Relationships between membranous organelles in amoebae studied by electron microscopic cytochemical staining

Summary The intracellular location of a variety of enzymes was studied in Amoeba proteus with the use of electron microscopic cytochemical methods, in an attempt to assess the relationships between different membranous organelles. One group of enzymes, including nucleoside diphosphatases (IDPase, UDPase, GDPase, ADPase), carbamoyl phosphatase, alkaline phosphatase, and BAXD oxidase was localized mainly in the rough endoplasmic reticulum, nuclear envelope, and convex side of the Golgi apparatus. Esterase activity had a similar localization except that the Golgi apparatus was "stained" throughout most of its extent. A second group of enzymes was found in Golgi cisternae and vesicles, and in some vacuoles. This group included acid phosphatase, thiamine pyrophosphatase, and aryl sulfatase. Some enzymes previously detected in cytoplasmic membranes of other cells, including glucose-6-phosphatase, showed little or no activity in amoebae. The results suggest that there are chemical similarities and probable functional relationships between the rough endoplasmic reticulum, the nuclear envelope, and the convex side of the Golgi apparatus. On the other hand, the concave pole of the Golgi apparatus, aggregates of smooth tubules and vesicles, and the cell surface appear more closely related to one another than to the endoplasmic reticulum and the convex side of the Golgi apparatus. The cytochemical similarity between the Golgi apparatus and certain vacuoles such as food vacuoles may reflect the role of the Golgi apparatus in the formation of lysosomes. The locations of reaction products of the various enzymes in amoebae are compared with observations reported for other cell types.Supported by a research grant (VC-169) from the American Cancer SocietyThe author is indebted for technical assistance to Mrs. Sue Thompson and Mrs. Christine Folsom-Kovarik     

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.     

An ultrastructural study of phosphatases and aryl sulphatase in BHK and CHO cells grown in culture

Summary The ultrastructural distribution of a number of phosphatases and aryl sulphatase has been studied in BHK 21/C 13, BHK21/J 1 and CHO cells grown in culture. In all three cell lines acid -glycerophosphatase and aryl sulphatase appear to be confined to lysosomes and elements of the Golgi apparatus and glucose-6-phosphatase to the endoplasmic reticulum. With thiamine pyrophosphate at pH 7.0 in CHO cells reaction product is present in lysosomes, the Golgi apparatus, the endoplasmic reticulum and on the cell surface. Preincubation at acid pH reduces the reactions in the endoplasmic reticulum but enhances the surface activity. At pH 5.0 and pH 7.0 in CHO cells p-nitrophenylphosphatase is present in lysosomes, the Golgi apparatus and the endoplasmic reticulum and this activity is inhibited by sodium fluoride. p-nitrophenylphosphatase activity is also present on the cell surface of CHO cells and this activity is not inhibited by sodium fluoride. No activity could be demonstrated in any cells at pH 9.O. The significance of these results is discussed with respect to the possible role of surface acid phosphatase in the process of transformation.     

Appearance of acid phosphatase activity in the developing anterior pituitary of the rat

The ultrastructure of the anterior pituitary gland in developing rats was investigated according to Gomori's method for acid phosphatase. During the earlier period of development (day 14 to 16 of gestation), enzyme activity could not be found, although nonspecific deposits of lead were observed within the nuclear envelope, ER, and Golgi cisternae. This facilitated observation of the topographical relationship of the intracellular membrane system and suggestive evidence was obtained that the nuclear envelope in the pituitary anlage is involved in formation of the Golgi apparatus.During days 17 and 18 of gestation, when granule formation begins, little acid phosphatase activity was detectable in the Golgi apparatus and in the secretory granules. A polarized distribution of acid phosphatase was first detected in the Golgi apparatus on day 20 of gestation, with a concomitant increase of lysosomes.From these findings it seems that acid phosphatase begins to contribute to the secretory process a few days after granule formation has started.     

Affinity cytochemical differentiation of glycoconjugates of small intestinal absorptive cells using Pisum sativum and Lens culinaris lectins

We studied the subcellular localization of glycoconjugates recognized by the garden pea and lentil lectins (Pisum sativum, PSA; Lens culinaris, LCA) in mature absorptive cells of duodenum and jejunum of fasted rats. PSA and LCA are mannose-, glucose-, and N-acetyl-glucosamine-recognizing lectins that bind with high affinity to fucosylated core regions of N-glycosidically linked glycans. The binding reactions were cytochemically demonstrated in a pre-embedment incubation system using peroxidase-labeled lectins. Both pea and lentil lectins bound with constituents of nuclear envelope and endoplasmic reticulum, cisternae of the Golgi apparatus, several Golgi-associated vesicles, lysosomes, and portions of the plasma membrane. PSA and LCA label was non-homogeneous in the endoplasmic reticulum; in the Golgi apparatus the reactions were most intense in the cis and medial cisternae of the stacks. For inhibition of the intense reactions apparent in the Golgi apparatus, in lysosomes, and at the plasma membrane, considerably higher concentrations of competitive sugars were necessary than for abolition of the endoplasmic reticulum label. This indicates that endoplasmic reticulum glycoconjugates bind at low affinities with pea and lentil lectins, and that high-affinity PSA/LCA-binding glycoconjugates, which may correspond to corefucosylated N-linked glycans, predominate in cis and medial Golgi cisternae, lysosomes, and at the plasma membrane.     

