rat small intestinal absorptive cells. II. Morphology and cytochemical staining pattern during cell differentiation. J. Submicrosc. Cytol., 14: 587-596. Ellinger, A., and Pavelka, M. (1985)Post-embedding localization of glycoconjugates by means of lectins on thin sections o f tissues embedded in LR white. Histochem. J., 17:1321-1336. Ellinger, A., and Pavelka, M. (1988)Localization of fucosyl restdues in cellular compartments of rat duodenal absorptive enterocytes and goblet cells. Eur. J. Cell Biol., 4752-71. Farquhar, M.G. (1985)Progress in unraveling pathways of the Golgi traffic. Annu. Rev. Cell Biol., 1:447-488. Farquhar, M.G., and Palade, G.E. (1981)The Golgi apparatus (cornplex)-(1954-1981)-from artifact to center stage. J. Cell Biol., 91: 77s- 103s . Frens, G. (1973)Controlled nucleation for the regulation of particle size in monodisperse gold suspensions. Nature, Phys. Sci., 2412022. Friend, D.S., and Murray, M . J . (1965)Osmium impregnation of the Golgi apparatus. Am. J. Anat., 117:135-150. Geuze, H.J., Slot, J.W., Strous, G.J.A.M., Hasilik, A,, and von Figura. K. (1985)Possible pathways for lysosomal enzyme delivery. J. Cell Biol., 101:2253-2262. Goldberg, D.E., and Kornfeld, S. (1983)Evidence for extensive subcellular organization of asparagine-linked oligosaccharide process-

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