Detection of pinocytic activity using selective staining with phosphotungstic acid

Summary Post-staining with phosphotungstic acid has been investigated using thin sections of isolated tomato fruit protoplast material. Certain areas of the plasmalemma and some cytoplasmic vesicles were found to stain intensely, and this selectivity of staining has been shown to be associated with pinocytic activity.This work was supported by a special grant from the S.R.C. One of us (MAM) acknowledges the award of an S.R.C. Research Studentship. This work is part of a thesis approved for the degree of Ph.D. in the University of Nottingham (MAM).     

The mechanism of infection of plant protoplasts by viruses

Summary The process of virus infection of protoplasts isolated from tobacco leaves has been examined by means of electron microscopy. Immediately after inoculation, virus particles appear at two types of site: trapped in complex surface lesions of the plasmalemma, or in peripheral cytoplasmic vesicles. The complex lesions are only visible after treatment of the protoplasts with inocula containing poly-l-ornithine. With infection by tobacco mosaic virus and cowpea chlorotic mottle virus, which require poly-l-ornithine, the majority of virus particles occur at lesion sites. Pea enation mosaic virus, which does not require poly-l-ornithine for infection to become established, is found predominantly and in high numbers in peripheral vesicles. The behaviour of these three viruses is discussed in terms of a probable mechanism for infection of the protoplasts.     

Membrane deletion during plasmolysis in hardened and non-hardened plant cells

Abstract Video recordings of interference phase contrast microscopy were used to study plasmalemma deletion during plasmolysis in hardened and non-hardened suspension cultured cells of Brassica napus, alfalfa, and cells isolated from rye seedlings. Although different hardening regimes and different cells were used, the responses to plasmolysis were consistent. Hardened cells uncoupled the volume to surface area ratio during plasmolysis both by forming a large number of strands between the cell wall and protoplast and by leaving rivulet-like networks of membranes on the cell wall surface. Tonoplast membrane was deleted as sac-like intrusions into the vacuole. Non-hardened cells produced few strands during plasmolysis. They also deleted plasmalemma and tonoplast into the vacuole as endocytotic vesicles. During deplasmolysis of hardened cells both the individual membrane strands and the rivulets of membrane material vesiculated into strings of vesicles. The vesicles were osmotically active and were re-incorporated into the expanding protoplast. Conversely, deplasmolysis in non-hardened cells resulted in few osmotically active vesicles and many broken strands. The vacuolar sac-like intrusions in hardened cells were re-incorporated into the vacuole whereas the endocytotic vesicles in non-hardened cells were not re-incorporated. Therefore, the non-hardened cells underwent expansion-induced lysis.     

An Alternative Mode of Elimination of Resin from Epithelial Cells of Resin Ducts in Pinus sylvestris

The fine structure of resin secreting cells of the cortical resin ducts in stem of Pinus sylvestris Carr. was studied with the help of high pressure freezing and freeze-substitution techniques. An unprecedented mode of resin elimination was observed. It seems that the hydrolase-contained Golgi vesicles take part in the dissolution of plasmalemma, and a passage is formed, through which resin material may quickly pass outside of the protoplast. Then, the ruptured plasmalemma was repaired by some vesicles, possibly derived from Golgi bodies.     

Osmotically induced fluid-phase uptake of fluorescent markers by protoplasts ofChenopodium album

Summary Protoplasts fromChenopodium album suspension culture show large, up to 5-fold, changes in surface area upon hypertonic or hypotonie treatment. These surface area variations cannot be explained by elastic stretching of the plasmalemma. An exchange of membrane material between the plasmalemma and an internal membrane source takes place. Fluid-phase uptake experiments with the fluorescence dyes 5, 6-carboxyfluorescein and Lucifer yellow CH demonstrated that osmotic shrinkage of protoplasts is accompanied by vesicular uptake of the external medium into protoplast cytoplasm. Confocal laser scanning microscopy, as well as conventional fluorescence microscopy, revealed the number, diameter and distribution of the osmocytotic vesicles at different osmotic levels. The rate of osmocytotic vesicle uptake was higher in the presence of calcium chloride than in the presence of EDTA in the external medium. At 6.9 mM calcium chloride we observed a loss of vesicular fluorescence upon returning protoplasts to 0.4 M from 0.8 M sorbitol.     

