The behavior of the plasma membrane following osmotic contraction of isolated protoplasts: Implications in freezing injury

Summary Following osmotic contraction of isolated rye protoplast (Secale cereale L. cv. Puma) that results in nearly a 50% reduction in volume, the plasma membrane was smooth, with no folding or pleating. Instead, deletion of plasma membrane occurred and numerous cytoplasmic vesicles were observed. As a result, the area of the plasma membrane was reduced by approximately 40%. Thin sections revealed that the cytoplasmic vesicles were membrane bound and not merely voids in the cytoplasm. High resolution video microscopy revealed the extent of vesiculation showing large clusters of cytoplasmic vesicles following osmotic contraction. Labeling the plasma membrane with fluorescein-Con-A prior to hypertonic contraction suggested that the cytoplasmic vesicles were derived from the plasma membrane. Freeze-fracture particle density on both the protoplasmic (PFp) and exoplasmic face (EFp) of the plasma membrane remained unchanged following contraction, which is consistent with a unit-membrane deletion into cytoplasmic vesicles. Upon partial re-expansion of the protoplasts, thin sections showed that the vesicles remained in the cytoplasm. These results using osmotic manipulation confirm earlier observations of isolated protoplasts at the light microscope level. Upon contraction plasma membrane is deleted into cytoplasmic vesicles, which are not readily reincorporated into the plasma membrane upon expansion. Lysis occurs before the original volume and surface area are regained.Department of Agronomy Series Paper no. 1456.     

The morphology of multivesicular bodies in soybean protoplasts and their role in endocytosis

Summary Multivesicular bodies (MVBs) in soybean protoplasts are distinct organelles (generally 250–500 nm in diameter) consisting of a limiting membrane and a number of smaller internal vesicles (generally 40–100 nm in diameter). MVBs of soybean protoplasts are morphologically similar to MVBs of animal cell systems. They can have tubular protuberances which extend from the main body of the organelle and a lamellar plaque on the cytoplasmic surface of their limiting membrane. In addition, the internal vesicles can be labeled by a zinc iodide-osmium tetroxide postfixation and may form via invagination of the limiting membrane.The MVBs of soybean protoplasts are a major compartment in the endocytotic pathway. They accumulate, over time, exogenously applied cationized ferritin and may deliver it to the major lysosomal or lytic compartment of the plant cell, namely, the vacuoles.     

Cytolocalization of ion-channel antagonist binding sites in sunflower protoplasts during the early steps of culture

Summary A fluorescently labeled phenylalkylamine (PAA), DM-Bodipy PAA, was used as a probe for in vivo labeling of PAA binding sites in sunflower hypocotyl protoplasts in culture. Verapamil, a PAA known as a calcium channel antagonist in plants, lowers the division rate of sunflower protoplasts in culture. The binding specificity of DM-Bodipy PAA was established at various culture times by competition experiments with (–)bepridil. Studies on the Cytolocalization of DM-Bodipy PAA binding sites by confocal imaging showed that in freshly isolated protoplasts PAA receptors were organized into clusters uniformly distributed over the cell surface. During protoplast culture, the fluorescence labeling pattern evolved from peripheral to cytoplasmic. After a few days of culture, PAA binding sites were present inside the cell, along cytoplasmic strands, on the membrane of vesicles and vacuoles, and were highly concentrated around the nucleus. After protoplast division, the labeling was mainly restricted to a zone close to the new cell wall. On symmetrical division, binding sites were uniformly distributed on both sides of the new cell wall. With asymmetrical division, binding sites were concentrated in a ring surrounding the new cell plate.Abbreviations PAA phenylalkylamine - DHP dihydropyridine - FDA fluorescein diacetate     

New Procedure for the Isolation of Membrane Vesicles of Bacillus subtilis and an Electron Microscopy Study of Their Ultrastructure

