Secretory organelle biogenesis: Trichocyst formation in a ciliated protozoan

Summary— By classical electron microscopy and immunoelectron microscopy, the biogenesis of trichocyst secretory granules has been followed in the ciliated protozoan Pseudomicrothorax dubius. The very early pre-trichocysts form by fusion of bristle-coated, electron-dense vesicles (dense vesicles) with electron-translucent vesicles (clear vesicles), both of which originate in a well-developed trans-Golgi network (TGN). The pre-trichocyst grows by further fusion with dense and clear vesicles as well as with other pre-trichocysts until it reaches its maximum diameter of about 2 μm. Dense and clear vesicle formation from the TGN has been followed, and the fusion sequence of dense vesicles with the pre-trichocyst has been documented. The contents of the dense vesicles are the precursors of the trichocyst tip, which is composed of four arm-like rods, whereas the shaft precursors are supplied by the clear vesicles. The first evidence of trichocyst shaft formation is the appearance of a paracrystalline, dense core condensation center in the pre-trichocyst. Following shaft formation, the trichocyst tip forms by fusion and condensation of the dense arm precursors along each of the four sides of the shaft. Docking of the fully formed trichocyst in the cell cortex is described. Pre-trichocyst biogenesis in cells grown with and without Se is compared.     

Characterization of the bioluminescent organelles in Gonyaulax polyedra (dinoflagellates) after fast-freeze fixation and antiluciferase immunogold staining

To characterize the microsources of bioluminescent activity in the dinoflagellate Gonyaulax polyedra, an immunogold labeling method using a polyclonal antiluciferase was combined with fast-freeze fixation and freeze substitution. The quality of the preservation and the specificity of the labeling were greatly improved compared to earlier results with chemical fixation. Two organelles were specifically labeled: cytoplasmic dense bodies with a finely vermiculate texture, and mature trichocysts, labeled in the space between the shaft and the membrane. The available evidence indicates that the dense bodies are the light-emitting microsources observed in vivo. The dense bodies appear to originate in the Golgi area as cytoplasmic densifications and, while migrating peripherally, come into contact with the vacuolar membrane. Mature organelles protrude and hang like drops in the vacuolar space, linked by narrow necks to the cytoplasm. These structural relationships, not previously apparent with glutaraldehyde fixation, suggest how bioluminescent flashes can be elicited by a proton influx from a triggering action potential propagated along the vacuolar membrane. Similar dense bodies were labeled in the active particulate biochemical fraction (the scintillons), where they were completely membrane bound, as expected if their necks were broken and resealed during extraction. The significance of the trichocyst reactivity remains enigmatic. Both organelles were labeled with affinity-purified antibody, which makes it unlikely that the trichocyst labeling is due to a second antibody of different specificity. But trichocysts are not bioluminescent; the cross-reacting material could be luciferase present in this compartment for some other reason, or a different protein carrying similar antigenic epitopes.     

Light- and electron-microscopic observations of the spinal ganglion cells of mice

Under the light microscope, large and small types of spinal ganglion cells can be seen in the mouse. Among those of the small type bipolar cells are observed. By electron microscopy, the cells of the large type have a low electron density and those of the small type a high electron density. Bipolar cells continue to be observed among the latter. The cells of the spinal ganglia contain different cellular apparatus in enormous quantity. There are four kinds of lysosomes in the GERL, i.e. the coated vesicles, dense bodies, multivesicular bodies and autophagic vacuoles.     

Electron microscope observations on intracellular virus-like particles associated with the cells of the Lucké renal adenocarcinoma