The development and ultrastructure of gum ducts inCitrus plants formed as a result of brown-rot gummosis

Summary Young stems ofCitrus plants were infected with the fungusPhytophthora citrophthora. The effect of the infection on gum duct development was studied. The following sequence of structural changes was observed in the cambial zone: 1. The middle lamellae between layers of xylem mother cells dissolve forming duct cavities. 2. The cells around the duct cavities differentiate into epithelial cells rich in cytoplasm. 3. The amount of Golgi bodies and associated vesicles increases. The vesicles and small vacuoles, some of which seem to originate from the fusion of Golgi vesicles, contain fibrillar material that stains for polysaccharides. Vesicles and vacuoles appear to fuse with the plasmalemma. Material staining positively for polysaccharides accumulates between the plasmalemma and cell wall, and penetrates the latter. 4. The protoplast shrinks and the space below the cell wall, which contains polysaccharides, increases in volume. 5. After a period of 10 days or more the gum ducts become embedded in the xylem, and the activity of the epithelial cells ceases. The cell walls of many of them break, and the gum still present in the cells is released.     

Developmental studies on the loricate choanoflagellateStephanoeca diplocostata ellis

Summary Cells ofStephanoeca diplocostata comprise a colourless, flagellated, protoplast lodged in a lorica made of siliceous costae. The single anterior flagellum creates a water current from which bacteria and other food particles are filtered by the collar and ingested by linguiform pseudopodia that arise from the protoplast at the base of the collar. A waist divides the lorica into two chambers, the anterior of which contains three transverse and 17–20 longitudinal costae whereas the posterior chamber comprises two systems of spirally deflected costae and on some cells a pedicel at the hind end. Between 150–185 costal strips of similar length form the lorica. A thin investment covers the inner surface of the posterior chamber and lower part of the anterior chamber and joins with the protoplast at the level of the waist. Costal strips are produced within membrane-bounded vesicles in the peripheral cytoplasm and, although the origin of these vesicles is unknown, there is usually a close association with the Golgi apparatus. Once complete, strips are apparently released sideways through the plasmalemma into the cavity of the posterior lorica chamber. Later, bundles of strips are transferred to the top of the inner surface of the collar where they collectively form a horizontal ring. When sufficient strips to form a lorica have been accumulated at the top of the collar, cell division proceeds.     

Unusual transfer cells in the epithelium of the nectaries ofAsclepias curassavica L.

Summary The secretory cells of the nectaries ofAsclepias curassavica form a glandular epithelium in the inner parts of the stigmatic chambers. They resemble transfer cells in having many infoldings of the plasmalemma. The wall protuberances, however, are poorly developed and often lacking. The plasmalemma is highly convoluted and forms, in places, external compound membranes where the extracytoplasmic space is collapsed completely. Active glands contain dilated cisternae of the ER and large vesicles which are mainly associated with the cis face of the dictyosomes. In addition, small vesicles are observed in high number. It is discussed whether the secretion is granulocrine or eccrine and whether the enlargement of the plasmalemma is the cause or the consequence of the high secretory activity. After the secretory phase the outer peripheral part of the cytoplasm disintegrates. The remaining part of the protoplast is covered by a new plasmalemma.     

Mechanism of viral infection of macrophages by an agent of hemorrhagic fever with renal syndrome: ultrastructural aspects

Monocytes/macrophages are one of the first cells subjected to the infectious effect of viruses. The present paper analyses for the first time the ultrastructural changes in macrophages caused by an agent of hemorrhagic fever with renal syndrome (HERS)--hantavirus (HV). After a local fusion with the host cell plasmalemma and its adsorption on the cell surface, the HV penetrates through the macrophage membrane. This process occurred without destruction of cell plasmalemma. HV viral particles were observe within the macrophage cytoplasm mostly on the smooth granular endoplasmic reticulum vesicles. Viroplasts were defined in macrophages after a 2 h incubation, with synthesis of viral nucleoproteins and primary covers being observed on the surface of viroplasts. Viral particles left macrophages in the process of budding on the phagocyte surface. Thereby HV, similar to other enveloped viruses, realizes entrance and egress from the target cell without damaging its plasmalemma. This accounts for the viral ability to reproduce in macrophages for a long time without any cytopathological effect. Consequently, in the absence of obvious destruction changes, mononuclear phagocytes can serve as a long-term storage of viruses, and thus being involved in HV dissemination during HERS.     