A rapid procedure for the isolation of membrane vesicles of Bacillus subtilis is described that minimizes the action of proteolytic enzymes, excreted by this organism, on the membrane proteins. The membrane vesicles obtained have, in addition to a low endogenous respiration rate, a low endogenous activity for transport of amino acids and carboxylic acids. In the presence of the electron donor, ascorbate-phenazine methosulfate, the transport activities for these compounds were comparable to the activities of intact cells. In addition, these activities were retained for a prolonged period of time. Electron microscopy examination of thin sections of the vesicles showed that the preparation consisted almost exclusively of membrane vesicles which were not contaminated with other cell components. The membrane vesicles, which are six to seven times smaller in diameter than protoplasts, often enclosed smaller vesicles. Freeze-etching of intact cells, protoplasts, and membrane vesicles showed that the orientation of the membrane of the vesicles was identical to the orientation of the plasma membrane in intact cells and protoplasts. This also held for the majority of the membranes of the enclosed vesicles, only 15% having the opposite orientation.     

Formation of inside-out vesicles of Bacillus licheniformis. Dependence on buffer composition and lysis procedure.

1. The extent to which the cytoplasmic membrane of the Gram-positive bacterium Bacillus licheniformis formed inside-out vesicles was studied with the freeze-fracture technique. The membrane orientation appeared to be dependent on the buffer compositon as well as on the lysis procedure used. 2. By manipulating these conditions, membrane preparations were obtained with the percentage of inside-out vesicles varying from 15 to 80%. 3. More vesicles had the opposite orientation when the cells were lysed in potassium phosphate buffer than when they were lysed in sodium phosphate buffer. Tris-HCl buffer favoured the formation of inside-out vesicles more than phosphate buffer. 4. Lysis of protoplasts in hypotonic buffers resulted in more inside-out vesicles than did direct lysis of cells in hypotonic media. 5. In an attempt to explain the observed differences, experiments were performed in which the morphology of thin-sectioned lysing cells in sodium phosphate buffer was compared with that in potassium phosphate buffer. The results from these experiments indicate that the formation of inside-out vesicles is brought about by an effect on the membrane itself rather than on the cell wall, on the cell wall membrane association, or on the cytoplasm.     

Electric pulse induced membrane permeabilization. Spatial orientation and kinetics of solute efflux in freely suspended and dielectrophoretically aligned plant mesophyll protoplasts

Asymmetric breakdown (occurring in only one hemisphere of the cell) was induced in freely suspended and dielectrophoretically aligned vacuole-containing or evacuolated plant protoplasts as well as in isolated vacuoles. In suspended cells breakdown was restricted to the hemisphere facing the anode and in isolated vacuoles to the opposite hemisphere. This difference in the orientation of the asymmetric breakdown can be explained by the opposite direction of the intrinsic membrane potentials of isolated vacuoles and of cells on which the generated potential difference is superimposed. The ensuing permeabilization of the membrane was microscopically monitored by dye uptake and by release of chloroplasts and of cytoplasmic and/or vacuolar solutes. The asymmetric release of intracellular substances (organic acids and/or amino acids) was detected by accumulation of chemotactic bacteria (Pseudomonas aeruginosa) close to the permeabilised membrane area of the cells or vacuoles. Maximum bacteria accumulation required about 5 min and subsequently disappeared after a further 20 min presumably because of the restoration of the original membrane impermeability. With vacuoles retention of the accumulated bacteria was shorter indicating that the resealing process of the tonoplast membrane was faster than that of the plasmalemma. From the kinetics of bacteria accumulation and retention it is therefore possible to deduce information about the life-span and the resealing properties of electropermeabilized membrane areas on the single-cell level. Symmetric breakdown in both hemispheres of the cells could be achieved by electric field-mediated cell rotation of about 180 degrees between two pulses of the same polarity or by application of two pulses of alternating polarity. In dielectrophoretically aligned protoplasts of comparable diameter, breakdown occurred in both hemispheres, even though the breakdown was still asymmetric. It could be demonstrated by the uptake of the vital dye neutral red that the size of the membrane area which was permeabilized was much larger in that hemisphere oriented to the anode than in the other one. The relevance of these observations for further improvement of electroinjection of macromolecules and of electrofusion is discussed. In particular, it is pointed out that positioning of differently sized cells in electric field-mediated hybridisation and the polarity of the breakdown pulse is of great importance with respect to hybrid yield.     