The common renal adenocarcinoma of the leopard frog was studied in thin sections with the electron microscope. Approximately a third of the tumors examined were found to contain spheroidal bodies of uniform size and distinctive morphology that are believed to be virus particles. These consist of hollow spheres (90 to 100 mmicro) having a thick capsule and a dense inner body (35 to 40 mmicro) that is eccentrically placed within the central cavity (70 to 80 mmicro). Virus particles of this kind occur principally in the cytoplasm but occasionally they are also found in the nucleus and in the extracellular spaces of the tumor. The intranuclear inclusion bodies that are visible with the light microscope are largely comprised of hollow, spherical vesicles with thin limiting membranes. These are embedded in a finely granular matrix. A few of the thin walled vesicles contain a dense inner body like that of the cytoplasmic virus particles. This suggests that they may be immature virus particles. The inclusion bodies are believed to be formed in the course of virus multiplication but they usually contain very few mature virus particles. Bundles of dense filaments and peculiar vacuolar inclusions also occur in the cytoplasm of the tumor cells. These seem to be related in some way to the presence of virus but their origin and significance remain obscure. These findings are discussed in relation to previous work suggesting that the Lucké adenocarcinoma is caused by an organ-specific filtrable agent. It is concluded that the "virus particles" found in electron micrographs of the tumor cells may be the postulated tumor agent. On the other hand, the possibility remains that the particles described here are not those that are causally related to the tumors.     

ELECTRON MICROSCOPE OBSERVATIONS ON INTRACELLULAR VIRUS-LIKE PARTICLES ASSOCIATED WITH THE CELLS OF THE LUCKÉ RENAL ADENOCARCINOMA

The common renal adenocarcinoma of the leopard frog was studied in thin sections with the electron microscope. Approximately a third of the tumors examined were found to contain spheroidal bodies of uniform size and distinctive morphology that are believed to be virus particles. These consist of hollow spheres (90 to 100 mµ) having a thick capsule and a dense inner body (35 to 40 mµ) that is eccentrically placed within the central cavity (70 to 80 mµ). Virus particles of this kind occur principally in the cytoplasm but occasionally they are also found in the nucleus and in the extracellular spaces of the tumor. The intranuclear inclusion bodies that are visible with the light microscope are largely comprised of hollow, spherical vesicles with thin limiting membranes. These are embedded in a finely granular matrix. A few of the thin walled vesicles contain a dense inner body like that of the cytoplasmic virus particles. This suggests that they may be immature virus particles. The inclusion bodies are believed to be formed in the course of virus multiplication but they usually contain very few mature virus particles. Bundles of dense filaments and peculiar vacuolar inclusions also occur in the cytoplasm of the tumor cells. These seem to be related in some way to the presence of virus but their origin and significance remain obscure. These findings are discussed in relation to previous work suggesting that the Lucké adenocarcinoma is caused by an organ-specific filtrable agent. It is concluded that the "virus particles" found in electron micrographs of the tumor cells may be the postulated tumor agent. On the other hand, the possibility remains that the particles described here are not those that are causally related to the tumors.     

The Trichocysts of Chilomonas paramecium*

SYNOPSIS Axenic cultures of Chilomonas paramecium were grown in media lacking a C-source, resulting in breakdown in autophagosomal vesicles of large numbers of trichocysts. Return of the starved organisms to complete media was followed by a wave of trichocyst formation. Stages in the degeneration and subsequent reformation of trichocysts are described as well as attempted labeling of the developing organelles with ³H-thymidine. A modification of the method of Anderson et al. (2) was used for isolating quantities of exploded trichocysts from Chilomonas. Attempts at isolation of the trichocyst in its coiled state were unsuccessful. Isolated trichocysts mounted on electron microscope grids were subjected to various types of enzymatic digestions.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Membrane behavior of exocytic vesicles. II. Fate of the trichocyst membranes in Paramecium after induced trichocyst discharge.

A specific exocytic process, the discharge of spindle trichocyts of Paramecium caudatum, was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,000) of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for the length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. The entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for Tetrahymena.     

Fine structure of synapses in the hippocampus of the rabbit with special reference to dark presynaptic endings

Summary The stratum radiatum of h ₃ and h ₄ in the hippocampus of the rahbit, where the mossy fiber endings are distributed, was investigated under the electron microscope. These regions contain a certain number of electron dense presynaptic endings. These are characterized by highly dense synaptic vesicles and mitochondrial matrices. The dense endings are not considered as degenerated. Electron dense silver particles, substituted for zinc, occurred on the synaptic vesicles of these dense terminals as well as the mossy fiber endings after the application of Timm's histochemical method modified for electron microscopy. It is concluded that the dark synaptic endings observed might represent mossy fiber terminals in a special functional phase, or might be the result of structural alteration in the course of tissue preparation. The zinc localized in the synaptic vesicles is thought to be associated with the neurotransmitter present in these endings.     