Topography and functional information of plasma membrane

By using atomic force microscope (AFM), the topography and function of the plasmalemma surface of the isolated protoplasts from winter wheat mesophyll cells were observed, and compared with dead protoplasts induced by dehydrating stress. The observational results revealed that the plasma membrane of living protoplasts was in a state of polarization. Lipid layers of different cells and membrane areas exhibited distinct active states. The surfaces of plasma membranes were unequal, and were characterized of regionalisation. In addition, lattice structures were visualized in some regions of the membrane surface. These typical structures were assumed to be lipid molecular complexes, which were measured to be 15.8±0.09 nm in diameter and 1.9±0.3 nm in height. Both two-dimensional and three-dimensional imaging showed that the plasmalemma surfaces of winter wheat protoplasts were covered with numerous protruding particles. In order to determine the chemical nature of the protruding particles, living protoplasts were treated by proteolytic enzyme. Under the effect of enzyme, large particles became relatively looser, resulting that their width was increased and their height decreased. The results demonstrated that these particles were likely to be of protein nature. These protein particles at plasmalemma surface were different in size and unequal in distribution. The diameter of large protein particles ranged from 200 to 440 nm, with a central micropore, and the apparent height of them was found to vary from 12 to 40 nm. The diameter of mid-sized protein particles was between 40―60 nm, and a range of 1.8―5 nm was given for the apparent height of them. As for small protein particles, obtained values were 12―40 nm for their diameter and 0.7―2.2 nm for height. Some invaginated pits were also observed at the plasma membrane. They were formed by the endocytosis of protoplast. Distribution density of them at plasmalemma was about 16 pits per 15 μm2. According to their size, we classified the invaginated pits into two types―larger pits measuring 139 nm in diameter and 7.2 nm in depth, and smaller pits measuring 96 nm in diameter and 2.3 nm in depth. On dehydration-induced dead pro-toplasts, the degree of polarization of plasma membranes decreased. Lipid molecular layers appeared relatively smooth, and the quantity of integral proteins reduced a lot. Invaginated pits were still de-tectable at the membrane surface, but due to dehydration-induced protoplast contraction, the orifice diameter of pits reduced, and their depth increased. Larger pits averagely measuring 47.4 nm in di-ameter and 31.9 nm in depth, and smaller pits measuring 26.5 nm in diameter and 43 nm in depth at average. The measured thickness of plasma membranes of mesophyll cells from winter wheat examined by AFM was 6.6―9.8 nm, thicker in regions covered with proteins.     

Magnetic separation of pinocytic vesicles of defined age from Entamoeba histolytica

We describe a rapid and simple method to isolate pinocytic vesicles of defined age (residing time within the cell) from Entamoeba histolytica. Amoebas are allowed to pinocytize for greater than 5 min a suspension of superparamagnetic iron oxide particles, washed, and resuspended for predetermined periods (up to 150 min) in iron oxide-free medium. Subsequently, the cells are homogenized and iron oxide-containing vesicles are separated magnetically. Recovery of vesicles (estimated with fluorescein isothiocyanate-dextran as a quantitative marker for pinocytosis) was 20-40%. Contamination with "older" vesicles or with plasma membrane (estimated with fluorescein isothiocyanate-dextran and with fluorescein isothiocyanate-conjugated, succinylated concanavalin A, respectively) was negligible. Using this method we obtained evidence that in E. histolytica, contrary to the situation in animal cells, pinocytic vesicles within 150 min after invagination neither shrunk nor fused with each other to any significant extent. The method should be generally applicable to protozoa for the isolation of pinocytic vesicles and digestive vacuoles.     

Cellular uptake and infection by canine parvovirus involves rapid dynamin-regulated clathrin-mediated endocytosis, followed by slower intracellular trafficking

Canine parvovirus (CPV) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. We used a variety of approaches to define the early stages of cell entry by CPV. Electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. Overexpression of a dominant interfering mutant of dynamin in the cells altered the trafficking of capsid-containing vesicles. There was a 40% decrease in the number of CPV-infected cells in mutant dynamin-expressing cells, as well as a approximately 40% decrease in the number of cells in S phase of the cell cycle, which is required for virus replication. However, there was also up to 10-fold more binding of CPV to the surface of mutant dynamin-expressing cells than there was to uninduced cells, suggesting an increased receptor retention on the cell surface. In contrast, there was little difference in virus binding, virus infection rate, or cell cycle distribution between induced and uninduced cells expressing wild-type dynamin. CPV particles colocalized with transferrin in perinuclear endosomes but not with fluorescein isothiocyanate-dextran, a marker for fluid-phase endocytosis. Cells treated with nanomolar concentrations of bafilomycin A1 were largely resistant to infection when the drug was added either 30 min before or 90 min after inoculation, suggesting that there was a lag between virus entering the cell by clathrin-mediated endocytosis and escape of the virus from the endosome. High concentrations of CPV particles did not permeabilize canine A72 or mink lung cells to alpha-sarcin, but canine adenovirus type 1 particles permeabilized both cell lines. These data suggest that the CPV entry and infection pathway is complex and involves multiple vesicular components.     