Cytoplasmic microvesicles in chromophobe cell renal carcinoma demonstrated by freeze fracture

In the chromophobe cell type of renal carcinoma, cytoplasmic microvesicles (frequently with "inner vesicles") demonstrable by transmission electron microscopy are one of the most important diagnostic features. The present paper reports on these microvesicles in freeze fracture replicas. Their diameter is mainly between 140 and 300 micron, but smaller and very much larger vesicles may also occur. The vesicle membrane is devoid of, or contains only scanty intramembranous particles. Cytoplasmic invaginations, probably the precursors of "inner vesicles" can also be detected. Connections with the agranular endoplasmic reticulum, mitochondria or other cell components could not be documented. Larger vacuoles limited by a membrane and containing large numbers of microvesicles have been interpreted as autophagic vacuoles. In freeze fracture replicas, there are marked differences between the chromophobe cell and the clear cell type of renal carcinoma, providing further evidence that the chromophobe cell type is a distinct entity.     

Orientation of succinate dehydrogenase and cytochrome b558 in the Bacillus subtilis cytoplasmic membrane.

The orientation of the three subunits of the membrane-bound succinate dehydrogenase (SDH)-cytochrome b558 complex in Bacillus subtilis was studied in protoplasts ("right side out") and isolated membranes (random orientation), using immunoadsorption and surface labeling with [35S]diazobenzenesulfonate. Anti-SDH antibodies were adsorbed by isolated membranes but not by protoplasts. The SDH Mr 65,000 flavoprotein subunit was labeled with [35S]diazobenzenesulfonate in isolated membranes but not in protoplasts. The flavoprotein subunit is thus located on the cytoplasmic side of the membrane. The location of the SDH Mr 28,000 iron-protein subunit was not definitely established, but most probably the iron-protein subunit also is located on the cytoplasmic side of the membrane. Antibodies were not obtained to the hydrophobic cytochrome b558. The cytochrome was strongly labeled with [35S]diazobenzenesulfonate in protoplasts, and labeling was also obtained with isolated membranes. Cytochrome b558 is thus exposed on the outside of the membrane. In B. subtilis SDH binds specifically to cytochrome b558, which suggests that the cytochrome is exposed also on the cytoplasmic side of the membrane. The results obtained suggest that the B. subtilis SDH is exclusively located on the cytoplasmic side of the membrane where it is bound to cytochrome b558, which spans the membrane.     

A comparative study of submicroscopic structures of protoplasts of various yeast species

A submicroscopic structure was studied of protoplasts of five different yeast species multiplying by budding, formation of cross septum and by a division typical for apiculate yeasts. The protoplasts retain their species specificity. Most considerable changes typical for the conversion of a cell to protoplast are found in membrane cell systems. The reduction of membranes of the endoplasmic reticulum is particularly striking. Both membrane units are frequently separated from each other by lenticular pseudovacuoles. Mitochondria in protoplasts are swollen and their number is reduced approximately two-fold. Defects are often observed in a nuclear membrane. The perinuclear space is usually extended by lenticular pseudovacuoles. A large number of vacuoles is observed in the basic protoplast cytoplasm. The surface of the protoplasts of all species studied is formed only by a cytoplasmic membrane. A partially digested original cell wall often adheres to protoplasts ofSchizosaccharomyces pombe.     

Purification of Functionally Sealed Cytoplasmic Side-out Plasma Membrane Vesicles from Saccharomyces cerevisiae

Highly purified plasma membrane vesicles were prepared from yeast protoplasts by a combination of osmotic lysis, differential centrifugation, and separation in an aqueous dextran/polyethylene glycol two-phase system. The vesicles were predominantly (85-90%) of cytoplasmic side-out orientation and displayed large ATP-dependent proton pumping activity which was inhibited by vanadate (100 μM) but not by bafilomycin or nitrate. The preparation presented a distinct polypeptide profile with respect to the total membrane fraction and was enriched in the 110-kDa polypeptide corresponding to the plasma membrane H⁺-ATPase. This preparation of native plasma membranes vesicles is especially suitable for functional studies in vitro.     