The fine structure and ontogeny of trichocysts in marine dinoflagellates

Long, rigid, rod-like structures found in the culture medium of several marine dinoflagellates are shown in this report to have fine transverse bandings characteristic of extruded trichocysts. These structures in genera such asGonyaulax are believed to pass through the heavily plated surface via narrow pores. In the resting or charged form, trichocysts are found to have an elaborate crystalline core connected by a series of fibers and still finer fibrils to the apex of an enclosing sac. The walls of this sac consist of a single membrane and fine thread-like hoops or spirals. The design of the whole charged trichocyst is suggestive of a mechanical sensing device. Trichocysts are found to originate in membrane-limited vesicles which are localized within a spherical shell composed of Golgi bodies. Initially these vesicles contain homogeneous materials, but with increasing development a crystal lattice appears and ultimately the resting trichocyst core evolves. At this point the trichocyst leaves the Golgi area and migrates elsewhere in the cytoplasm. The charged trichocyst core is found to be waterbut not acetone-soluble in contrast to the discharged trichocyst which is unaffected by either solvent. These facts together with the finding of shafts apparently polymerizing from amorphous contents are interpreted as supporting the hydration theory of trichocyst discharge. Finally, the striking similarities between the origin and structure of extruded trichocyst shafts and the origin and structure of collagen fibers are discussed briefly.     

The variety of synaptic vesicles in the interpeduncular nucleus (ITP) of the frog.

The electron microscope has revealed a large variety of synaptic vesicles in the interpeduncular nucleus (ITP) of the frog "Rana esculenta". They vary in shape, size and electron density. There are two types of synapses which show only translucent spherical vesicles: in one type the vesicles are 40 nm, in the other type they are 70 nm in diameter. In other types of synapses the translucent vesicles may be mixed with those with dense core. Large granules, 160 nm in diameter, already reported in the ITP (KEMALI 1977a), are also shown as well as tiny flat mixed with large flat dense core vesicles of dumb-bell shape. Two types of axo-axonic synapses are illustrated while no crest synapses have been demonstrated. The results suggest that the afferents to the ITP might be more numerous than those reported in the literature or that--as in the case of the habenular afferents which consist of cholinergic and peptergic fibres--each projecting nucleus to the ITP has different types of fibres with more than one type of transmitter. Furthermore, due to the vesicles sizes, we may consider the ITP as a site in the vertebrate central nervous system where conventional neurotransmitter structures coexist with probable neurohumoral elements.     

Cytoskeleton-secretory vesicle interactions during the docking of secretory vesicles at the cell membrane in Paramecium tetraurelia cells

Stationary-phase cells of Paramecium tetraurelia have most of their many secretory vesicles ("trichocysts") attached to the cell surface. Log-phase cells contain numerous unoccupied potential docking sites for trichocysts and many free trichocysts in the cytoplasm. To study the possible involvement of cytoskeletal elements, notably of microtubules, in the process of positioning of trichocysts at the cell surface, we took advantage of these stages. Cells were stained with tannic acid and subsequently analyzed by electron microscopy. Semithin sections allowed the determination of structural connections over a range of up to 10 micrometer. Microtubules emanating from ciliary basal bodies are seen in contact with free trichocysts, which appear to be transported, with their tip first, to the cell surface. (This can account for the saltatory movement reported by others). It is noteworthy that the "rails" represented by the microtubules do not directly determine the final attachment site of a trichocyst. Unoccupied attachment sites are characterized by a "plug" of electron-dense material just below the plasma membrane; the "plug" seems to act as a recognition or anchoring site; this material is squeezed out all around the trichocyst attachment zone, once a trichocyst is inserted (Westphal and Plattner, in press. [53]). Slightly below this "plug" we observed fasciae of microfilaments (identified by immunocytochemistry using peroxidase labeled F(ab) fragments against P. tetraurelia actin). Their arrangement is not altered when a trichocyst is docked. These fasciae seem to form a loophole for the insertion of a trichocyst. Trichocyst remain attached to the microtubules originating from the ciliary basal bodies--at least for some time--even after they are firmly installed in the preformed attachment sites. Evidently, the regular arrangement of exocytotic organelles is controlled on three levels: one operating over a long distance from the exocytosis site proper (microtubules), one over a short distance (microfilament bundles), and one directly on the exocytosis site ("plug").     