Response of the resident macrophage to concanavalin A : Alterations of surface morphology and anionic site distribution

We have characterized certain resident guinea-pig peritoneal macrophage surface alterations following treatment with concanavalin A (ConA) and succinyl-ConA (S-ConA). Studies employing scanning electron microscopy (SEM) have demonstrated that ConA, a tetrameric lectin, decreases dramatically the number of macrophage surface folds and ruffles although S-ConA, a dimeric derivative, is apparently not active. However, incubation of S-ConA-treated cells with rabbit anti-ConA (anti-ConA) restored this effect. The decrease in surface folds could not be observed in the presence of α-methyl- -mannoside (αMM), a hapten sugar of ConA. We have performed several receptor-labeling transmission electron microscopy (TEM) studies employing ferritin-conjugated ConA (FT-ConA) and cationized ferritin (CF). These experiments indicate that the ConA receptor-FT-ConA complexes form (1) clusters on the plasmalemma and (2) adsorptive pinocytic vesicles lined with the ferritin label. At the same time scale, we have observed a redistribution of macrophage surface anionic sites following treatment with ConA or S-ConA plus anti-ConA but not S-ConA alone. The redistribution of anionic sites is abolished in the presence of αMM. The specificity of the CF label was checked by pre-incubating cells with poly- -lysine (PLL) or neuraminidase and by employing normal ferritin. These studies provide evidence which support the concept of a directed movement of surface charges during adsorptive pinocytosis. We discuss the possible relevance of this concept with regard to regional alterations of pH at the plasmalemma and the contribution of these anions to the trans-pinosomal pH gradient through the Donnan potential.     

Cellulose microfibril deposition at the plasmalemma surface of regenerating tobacco mesophyll protoplasts: A deep-etch study

Summary The reappearance of cellulose microfibrils at the naked surface of protoplasts enzymatically isolated from tobacco (Nicotiana tabacum L. var. Xanthi) mesophyll tissue has been closely studied using the techniques of thin-sectoining and the deep-etch modification of the freeze fracture procedure.A 16 h lag period was recorded between the time of isolation and the sudden appearance of considerable lengths of cellulose microfibril at the outer protoplast surface. The microfibrils were not associated with any structured particles or apparently differentiated regions of the plasmalemma. Terminal regions of the microfibrils appeared to have tapering ends, or else be sinking into the membrane substance. There was no evidence to suggest transport of intact microfibrils in vesicles through the cytoplasm to the plasmalemma.The reported observations have been discussed with respect to the various working hypotheses which have been proposed for the in vivo construction of cellulose microfibrils.     

Fine Structure of Botrytis fabae Sardina Conidia

The fine structure of Botrytis fabae conidia was studied usinga variety of electron-microscope techniques. The spore walllacks conspicuous ornamentation and consists of microfibrilsembedded in a granular matrix. The two distinct wall layersseen in chemically fixed sections cannot be detected in cross-fracturedreplicas; the two layers are probably structurally similar.The outer surface of the plasmalemma is covered with branchedinvaginations and two kinds of particles. Three distinct typesof particles are present on the inner surface of the plasmalemma.In freeze-etched replicas nuclei, vacuoles, and other organellesalways appear smoothly rounded. Small vesicles pass throughthe plasmalemma into the cell wall. Particles approximately10 nm in diameter occur in compact rows on the cristae of cross-fracturedmitochondria: dense spherical particles, probably of calciumphosphate, are present in chemically fixed mitochondria. Prevacuolesand vesicles with membranous inclusions can be seen in bothcross-fractured replicas and chemically fixed sections. In cross-fracturedreplicas vacuoles and lipid bodies are frequently joined bystrands of endoplasmic reticulum.     

