Comparison of the ultrastructure of adrenaline and noradrenaline storage granules of bovine adrenal medulla

The ultrastructure of the membranes of noradrenaline (NA) and adrenaline (A) granules of the bovine adrenal medulla (Terland, O., T. Flatmark, and H. Kryvi, Biochim, Biophys. Acta 553, 460--468 (1979)) was analyzed by transmission, negative staining and freeze-etch electron microscopy. The two types of storage granules can be distinguished mainly by two morphological criteria: (a) The NA-granules have a more electron dense matrix core than the A-granules, (b) the NA-granules revealed less asymmetry in the distribution of intramembrane particles (nPF:nEF = 4,5:1) than the A-granules (nPF:nEF = 9:1). Thus, the trilaminar structure, negative staining pattern and size distribution of the intramembrane particles of the two fracture faces on freeze-etch electron microscopy were very similar for the two types of granules. Freeze-etching revealed a wide range of the particle size distribution for both fracture faces in both types of granules, with an average diameter of 12.6 +/- 2.7 nm (A-granules) and 10.2 +/- 2.8 nm (NA-granules) for the E-fracture faces and 11.4 +/- 2.7 nm (A-granules) and 9.8 +/- 2.4 nm (NA-granules) for the P-fracture faces. Some of the particles on the P-fracture face (outer surface of the membrane) revealed a subunit structure, most clearly seen in the specimens of NA-granules. Morhpometric analyses of sectioned bovine adrenal medulla revealed that the chromaffin granules on an average account for approx. 13.5% of the cytoplasmic volume in the total population of chromaffin cells.     

Immunocytochemical Study of Microtubules in Chromaffin Cells in Culture and Evidence That Tubulin Is Not an Integral Protein of the Chromaffin Granule Membrane

Abstract Bovine adrenal chromaffin cells were maintained in culture in Dulbecco's modified Eagle's medium containing 20% foetal calf serum and 10 units per ml of Nerve Growth Factor. Under these conditions, chromaffin cells developed up to five neurites per cell. The neurites showed lateral branches and varicosities along their trunk which ended with thick growth cone-like structures. Cultures of chromaffin cells were stained by indirect immunofluorescence with antibodies against (a) chromogranin A to follow the distribution of chromaffin granules, the catecholamine-storing organelles, and (b) tubulin, to study the microtubular system during outgrowth of neurites. Chromogranin A antibodies showed a very intensely staining punctate pattern, not randomly distributed but localized in neurites. Chromaffin granules were found to migrate from the cell body to reach neurite endings where they were densely packed. Intense staining was also observed in varicosities; a linear arrangement of granules was evident along neurite trunks. Tubulin antibodies decorated a complex network, clearly visible at the cell periphery and also in the growth cone-like structures, in the palm region of the growth cone. Colchicine treatment effected retraction of neurites and disappearance of organized microtubule networks; chromaffin granules were found in the perinuclear region of the cell. Some tubulin (0.2% of total membrane proteins) was found in the purified chromaffin granule membrane preparation; however, this tubulin is probably associated with contaminating plasma membranes. By the criteria of morphology and staining with antitubulin antibodies, adult bovine chromaffin cells in culture display characteristics similar to those of sympathetic neurones. In addition, they showed an exaggerated transport of granules. Adult bovine chromaffin cells in culture offer an excellent model for studying the role of microtubules and the contractile apparatus in relation to cell morphological changes and neurosecretion.     

Surface structure of Uukuniemi virus.

Uukuniemi virus, grown in chicken embryo fibroblasts, has been studied by electron microscopy using negative staining, thin sectioning, and freeze-etching techniques. The spherical virus particle measures about 95 nm in diameter. Its envelope consists of a 5-nm thick membrane covered by 8- to 10-nm long surface projections. These are composed of two polypeptides species of about the same size. Both of them can be removed by digestion with the proteolytic enzyme thermolysin except for a small fragment. The enzyme-treated particles are smooth surfaced and extremely deformable. The glycopolypeptides are clustered to form hollow cylindrical morphological units, 10 to 12 nm in diameter, with a 5-nm central cavity. Both negative staining and freeze-etching suggest that these units are penton-hexon clusters arranged in a T = 12, P = 3, icosahedral surface lattice. The membrane to which the surface subunits are attached is probably a lipid bilayer as evidenced by its double-track appearance in thin sections and the tendency of the freeze fracturing to occur within it. The strand-like nucleoprotein appears from thin-sectioning results to be to a large part located in a zone underneath the membrane.     