The fine localization of acid phosphatase activity in the unvacuolated notochordal cells of the early chick embryo

Summary The electron microscopical localization of acid phosphatase activity was investigated in ultra-thin and semi-thin sections of unvacuolated notochordal cells of chick embryos from stages 9 to 14 (as defined by Hamburger & Hamilton). At stage 9, many notochordal cells show a lightly positive reaction for acid phosphatase activity. Thereafter, the acid phosphatase-positive cells of the notochord increase in number and, at stage 14, the reaction products for the enzyme are distributed throughout almost all the cisternae of the nuclear envelope and a well-differentiated endoplasmic reticulum, the parallel cisternal and reticular parts of the Golgi complex, and various lysosomes in nearly all notochordal cells. In the cisternae of the nuclear envelope and endoplasmic reticulum, the acid phosphatase reaction products are in a fine granular form. In the outermost layer of the cisternal parts of the Golgi complex, faint lead deposits similar to those in the endoplasmic reticulum are found, but in other cisternal and reticular regions which may correspond to the GERL, considerable amounts of reaction products are present. Knob-like projections are also seen protruding from the reticular parts of the Golgi complex. These results suggest that, at least up to stage 14, the notochordal cells are actively synthesizing acid phosphatase which is directly transported from the endoplasmic reticulum to the Golgi complex. The enzyme may be accumulated by the Golgi complex from which primary lysosomes are formed. Furthermore, the pattern of the ultrastructural localization of acid phosphatase activity in embryonic notochordal cells of the chick differs from that of adult cells of other animals.     

Cytochemical characterization of the endomembranous system during oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The endomembranous system of Serrasalmus spilopleura oocyte secondary growth was analysed using structural and ultrastructural cytochemical techniques. In vitellogenic oocytes, the endoplasmic reticulum components, the nuclear envelope intermembranous space, some Golgi dictiossomes, lysosomes, yolk granules, regions of the egg envelope and sites of the follicle cells react to acid phosphatase detection (AcPase). The cortical alveoli, some heterogeneous cytoplasmic structures, regions of the egg envelope, and sites of the follicle cells are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). The endoplasmic reticulum components, some vesicles, and sites of the follicle cells also react to osmium tetroxide and potassium iodide impregnation (KI). The biosynthetic pathway of lysosomal proteins, such as acid phosphatase, required for vitellogenesis, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes, and, finally, lysosomes. In S. spilopleura oocytes at secondary growth, the endomembranous system takes part in the production of the enzymes needed for vitellogenesis, and in the metabolism of yolk exogenous components (AcPase detection). The endomembranous system compartments also show reduction capacity (KI reaction) and are involved in the metabolism of proteins rich in SH‐groups (ZIO reaction).     

Subcellular fractionation studies on the post-translational processing of pro-adrenocorticotropic hormone/endorphin in rat intermediate pituitary

The subcellular localization of the post-translational processing steps which occur in the conversion of pro-adrenocorticotropic hormone (ACTH)/endorphin into beta-endorphin-sized molecules in rat intermediate pituitary has been studied. Primary cell cultures were incubated in radioactively labeled amino acids, and a subcellular fraction containing secretory granules was separated from a subcellular fraction containing rough endoplasmic reticulum and Golgi apparatus by centrifugation of homogenates on gradients on Percoll (Pharmacia Fine Chemicals). The radiolabeled beta-endorphin-related material in the granule and rough endoplasmic reticulum/Golgi apparatus fractions was quantitated by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis. A pulse-chase labeling experiment demonstrated that newly synthesized beta-endorphin-related material first appeared in the rough endoplasmic reticulum/Golgi apparatus fraction and after longer incubations (chase) appeared in the secretory granule fraction. After 2 h of chase incubation, about 85% of the beta-endorphin-related material synthesized during the 30-min pulse incubation had been transferred from the rough endoplasmic reticulum/Golgi apparatus to the secretory granule fraction. The conversion of most of the newly synthesized pro-ACTH/endorphin into beta-lipotropin occurred in the rough endoplasmic reticulum/Golgi apparatus fraction, whereas the conversion of most of the beta-lipotropin into beta-endorphin-sized molecules occurred in the secretory granule fraction.     

