Immunocytochemical visualization of the Golgi apparatus in several species, including human, and tissues with an antiserum against MG-160, a sialoglycoprotein of rat Golgi apparatus

We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain. By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12). The monoclonal antibody (MAb) reacted only with rat tissues. Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues. Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography. Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD. Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD. Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD. Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus. Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus. The cisternae of the trans Golgi network were not stained. These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae. The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.     

Subfractionation of rat liver Golgi apparatus by free-flow electrophoresis

Using the technique of preparative free-flow electrophoresis, cisternae of unstacked rat liver Golgi apparatus were separated into a series of fractions of increasing content of sialic acid, thiamine pyrophosphatase and 5'-nucleotidase, markers regarded as being concentrated toward the mature Golgi apparatus face. These same fractions showed a decreasing content of nucleoside diphosphatase, an endoplasmic reticulum marker. Fractions enriched in sialic acid also were enriched in cisternae from the mature or trans face of the Golgi apparatus as deduced from cytochemical criteria. Those fractions least enriched in sialic acid contained cisternae that accumulated deposits of reduced osmium under standard conditions, a test used to mark the opposite, forming or cis-face. Thus subfractionation along the functional polarity axis of the Golgi apparatus with separation of cis and trans face cisternae has been achieved.     

Cisterna-specific localization of glycosylation-related proteins to the Golgi apparatus

The Golgi apparatus is an intracellular organelle playing central roles in post-translational modification and in the secretion of membrane and secretory proteins. These proteins are synthesized in the endoplasmic reticulum (ER) and transported to the cis-, medial-and trans-cisternae of the Golgi. While trafficking through the Golgi, proteins are sequentially modified with glycan moieties by different glycosyltransferases. Therefore, it is important to analyze the glycosylation function of the Golgi at the level of cisternae. Markers widely used for cis-, medial- and trans-cisternae/trans Golgi network (TGN) in Drosophila are GM130, 120 kDa and Syntaxin16 (Syx16); however the anti-120 kDa antibody is no longer available. In the present study, Drosophila Golgi complex-localized glycoprotein-1 (dGLG1) was identified as an antigen recognized by the anti-120 kDa antibody. A monoclonal anti-dGLG1 antibody suitable for immunohistochemistry was raised in rat. Using these markers, the localization of glycosyltransferases and nucleotide-sugar transporters (NSTs) was studied at the cisternal level. Results showed that glycosyltransferases and NSTs involved in the same sugar modification are localized to the same cisternae. Furthermore, valuable functional information was obtained on the localization of novel NSTs with as yet incompletely characterized biochemical properties.     

Attachment of terminal N-acetylglucosamine to asparagine-linked oligosaccharides occurs in central cisternae of the Golgi stack

Using monoclonal antibodies and electron microscopy, we have localized N-acetylglucosamine transferase I within the Golgi apparatus. This enzyme initiates the conversion of asparagine-linked oligosaccharides to the complex type. We have found that the enzyme is concentrated in the central (or medial) cisternae of the Golgi stack. Cisternae at the cis and trans ends of the Golgi complex appear to lack this protein. These experiments establish a function for the medial portion of the Golgi and imply that the Golgi is partitioned into at least three biochemically and morphologically distinct cisternal compartments.     

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.     

The Golgi apparatus ofPhytophthora cinnamomi makes three types of secretory or storage vesicles concurrently

Summary The formation of three types of vesicles in the oomycetePhytophthora cinnamomi was investigated using ultrastructural and immunocytochemical techniques. All three vesicles are synthesised at the same time; one type serves a storage role; the others undergo regulated secretion. A monoclonal antibody Lpv-1 that is specific for glycoproteins contained in the storage vesicles labelled the endoplasmic reticulum (ER), elements in the transition region between ER and Golgi stack, and cis, medial and trans Golgi cisternae. Cpa2, a monoclonal antibody specific for glycoproteins contained within secretory dorsal vesicles labelled the transition region, cis cisternae and a trans-Golgi network. Vesicles possessing a structure characteristic of mature secretory ventral vesicles were observed in close association with the trans face of Golgi stacks. The results suggest that all three vesicles are formed by the Golgi apparatus. Double immunogold labelling with Lpv-1 and Cpa-2 showed that these two sets of glycoproteins occurred within the same Golgi cisternae, indicating that both products pass through and are sorted concurrently within a single Golgi stack.     