Chromaffin granule H(+)-ATPase has F1-like structure.

Isolated H(+)-ATPase from chromaffin granules was reconstituted into liposomes and the resultant proteoliposomes were further purified by Ficoll density gradient centrifugation. Studies by electron microscopy showed that proteoliposomes had particle structures (average diameter, about 10 nm) on their outer surface. These particles could be removed from the proteoliposomes by cold treatment. Immuno-electron microscopy showed that these particles were recognized by antibodies against the hydrophilic sector of the enzyme. These results indicate that the H(+)-ATPase has a peripheral membrane structure similar to that of F1-ATPase.     

Electron-Microscopic Demonstration of a “Head and Stalk” Structure of the Leaf Vacuolar ATPase in Mesembryanthemum crystallinum L.

The structure of the vacuolar ATPase from mesophyll tonoplasts of Mesembryanthemum crystallinum has been studied by electron microscopy using negatively stained specimens of membrane-bound and detergent-solubilized ATPase molecules. We observed a high density of particles on the surface of tonoplast vesicles and “head and stalk” structures on the edge of the membrane, similar to the F₀F₁-ATPases of mitochondrial and chloroplast membranes. The staining conditions, which are often critical for such small objects, were improved by using methylamine tungstate as negative stain for the membrane-bound ATPase. Compared to other staining solutions generally applied, dissociation of the F₁-like enzyme complex from the membrane was best prevented and structural damage of the vesicles was least observed with methylamine tungstate. In freeze-fracture electron microscopy of tonoplast vesicles, where dissociation never occurs since no detergent is used, we also observed “head and stalk” structures on the edge of the membranes, beside many particles on the fracture faces. The detergent-solubilized ATPase forms string-like structures, caused by the aggregation of the hydrophobic membrane-embedded F₀-like part of the enzyme. After negative staining the F₁-like enzyme complex is arranged alternately along both sides of the string and connected by a narrow stalk.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.     

Release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells

Summary The release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells was studied by electron microscopy. The treatment of the cells with SEB at the concentration of 20 μg/ml caused marked increase of the chromaffin granules that either bound to the plasma membrane by the characteristic rods, measuring 15 to 20 nm in length and showing a tubular structure, or budded off at the free cell surface, surrounded by a layer of rod-containing cytoplasm and enclosed by the plasma membrane. The binding between the granular and plasma membranes by the rods did not lead to membrane fusion and exocytosis of the granular content. Many of the bound granules showed vesiculation with loss of the electron-dense core material; at the same time, some of the binding rods contained intraluminal electron-dense material similar to the granular core material. These findings suggested that the electron-dense material (i.e., norepinephrine) of the bound granules was released extracellularly through channels within the rods. Although the granules were bound to the plasma membrane with equal frequency at the free and contiguous cell surfaces, the granular budding occurred only at the free cell surface, indicating that it occurred incidentally to some granules bound at the free cell surfaces. On the basis of the morphological observations, it is postulated that the electron-dense material of the bound granule is selectively released extracellularly through the rods, leaving the vesiculated granules behind in the cytoplasm. The same mode of release of the granular content was observed, though less frequently, in the untreated control cells. No morphological evidence that indicated that the granular content was released extracellularly by exocytosis was found in the treated and control cells. The present observations indicated that the SEB treatment of PC12 cells stimulated the binding of chromaffin granules to the plasma membrane by the rods and the budding of the bound granules at the free cell surface.     

Membrane recycling after exocytosis: An ultrastructural study of cultured chromaffin cells

When exocytosis of granule contents is induced by nicotine stimulation, glycoprotein III (a chromaffin granule membrane constituent) is exposed on the surface of cultured chromaffin cells, where it may be labeled with an immunocytochemical tracer. The subsequent fate of this glycoprotein after endocytosis was followed at the ultrastructural level using immunogold methods and was analyzed by morphometry. After stimulation exocytosis membranes newly inserted into the plasma membrane labeled with gold particles for glycoprotein III were found to be endocytosed via coated vesicles and finally found in organelles devoid of chromogranin A, the major secretory granule protein. At intervals between 30 min and 24 h after cell stimulation and immunolabeling, most labeled structures were identified by two different morphological approaches as prelysosomes and lysosomes. In contrast with results obtained on freshly isolated chromaffin cells, it is thus concluded that in cultured cells granule membrane recycling into new granules does not occur. It is suggested that the fate of granule membrane endocytosed after cell stimulation may be influenced by the external conditions to which cells are previously exposed.     