The demonstration of acid phosphatase in cultured 3T3 mouse cells.

Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium beta-glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.     

Phosphatase cytochemistry with cerium as trapping agent. Verification of acid phosphatase and glucose-6-phosphatase reactive sites

Lead is prevalently replaced by cerium as trapping agent in phosphatase cytochemistry to prevent non-specific precipitation. Recently, substrate specific but artefactual lead precipitates have been described in the nuclear envelope (NE) and rough endoplasmic reticulum (RER) due to a local matrix effect. In the present study a verification was carried out of the localization of acid phosphatase and glucose-6-phosphatase in the NE and RER of rat peritoneal macrophages and hepatocytes respectively with cerium. It appeared that precipitates of cerium phosphate in NE and RER of peritoneal macrophages do not represent sites of acid phosphatase activity but are due to the matrix effect. However, in rat hepatocytes these organelles demonstrate true reactive sites for glucose-6-phosphatase.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Summary Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum and nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

The enzyme cytochemistry of the intracellular organelles in the rat choroid plexus epithelial cell

Electron microscopic cytochemical studies on the rat choroid plexus epithelium have revealed enzymatic sites for the activities of acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase on different organelles. Only the activity of acid phosphatase has been previously described. Acid phosphatase, glucose-6-phosphatase and thiamine pyrophosphatase were respectively situated mainly in the lysosomes, in the endoplasmic reticulum an nuclear envelope, and in the Golgi complex. These three enzymes can thus be considered as marker enzymes for their respective organelles in the choroid plexus epithelial cells as well as in other tissue cells. The possible function of these enzymes in the choroid plexus epithelial cells is also briefly discussed.     

The relationships between the Golgi apparatus, GERL, and lysosomes of fetal rat liver Kupffer cells examined by ultrastructural phosphatase cytochemistry

Kupffer cells are the sinusoidal macrophages of the liver. Using ultrastructural phosphatase cytochemical methods, we examined the relationship between the Golgi apparatus, GERL, and lysosomes of Kupffer cells in fetal rat livers identified, in part, by their ability to phagocytize intravenously injected latex spheres. Thiamine pyrophosphatase (TPPase) activity was localized to the inner Golgi saccules and some vesicles in the Golgi region but not to GERL. A TPPase-like activity, demonstrable in lysosomes, was abolished by sodium fluoride but not suppressed by the alkaline phosphatase inhibitors L-cysteine and L-p-bromotetramisole. Acid phosphatase (AcPase) was localized by GERL, some coated vesicles, and in lysosomes, but not to the Golgi stacks. Continuities between GERL and lysosomes were observed. Phagosomes containing internalized latex spheres received TPPase and AcPase sequentially. TPPase was localized in phagosomes immediately after latex administration. AcPase activity was not found here until at least 10 minutes following the injection of the particulates. Our findings indicate that Kupffer cell lysosomes are derived from GERL, but also suggest that phagosomes may receive material packaged by the Golgi apparatus as well as GERL.     

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

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

Fine structural and cytochemical studies on the hamster subcommissural organ

The subcommissural organ (SCO) of the golden hamster (Mesocricetus auratus) was studied by conventional electron microscopy, freeze-fracture technique, zinc-iodide-osmium (ZIO) and acid phosphatase cytochemical reactions. The ultrastructure of hamster SCO cells shows a few flattened cisternae of rough endoplasmic reticulum (ER) without dilated ones in the cytoplasm. The Golgi apparatus is very well developed. Freeze-fracture studies also indicate only short profiles of flattened ER in the cytoplasm endorsing the absence of dilated ER cisternae. After the treatment with ZIO mixture, reaction products were observed over flattened cisternae of the ER and the nuclear envelope. The Golgi apparatus was also reactive toward the ZIO mixture. Acid phosphatase activities are localized in the inner one or two saccules of the Golgi apparatus and dense bodies. From these results we suggest that (1) hamster SCO cells do not accumulate secretory material in the cytoplasm in the form of discrete secretory granules or dilated cisternae of ER, and (2) hamster SCO cells may possess extremely high secretory activity or may not be actively involved in secretory function at all as in rats or other rodents.     

Intracellular labeling with ferritin conjugates. A specificity problem due to the affinity of unconjugated ferritin for selected intracellular sites.