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.     

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.     

Monoclonal antibodies as markers of the endocytic and secretory pathways

A galactosyltransferase-rich subcellular fraction and wheat germ agglutinin(WGA)-binding microsomal proteins from rat myeloma cells have been used to immunize BALB/c mice. Fusion of the corresponding spleen cells with the Sp2/0 mouse myeloma has lead to the production of hybridomas secreting monoclonal antibodies directed against four proteins of the Golgi complex (GC) and other smooth membranes (SM). Subcellular fractionation of myeloma cells and rat liver, Triton X-114 partitioning, protease treatment and lectin binding studies have permitted us to identify--by immunoblotting--the molecular weight of the proteins involved, their topology and their mode of association with membranes. Morphological analysis has been performed by immunocytochemistry at the light and electron microscopic level. Judging by these criteria, the GCII antigen is a protein of 44 kDa which is loosely associated with the endodomain of Golgi cisternae. GCIII is a detergent-binding glycoprotein of 130 kDa whose epitope is on the endodomain of Golgi cisternae. SMI is a detergent-binding glycoprotein of 58 to 90 kDa found at several stations along the endocytic path: in coated pits, coated vesicles, endocytic vesicles, but not in lysosomes. The epitope recognized by the corresponding antibody faces the ectodomain. When this antibody is added to living cells in culture, it is rapidly internalized. SMII is a detergent-binding glycoprotein of 140 kDa. The epitope recognized is restricted to membranes of Golgi complex cisternae and multivesicular bodies. These reagents should be useful for dissection and perturbation of vesicular traffic.     

The signal anchor and stem regions of the beta-galactoside alpha 2,6-sialyltransferase may each act to localize the enzyme to the Golgi apparatus.

The beta-galactoside alpha 2,6-sialyltransferase has been localized to the trans cisternae of the Golgi apparatus and the trans Golgi network where it transfers sialic acid residues to terminal positions on N-linked oligosaccharides. It is a type II transmembrane protein possessing a 9-amino acid amino-terminal cytoplasmic tail, a 17-amino acid signal anchor domain, and a 35-amino acid stem region which tethers the large luminal catalytic domain to the membrane anchor. Previous work has demonstrated that the soluble sialytransferase catalytic domain is rapidly secreted from Chinese hamster ovary cells. These results suggest that the signals for Golgi apparatus localization do not reside in the catalytic domain of the enzyme but must reside in the cytoplasmic tail, signal anchor domain, and/or stem region. To determine which amino-terminal regions are required for Golgi apparatus localization, mutant sialyltransferase proteins were constructed by in vitro oligonucleotide-directed mutagenesis, expressed in Cos-1 cells, and localized by indirect immunofluorescence microscopy. Signal cleavage-sialyltransferase mutants which consist of only the stem and catalytic domain of the enzyme are not rapidly secreted but are retained intracellularly and predominantly localized to the Golgi apparatus. However, deletion of either the stem region or the cytoplasmic tail of the membrane-bound sialyltransferase does not alter its Golgi apparatus localization. In addition, sequential replacement of the amino acids of the sialyltransferase signal anchor domain with amino acids from the signal anchor domain of a plasma membrane protein, the influenza virus neuraminidase does not alter the Golgi apparatus localization of the sialyltransferase. These observations suggest that sequences in the signal anchor region and stem region allow the Golgi apparatus localization of the membrane-bound and soluble forms of the sialytransferase, respectively, and that both regions may contain Golgi apparatus localization signals.     