Ultrastructural and cytochemical characterization of adrenal medullary plasma membrane vesicles and their interaction with chromaffin granules

Plasma membrane vesicles obtained by density gradient centrifugation of bovine adrenal medullary homogenates were analyzed by electron microscopic methods, including negative staining, ultrathin sections and freeze-fracture replicas. Rapid freezing showed the intramembrane structure of plasma membrane vesicles to be distinct from that of other organelle membranes, such as chromaffin granules. Cytochemical demonstration of acetylcholinesterase (EC 3.1.1.7) activity on most membrane profiles confirmed that plasma membrane vesicles are derived predominantly from plasma membranes. About half of the plasma membrane vesicles were smaller than 0.15 micron and almost none larger than 0.55 micron. Practically all were composed of single shells. Most vesicles were impermeable to cytochemical markers of the size of Ruthenium red (Mr 800) and none were permeable to markers larger than 40 kDa. Surface charge probes, concanavalin A binding and endogenous actin decoration with heavy meromyosin indicated that the major fraction of plasma membrane vesicles is oriented right-side-out. A minor population with opposite orientation could also be detected. Isotonic ionic media caused vesicle aggregation in suspensions of plasma membrane vesicles and chromaffin granules. Freeze-fracturing always revealed clusters of membrane-intercalated particles at the sites of contact between aggregated membranes.     

Structure of Escherichia coli After Freeze-Etching

Survival of Escherichia coli, quick-frozen under conditions similar to those employed for freeze-etching, is close to 100%. For determination of cell shrinkage, the diameters of freeze-etched E. coli cells (average, 0.99 mum) were compared with those of preparations after negative staining and after ultrathin sectioning. Negatively stained cells measured from 0.65 to 1.0 mum in diameter, and ultrathin sections showed average cell diameters of 0.70 mum. Freeze-etched replicas of logarithmically growing, as well as stationary, E. coli B cells revealed a smooth, finely pitted cell surface in contrast to cell surfaces seen with other preparative methods. The frozen cell wall may cleave in two planes, exposing (i) a smooth fracture face within the lipid layer and (ii) in rare instances an ill-defined particulate layer. Most frequently, however, cleavage of the envelope occurred between wall and protoplasmic membrane; large areas of the membrane were then exposed and showed a surface studded with predominantly spherical particles, an appearance which did not significantly change when the cells were fixed in formaldehyde and osmium tetroxide before freeze-etching. The distribution of these particles differed between logarithmically growing cells and stationary cells.     

Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling

The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.     

Membranes of chromaffin granules. Isolation and partial characterization of two proteins

Membranes of chromaffin granules were isolated from the adrenal glands of four different species. The solubilized membrane proteins could be resolved into several bands by polyacrylamide-gel electrophoresis (alkaline and acid gel systems). Two major protein components appeared to be common to the chromaffin granule membranes of ox, horse, pig and man. The various membrane proteins of bovine chromaffin granules were separated by filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. Two major membrane proteins (A and B) were obtained in purified form. Treatment of protein A with 2-mercaptoethanol before electrophoresis resulted in two more rapidly migrating subunits, whereas protein B was unaffected by mercaptoethanol treatment. The amino acid compositions of the two purified proteins were determined. They are very similar to that of the total membrane proteins but significantly different from that of the chromogranins, the soluble proteins of chromaffin granules.     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

ULTRASTRUCTURE AND CALCIUM TRANSPORT IN CRUSTACEAN MUSCLE MICROSOMES

Fragmented sarcoplasmic reticulum (FSR) from crustacean muscle was examined following preparation by a variety of electron microscopic techniques. The 30–40 A particles which appeared on the outer surface of FSR vesicles following negative staining were not observed following preparation by freeze-drying, freeze-etching, thin sectioning, or critical-point drying. Crustacean FSR exhibited high values of calcium uptake and extensive nodular formation in the presence of oxalate. 80–90 A diameter membrane particles were seen in freeze-etch preparations of both intact lobster muscle and FSR vesicles. Thin sections of FSR vesicles revealed a membrane thickness of 60–70 A. The membrane appeared to be triple layered, each layer having a thickness of 20–25 A.     

The distribution of Concanavalin A receptor sites on the membrane of chromaffin granules.