Myoneural junctions and neurosecretory endings on spermathecal muscle fibers of two weevils,Sitophilus granarius and Hypera postica

The fine structure of muscle fibers connecting the two arms of the spermatheca and their innervating axons was studied with the electron microscope. The muscle fibers appear to be a sub-set of skeletal and not visceral muscles. Neurosecretory axons with electron dense granules are adjacent to the muscle fibers in young females O-day post-eclosion but not in the ovipositing adult. The typical nerves form synaptic junctions with muscle fibers at all ages but the nerves are divided into two types based on the morphology of the synaptic vesicles they contain, either spherical or flattened.     

GABA and glutamate-like immunoreactivity at synapses received by dorsal unpaired median neurones in the abdominal nerve cord of the locust

Dorsal unpaired median (DUM) neurones in the abdominal ganglia of the locust were impaled with microelectrodes and some were injected intracellularly with horseradish peroxidase so that their synapses could be identified in the electron microscope. Simultaneous recordings from DUM neurones in different abdominal ganglia revealed that they received common postsynaptic potentials from descending interneurones. Post-embedding immunocytochemistry using antibodies against GABA and glutamate was carried out on ganglia containing HRP-stained neurones. GABA-like immunoreactivity was found in 39% (n=82) of processes presynaptic to abdominal DUM neurones and glutamate-like immunoreactivity in 21% (n=42) of presynaptic processes. Output synapses from the DUM neurites were rarely observed within the neuropile. Structures resembling presynaptic dense bars but not associated with synaptic vesicles, were seen in some large diameter neurites.     

PC12 Cells that Lack Synaptotagmin I Exhibit Loss of a Subpool of Small Dense Core Vesicles

Neurons communicate by releasing neurotransmitters that are stored in intracellular vesicular compartments. PC12 cells are frequently used as a model secretory cell line that is described to have two subpools of vesicles: small clear vesicles and dense core vesicles. We measured transmitter molecules released from vesicles in NGF-differentiated PC12 cells using carbon-fiber amperometry, and relative diameters of individual vesicles using electron microscopy. Both amperometry and electron micrograph data were analyzed by statistical and machine learning methods for Gaussian mixture models. An electron microscopy size correction algorithm was used to predict and correct for observation bias of vesicle size due to tangential slices through some vesicles. Expectation maximization algorithms were used to perform maximum likelihood estimation for the Gaussian parameters of different populations of vesicles, and were shown to be better than histogram and cumulative distribution function methods for analyzing mixed populations. The Bayesian information criterion was used to determine the most likely number of vesicle subpools observed in the amperometric and electron microscopy data. From this analysis, we show that there are three major subpools, not two, of vesicles stored and released from PC12 cells. The three subpools of vesicles include small clear vesicles and two subpools of dense core vesicles, a small and a large dense core vesicle subpool. Using PC12 cells stably transfected with short-hairpin RNA targeted to synaptotagmin I, an exocytotic Ca²⁺ sensor, we show that the presence and release of the small dense core vesicle subpool is dependent on synaptotagmin I. Furthermore, synaptotagmin I also plays a role in the formation and/or maintenance of the small dense core vesicle subpool in PC12 cells.     

Fine structure of monoamine-containing basal cells in the taste buds on the barbels of three species of teleosts

Summary The taste buds on the barbels in three species of teleosts (Cyprinus carpio, Misgurnus anguillicaudatus, Parasilurus asotus) were studied by means of fluorescence and electron microscopy. Intensely yellow-fluorescent cells, which are disk-shaped and located exclusively in a basal position, are observed in the barbel-buds of all fishes examined. The basal cells contain a large number of small clear vesicles approximately 40–60 nm in diameter, which show a tendency to aggregate in the cytoplasm facing the junction of the nerve terminals; chemically transmitting synapses are seen in the latter region. It is suggested from the present observations that the basal cells in the barbel-bud may originate from Schwann cells and have a dual function both as mechanoreceptors and paracrine elements. Since the administration of 5,6-DHT results in an appearance of small dense vesicles among the small clear vesicles, the possibility exists that the basal cell may be capable of taking up monoamines and storing them in the small clear vesicles.     