Glycoproteins of the lysosomal membrane

Three glycoprotein antigens (120, 100, and 80 kD) were detected by mono- and/or polyclonal antibodies generated by immunization with highly purified rat liver lysosomal membranes. All of the antigens were judged to be integral membrane proteins based on the binding of Triton X-114. By immunofluorescence on normal rat kidney cells, a mouse monoclonal antibody to the 120-kD antigen co-stained with a polyclonal rabbit antibody that detected the 100- and 80-kD antigens as well as with antibodies to acid phosphatase, indicating that these antigens are preferentially localized in lysosomes. Few 120-kD-positive structures were found to be negative for acid phosphatase, suggesting that the antigen was not concentrated in organelles such as endosomes, which lack acid phosphatase. Immunoperoxidase cytochemistry also showed little reactivity in Golgi cisternae, coated vesicles, or on the plasma membrane. Digestion with endo-beta-N-acetylglucosaminidase H (Endo H) and endo-beta-N-acetylglucosaminidase F (Endo F) demonstrated that each of the antigens contained multiple N-linked oligosaccharide chains, most of which were of the complex (Endo H-resistant) type. The 120-kD protein was very heavily glycosylated, having at least 18 N-linked chains. It was also rich in sialic acid, since neuraminidase digestion increased the pI of the 120-kD protein from less than 4 to greater than 8. Taken together, these results strongly suggest that the glycoprotein components of the lysosomal membrane are synthesized in the rough endoplasmic reticulum and terminally glycosylated in the Golgi before delivery to lysosomes. We have provisionally designated these antigens lysosomal membrane glycoproteins lgp120, lgp100, lgp80.     

Translocation of cytidine 5′-monophosphosialic acid across golgi apparatus membranes

Golgi apparatus, isolated from rat liver, incorporate [¹⁴C]sialic acid from CMP[¹⁴C]sialic acid into endogenous glycolipid and glycoprotein acceptors. Incorporation of [¹⁴C]sialic acid into endogenous glycoprotein acceptors was stimulated an average of 3-fold by Triton X-100 at an optimal concentration of 0.05% and was inhibited at higher concentrations. Incorporation of [¹⁴C]sialic acid into endogenous glycolipid acceptors was not stimulated by detergent. The major glycolipid product was identified by thin-layer chromatography as the ganglioside G_d3. SDS-polyacrylamide gel electrophoresis of the glycoprotein products demonstrated incorporation of [¹⁴C]sialic acid into 6–7 major bands. Neuraminidase studies determined that approximately 60% of the [¹⁴C]sialic acid incorporated into endogenous acceptors in the absence of detergent had a luminal orientation. Furthermore, electron microscopy studies showed that the isolated Golgi apparatus fraction consisted of intact membrane cisternae. Our results demonstrate that sialylation of cisternal acceptors located on the inside of the membrane occurs in the absence of detergent. They are consistent with carrier-mediated transport as a mechanism to allow CMPsialic acid to traverse the Golgi apparatus membrane and to be used to glycosylate endogenous glycoprotein and glycolipid acceptors.     

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

Summary 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 cisternac 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.     