The distribution of concanavalin A (con A) receptor sites on the membranes of chromaffin granules has been investigated by binding studies using 125I-labelled con A and by electron-microscope studies using ferritin-labelled con A. In both experiments con A was observed to bind to chromaffin granule membranes but not to intact granules. The ferritin-con A particles bind to only one of the two possible surfaces of the chromaffin granule membranes. These results are in agreement with previous observations concerning the asymmetric distribution of saccharide residues on the surfaces of a number of different plasma membranes. They suggest that for the intracellular membrane of the chromaffin granule the saccharide sites, like those in plasma membranes, are not exposed to the cell cytoplasm. Further work is necessary to establish whether these sites are on the inner surface of the membrane or whether they are unmasked during the conversion of granules to membrane ghosts.     

Periplasmic Structure of Frozen-Etched and Negatively Stained Cells of Bacillus licheniformis as Correlated with Penicillinase Formation

Bacillus licheniformis strain 749/C (constitutive for penicillinase formation) and uninduced cells of strain 749 (penicillinase-inducible) were examined after freezeetching. In the early stationary phase, strain 749/C organisms had clusters of vesicles (30 to 40 nm in diameter) on the outer surface of the plasma membrane. These are randomly distributed on the membrane, including the region of septum formation. The vesicles are not intimately associated with the plasma membrane, and their inner and outer surfaces are devoid of particles. Periplasmic vesicles were not detected by freeze-etching in strain 749 (uninduced) or in young cells of 749/C; however, the membrane of mid-logarithmic phase 749/C cells had a corrugated appearance. Negatively stained 749/C cells (logarithmic phase) also showed many vesicular and tubular bodies in the periplasm as well as septal and cytoplasmic mesosomes of typical morphology. The periplasmic structures appear to be formed either by evagination of plasma membrane or by migration of vesicular bodies from the membranous pockets of the cytoplasm. Stationary phase cells of 749/C still have many periplasmic vesicular bodies; however, the mesosomes are greatly reduced both in number and size. In sharp contrast, strain 749 organisms have very few structures similar to the periplasmic bodies of strain 749/C. These findings support our previous view that penicillinase-producing cells of 749/C have periplasmic membranous structures that are rare in the uninduced strain 749, though there is some lack of correspondence between freeze-etching, negative staining, and thin section data. These structures may be important for the retention or storage of penicillinase in the cell.     

Characterization and visualization of tetanus toxin acceptors on adrenal chromaffin granules

Tetanus toxin (TT), a potent neurotoxin which blocks neurotransmitter release in neuronal systems, also inhibits Ca2(+)-induced catecholamine release from digitonin-permeabilized chromaffin cells. In searching for intracellular targets for the toxin we studied the binding of affinity-purified TT to bovine adrenal chromaffin granules. TT bound in a neuraminidase-sensitive fashion to intact granules and to isolated granule membranes, as assayed biochemically and visualized by electron microscopic techniques. The binding characteristics of the toxin to chromaffin granule membranes are very similar to the binding of TT to brain synaptosomal membranes. We suggest that the TT binding site is a glycoconjugate of the G1b type which is localized on the cytoplasmic face of the granule membrane and might be involved in exocytotic membrane fusion.     

Fine Structures of Cell Envelopes of Chlamydia Organisms as Revealed by Freeze-Etching and Negative Staining Techniques

The cell walls of Chlamydia psittaci (meningopneumonitis strain) were examined by the freeze-etching and negative staining techniques. It was observed that the cleaved convex surface of the developmental, reticulate body was covered with numerous non-etchable particles 9 to 10 nm in diameter, these particles being rarely seen on the concave surface. Similarly, the convex surface of the mature, elementary body (EB) was covered with many particles but the concavity lacked these particles. After etching, the smooth concave surface of the EB appeared to have a hexagonally arrayed subunit structure, on which the button structure (B structure) was observed. Each B structure had a diameter of 27 nm and several B structures were grouped together in a hexagonal arrangement with a center-to-center spacing of 45 nm. In a limited area of the negatively stained EB cell wall, hexagonally arrayed rosette structures were present, with a center-to-center spacing similar to the B structures seen in the freeze-etched preparation. Each rosette, about 19 to 20 nm in diameter, appeared to be composed of a radial arrangement of nine subunits. The freeze-fractured cell wall-cytoplasmic membrane complexes indicated that the outer surface of the cytoplasmic membrane which appeared as the convex surface was covered with the fine particles, and thus it was likely that frozen EB was cleaved at the gap between the cell wall and ctyoplasmic membrane. On the cleaved inclusion, several groups of fine particles were observed. In each group, the particles were arranged hexagonally with the spacing ranging from 20 to 50 nm.     

The heterogeneity of ion channels in chromaffin granule membranes

Chromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic ⁸⁶Rb⁺ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.