Crystals in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) leaves

Crystal-containing cells (C-cells) are widely spread in plant tissues; however, the origin of the crystals and their functions remain a subject of discussion. In sugar beet leaves, the membrane vesicles seen in an electron microscope appear in the cytoplasm and penetrate the vacuole by pinocytosis with the participation of tonoplast. In a light microscope, the vesicles fluoresce like crystals in C-cells. These crystal vesicles also fill the C-cells. The content of crystal vesicles is electron-transparent at all stages of leaf development. It is suggested that both individual crystal vesicles in the cytoplasm and in vacuoles and their agglomerations in C cells, vascular bundles, and epidermal cells are lytic compartments. Later, true crystals seem to be formed.     

Electron microscopical demonstration of acid phosphatase in blood platelets

Summary Ultrastructures of human and rabbit thrombocytes reveal specific subcellular organelles within these elements. Serotonin granules are demonstrated containing extremely electron opaque material in vesicles with an average diameter of 1,700 Å and a considerable number of large dense bodies (average size 4,000 Å in diameter) is seen. The latter are less electron dense as compared to the serotonin granules. The appearance of serotonin granules in the human thrombocyte is rare, while rabbit platelets show a higher number of these granular vesicles.Acid phosphatase activity in the large dense bodies of human and rabbit platelets has been demonstrated by means of electron microscopy. Present results together with currently available biochemical information are briefly discussed in relation to the lysosomal activity within the thrombocytes.     

Observations of Measles Virus Infection of Cultured Human Cells : I. A Study of Development and Spread of Virus Antigen by Means of Immunofluorescence

The development of measles virus in cultures of both primary human amnion cells and H.Ep.-2 cells has been followed by means of the indirect fluorescent antibody technic and concurrent light and electron microscope observations. The immunofluorescence studies revealed that there is a latent period for development of demonstrable measles virus antigen. In amnion cells the latent period lasted for at least 3 days. In contrast, virus antigen could be detected in H.Ep.-2 cells as early as 12 hours following inoculation. In each cell system virus antigen was seen in either nucleus or cytoplasm of infected cells, or both. Early localization tended to be perinuclear. Intranuclear fluorescence was generally less bright and less widespread than cytoplasmic fluorescence. Giant cells and long cytoplasmic spindle-shaped processes appeared regularly in infected cultures. Infectious virus was liberated into the nutrient fluid but when extracellular virus was inhibited by antibody, spread of infection from cell to cell in the monolayer still continued. Results obtained in concurrent electron microscope studies will be presented separately. Correlation of the results of the immunofluorescence and electron microscope studies suggests the possibility that much of the immunofluorescence observed might be due to antigen in virus precursors or components.     

The ultrastructure of mouse lung: the alveolar macrophage

Free alveolar macrophages of normal mouse lung have been studied in the electron microscope. The tissue was obtained from several young adult white mice. One other animal was instilled intranasally with diluted India ink 1(1/2) hours prior to the removal of the lung. Thin sections of the osmium-fixed, methacrylate-embedded tissue were examined either in an RCA EMU 2 electron microscope or in a Siemens and Halske Elmiskop I b. A few thick sections obtained from the same embeddings were stained for iron. The normal alveolar macrophages, which are usually in contact with the alveolar epithelium, were found to contain a variety of inclusion bodies, along with the usual cytoplasmic components like mitochondria, endoplasmic reticulum, and Palade granules. Another typical component of the cytoplasm of these cells which appears as small ( approximately 6 mmicro) very dense granules of composite fine structure is interpreted as ferritin. It is assumed that this ferritin is formed from red blood cells ingested by the alveolar macrophages. The macrophages in the alveoli were found to phagocytize intranasally instilled India ink particles. Such cells, with engulfed India ink particles, were often of more rounded form and the particles were frequently seen lying inside membrane-bound vacuoles or vesicles of the cytoplasm. The membrane of a few vesicles containing India ink particles was seen as the invaginated portion of the cell plasma membrane, and in one instance these same vesicles were seemingly interconnected with a rough surfaced cisterna of the endoplasmic reticulum. The process of phagocytosis is recognized as related to the "normal" process of pinocytosis.