RCA I-binding patterns of the Golgi apparatus

The distribution in the Golgi apparatus of binding sites for the galactose-specific Ricinus communis I lectin (RCA I) was studied in differently specialized cells, including goblet cells and absorptive enterocytes of the rat small intestine as well as acinar cells of the rat embryonic pancreas and submandibular gland. For the purpose of localizing the binding reactions, a pre-embedment method using horseradish peroxidase for electron microscopic visualization, and a post-embedding technique making use of the colloidal gold system were employed. The reactions obtained, localizing cell constituents which contain saccharides with terminal or internal beta-D-galactosyl residues, labeled diverse Golgi subcompartments. The goblet cells showed intense RCA I staining of the cisternae of the trans side of the Golgi stacks. The reaction was weak in the medial cisternae and the cis side of the stacks mostly was devoid of label. In the absorptive cells, in addition to the RCA I reaction of trans Golgi elements, binding sites for this lectin were concentrated in the stacks' medial section. In the embryonic acinar cells, accessible galactosyl residues were either confined to the trans and/or medial cisternae, or distributed across elements of all the stacked saccules. In the latter stacks, the reactions mostly were weak in the cis cisternae and increased in intensity towards the trans side. As regards the respective labeling patterns, similar percentages were calculated for the early and late stages of development: they were approximately 62% for the pattern which showed RCA I label limited to trans/medial cisternae and approximately 38% for the "cis-to-trans"-distributed RCA I reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation

Sialyltransferase (Galβ1,4GlcNAc α2,6 sialyltransferase) was localized by immunoelectron microscopy in rat liver hepatocytes using affinity-purified antibodies. Immunoreactivity for sialyltransferase was found in the Golgi apparatus, where it was restricted to an interconnected system consisting of the trans-cisternae and the trans-tubular network. This region of the Golgi apparatus exhibited both TPPase and CMPase activity and was the intracellular site where sialic acid residues bound to glycoprotein were detected using the Limax flavus lectin. Sialyltransferase and sialic acid residues were not detected in medial and cis-cisternae of the Golgi apparatus. These findings suggest that in rat hepatocytes sialylation of N-linked glycoproteins occurs in the complex formed by the trans-cisternae and the trans-tubular network of Golgi apparatus.     

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.     

Localization of fucosyl residues in cellular compartments of rat duodenal absorptive enterocytes and goblet cells

We have determined the subcellular distribution of fucosyl residues in rat duodenal absorptive enterocytes and goblet cells, using the binding affinity of the lectin I of Ulex europaeus (UEA I). In absorptive enterocytes, UEA I-lectin gold complexes were detected at the brush border and at the basolateral plasma membrane; pits of the plasma membrane were labeled, as were small vesicles, multivesicular bodies, lysosomes, and the Golgi apparatus. In the Golgi stacks, about half of the cisternae showed gold marker particles: accessible fucosyl residues were sparse in the cis subcompartment, the cismost cisterna mostly remaining negative; more intense label was found in medial cisternae; reactions were concentrated in the trans and transmost Golgi subcompartments. Cisternae, tubules and vesicles located at the trans Golgi side were the most constantly and intensely stained Golgi elements. In goblet cells, mucin granules and trans Golgi cisternae were labeled. Rarely, UEA I-gold bound to cisternae of the medial subcompartment; the cis subcompartment remained unstained. In part, UEA I-gold particles were restricted to dilated portions of the transmost Golgi cisterna and to secretory granules.     

Involvement of cis and trans Golgi apparatus elements in the intracellular sorting and targeting of acid hydrolases to lysosomes

To delineate the traffic route through the Golgi apparatus followed by newly synthesized lysosomal enzymes, we subfractionated the Golgi apparatus of rat liver by preparative free-flow electrophoresis into cisternae fractions of increasing content of trans face markers and decreasing contents of markers for the cis face. NADPase was used to mark median cisternae. Beta-Hexosaminidase, the high mannose oligosaccharide processing enzyme, alpha-mannosidase II, the two enzymes involved in the biosynthesis of the phosphomannosyl recognition marker, and the phosphomannosyl receptor itself decreased in specific activity or amount from cis to trans. Additionally, these activities were observed in a fraction consisting predominantly of cisternae, vesicles and tubules derived from trans-most Golgi apparatus elements. These results, along with preliminary pulse-labeling kinetic data for the phosphomannosyl receptor, suggest that lysosomal enzymes enter the Golgi apparatus at the cis face, are phosphorylated, and appear in trans face vesicles by a route whereby the phosphomannosyl receptor bypasses at least some median and/or trans Golgi apparatus cisternae.     

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.