Membrane flow during pinocytosis. A stereologic analysis

HRP has been used as a cytochemical marker for a sterelogic analysis of pinocytic vesicles and secondary lysosomes in cultivated macrophages and L cells. Evidence is presented that the diaminobenzidine technique (a) detects all vaculoes containing encyme and (b) distinguishes between incoming pinocytic vesicles and those which have fused with pre-existing lysosomes to form secondary lososomes. The HRP reactive pinocytic vesicle spaces fills completely within 5 min after exposure to enzyme, while the secondary lysosome compartment is saturated in 45--60 min. The size distribution of sectioned (profile) vaculoe diameters was measured at equilibrium and converted to actual (spherical) dimensions using a technique modified from Dr. S. D. Wicksell. The most important findings in this study have to do with the rate at which pinocytosed fluid and surface membrane move into the cell and on their subsequent fate. Each minute macrophages form at least 125 pinocytic vesicles having a fractional vol of 0.43% of the cell's volume and a fractional area of 3.1% of the cell's surface area. The fractional volume and surface area flux rates for L cells were 0.05% and 0.8% per minute respectively. Macrophages and L cells thus interiorize the equivalent of their cell surface area every 33 and 125 min. During a 3-period, the size of the secondary lysosome compartment remains constant and represents 2.5% of the cell volume and 18% of the surface area. Each hour, therefore, the volume and surface area of incoming vesicles is 10 times greater than the dimensions of the secondary lysosomes in both macrophages and L cells. This implies a rapid reduction in vesicle size during the formation of the secondary lysosome and the egress of pinocytosed fluid from the vacuole and the cell. In addition, we postulate that membrane components of the vacuole are subsequently recycled back to the cell surface.     

A freeze-etch study of the phototactic zoospores ofPhlyctochytrium sp., a marine fungus

Summary The major difference between freeze-etched illuminated and non-illuminatedPhlyctochytrium zoospores was in the plasmalemma. The illuminated spores had abundant 15–18 nm freeze fracture particles on the PF surface of the plasmalemma in a region external to the rumposomal complex. Non-illuminated spores show fewer and smaller (6–12 nm) freeze fracture particles on the PF surface of the plasmalemma external to the rumposomal complex. Both illuminated and non-illuminated zoospores have rumposomal complexes, nuclear caps, mitochondria, nuclei, and a variety of cytoplasmic vesicles. The nuclear pores were not randomly distributed over the surface of the zoospore nucleus. Micrographs of nuclei in developing sporangia indicate pore formation in the region of the nuclear pockets.A portion of this study was carried out at the Virginia Institute of Marine Science, Gloucester Point, Virginia.     

Morphogenesis of Sindbis virus in three subclones of Aedes albopictus (mosquito) cells.

The morphogenesis of Sindbis virus in three Aedes albopictus subcloned cell lines was examined. Each line was distinguishable with respect to morphology, cytopathic response to infection, and progeny yield. C7-10 cells, which produced the highest titers of virus and exhibited the most severe cytopathic response, were characterized ultrastructurally by the presence of budding particles at the cell surface and at the membranes of internal vesicles. C6/36 cells, which displayed a moderate cytotoxic response, manifested similar features in response to Sindbis virus infection. Both cell types also produced a structure composed of an electron-dense matrix in which nucleocapsids were embedded. Internally matured virions were released by exocytosis from these cells. In addition to a lack of cytopathic effect, u4.4 cells also failed to exhibit obvious morphogenetic changes upon infection. Virus particles were occasionally seen within vesicles, but budding at the cell surface was not detected. The mechanism of release of internally matured virions was not apparent. These studies provide further evidence that these three subcloned mosquito cell lines represent different tissues in the larval or adult insect.     

The development of alkaline phosphatase in trichinous muscle.

The development of alkaline phosphatase during invasion and encystment of Trichinella spiralis in rat skeletal muscle fibres was studied at the ultrastructural level. On day 14 after infection, the enzymatic activity is found in proliferating parts of the T-tubular system and in parts of the plasmalemma. In cells, in which a strong hyperplasia of this system is noted. AlPase is present in the abundant network of stratified and concentric membranes from which a large number of pinocytic vesicles arise. From day 50 till 1 year after infection the enzyme activity was invariably present in the matrix surrounding the larvae and was confined to the enormous amounts of cytoplasmic membranes. The possible functional significance of this enzyme in the matrix, in view of its peculiar localization in the immediate vicinity of the parasite, is discussed. In the presence of 0.1 mM of the levamisole analogue, compound R 30402, which is a stereospecific inhibitor of AlPase, the activity is completely lost.     

Endocytosis of simian virus 40 into the endoplasmic reticulum

The endocytosis of SV-40 into CV-1 cells we studied using biochemical and ultrastructural techniques. The half-time of binding of [35S]methionine-radiolabeled SV-40 to CV-1 cells was 25 min. Most of the incoming virus particles remained undegraded for several hours. Electron microscopy showed that some virus entered the endosomal/lysosomal pathway via coated vesicles, while the majority were endocytosed via small uncoated vesicles. After infection at high multiplicity, one third of total cell-associated virus was observed to enter the ER, starting 1-2 h after virus application. The viruses were present in large, tubular, smooth membrane networks generated as extentions of the ER. The results describe a novel and unique membrane transport pathway that allows endocytosed viral particles to be targeted from the plasma membrane to the ER.