Nonspecific binding of ferritin to chromatin and the cytoplasmic aspect of the nuclear envelope was observed when nonantigenic, serum-washed hepatocyte nuclei were incubated in ferritin-antibody conjugates. This labeling was duplicated when nuclei from a wide range of species and cell types were exposed to unconjugated ferritin. Unconjugated ferritin binding to nuclei did not depend on a subpopulation of denatured molecules or on the ferritin purification procedure. Binding occurred equally on unfixed and formaldehyde-fixed nuclei, but no ferritin bound to glutaraldehyde-fixed nuclei. Inconjugated ferritin also bound to the cytoplasmic aspects of the rough endoplasmic reticulum and the plasma membrane. The tracer did not bind to lysosomes, mitochondria, Golgi vesicles, the extracellular surface of plasma membranes, or the intracisternal surfaces of ruptured nuclear envelopes. The addition of 0.4 M KCl or 0.7 M NaCl to ferritin solutions and washing media at neutral pH reduced the binding of conjugated and unconjugated ferritin to nuclei to about 3% of that seen in 0.10 M phosphate buffer alone. The added salts caused little extraction of nuclear contents from formaldehyde-fixed nuclei. The use of one of these salts in ferritin conjugates should considerably improve the specificity of intracellular labeling.     

Submicroscopic study of adenohypophyseal cells with the hypophysis incubated in a hypertonic solution. I. The distribution of acid phosphatase, thiamine pyrophosphatase and arylsulfatase activities

In the rat adenohypophyseal mammotrophs and somatotrophs, activities of acid phosphatase, thiamine pyrophosphatase and aryl sulphatase were defined after the incubation of the pituitary gland in the medium containing 1.65 M sucrose. Following 60--120 minutes of the incubation the above enzymes were found, in addition to their usual sites, in all cisternae of the Golgi apparatus and the rough endoplasmic reticulum as well as within the perinuclear cisternae. The mechanism of the above alterations is discussed, and a suggestion is put forward that the emergence of enzymatic activities in the above structures may reflect their biogenetic pathways in the cell.     

The demonstration of acid phosphatase in cultured 3T3 mouse cells

Summary Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium -glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.     

LYSOSOMES AND GERL IN NORMAL AND CHROMATOLYTIC NEURONS OF THE RAT GANGLION NODOSUM

The rat ganglion nodosum was used to study chromatolysis following axon section. After fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to develop) (31–33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the mitochondrial enzyme were studied by electron microscopy as well as light microscopy. In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in the center of the cell and segregation of the remainder to the cell periphery. Golgi apparatus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. GERL is prominent in both normal and operated perikarya. Electron microscopic images suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from focal areas dilated with granular or membranous material; (c) "multivesicular bodies" in which vesicles and other material are sequestered; (d) autophagic vacuoles containing endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and mitochondria. The number of autophagic vacuoles increases following operation.     

Ultrastructural characteristics of insect corpora allata in relation to larval diapause

Summary The ultrastructure of active and inactive corpora allata from last instar larvae of the southwestern corn borer, Diatraea grandiosella, was examined. Active glands were obtained from pre-, early, and mid-diapausing larvae; inactive ones from late and non-diapausing larvae. Each gland contains 13 to 18 cells which have the following common features: well developed smooth endoplasmic reticulum, scattered rough endoplasmic reticulum and free ribosomes, microtubules, vacuolated nucleoli, and interlocking plasma membranes. The gland contains intercellular deposits, and is supplied by regular and neurosecretory axons.Special ultrastructural features of the corpus allatum from the five groups of larvae examined were as follows: pre-diapause: extensive vesicular smooth endoplasmic reticulum, numerous cup-shaped mitochondria and Golgi bodies with stacked cisterns and vesicles, few small lipid droplets, large nuclei with dispersed chromatin, absence of lysosomes; early diapause: stacked, whorled, and vesicular smooth endoplasmic reticulum of equal abundance, numerous rod-shaped mitochondria, some Golgi bodies but without distinct stacks of cisterns, few lipid droplets and lysosomes, chromatin dispersed and also attached to the nuclear envelope; mid-diapause: similar to early diapause except for the presence of more stacked, smooth endoplasmic reticulum, chromatin in large chunks mostly attached to the nuclear envelope; late diapause: whorled smooth endoplasmic reticulum and rod-shaped mitochondria predominating, complicated Golgi bodies with stacks of cisterns and large empty sacs, few large lipid droplets, some lysosomes containing mainly whorled bodies, chromatin in large chunks attached to the nuclear envelope; non-diapause: similar to late diapause except for less extensive smooth endoplasmic reticulum, more abundant mitochondria, fewer intercellular deposits. Although these observations suggest that the smooth endoplasmic reticulum, and possibly mitochondria, and Golgi bodies are involved in juvenile hormone production, specific sites of synthesis or storage of the hormone were not revealed.Supported in part by grant no. PCM 74-18155 A01 from the National Science Foundation. Contribution from the Missouri Agricultural Experiment Station as journal series no. 8234. We thank Ms. L. Yin for her skillful assistance, and Dr. M.F. Brown of the College of Agriculture Electron Microscope Facility for his advice and the use of equipment.