The effect of high temperatures on leaf cells of Valerianella: relative heat stability of the tonoplast membrane of mesophyll vacuoles

The effect of short-term heat stress on the tonoplast membrane of lamb's lettuce (Valerianella locusta (L.) Betcke) mesophyll vacuoles has been investigated. The maintainance of a proton concentration difference (δpH) across the tonoplast membrane served as a criterion for the integrity of the vacuoles. After heat treatment, δpH was measured at room temperature using the fluorescent amine, 9-aminoacridine. It was found with this method that thermal damage to isolated vacuoles mainly occurred in the temperature range above 50°C. Compared with this results, the photosynthetic functions of isolated lettuce protoplasts proved to be markedly more thermolabile, e.g. photosynthetic CO₂ fixation and light-induced chlorophyll fluorescence were drastically reduced at temperatures between 40° and 50°C. Heating of whole leaves and protoplasts and subsequent isolation of vacuoles showed that tonoplast-membrane integrity is not affected by heat stress in situ up to 45°C. Measurement of 9-aminoacridine fluorescence in protoplasts, which allowed conclusions to be drawn regarding the integrity of the tonoplast membrane in its natural cytoplasmic environment, revealed that heat treatment up to 55°C did not significantly affect vacuolar compartmentation. The data provide evidence that the tonoplast membrane is relatively heat stable compared with photosynthetic membranes.     

The formation of multivesicular structures in the adipose cells of chick embryos

Observation of the cytogenesis of adipose tissue of the chick embryo revealed a quantity of multiversicular structures (MVs) which were found in the intercellular space. Some of them were attached to the adipocytes and others were independently located in the intercellular space. The origin of those MVs appeared to be part of the degenerating mitochondria. Centrally located vesicles and vacuoles in degenerating mitochondria formed a group of short tubules and vacuoles which protruded through the cytoplasmic membrane or bulged out at the edge of the cytoplasmic process. The MVs then spread over the cytoplasmic membrane and finally were discharged from the cell surface as in the manner of apocrine secretion. An invisible barrier between the mass of vesicles and the rest of the cytoplasmic structures appeared to segregate the extruding MVs from the intercellular components such as ribosomes, microtubules, and microfilaments.     

Immunoferritin labeling of respiratory nitrate reductase in membrane vesicles of Bacillus licheniformis and Klebsiella aerogenes

The indirect immunoferritin labeling method was used to localize the membrane-bound respiratory nitrate reductase in membrane vesicles and protoplasts or spheroplasts of Bacillus licheniformis and Klebsiella aerogenes, respectively. For a comparison of the labeling of the various vesicle preparations, which differed not only in size but also in the percentage of inside-out orientation, a quantification of the results was needed to circumvent the problem of non-specifically bound ferritin. From the results the sidedness of the nitrate reductase in the cytoplasmic membrane of the abovementioned bacteria was determined as being cytoplasmic in B. licheniformis and as transmembranous in K. aerogenes.Non-Standard Abbreviations PBS phosphate buffered saline - IgG immunoglobulin G     

Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION

Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply.     

Molecular cloning and further characterization of a probable plant vacuolar sorting receptor.

BP-80 is a type I integral membrane protein abundant in pea (Pisum sativum) clathrin-coated vesicles (CCVs) that binds with high affinity to vacuole-targeting determinants containing asparagine-proline-isoleucine-arginine. Here we present results from cDNA cloning and studies of its intracellular localization. Its sequence and sequences of homologs from Arabidopsis, rice (Oryza sativa), and maize (Zea mays) define a novel family of proteins unique to plants that is highly conserved in both monocotyledons and dicotyledons. The BP-80 protein is present in dilated ends of Golgi cisternae and in "prevacuoles," which are small vacuoles separate from but capable of fusing with lytic vacuoles. Its cytoplasmic tail contains a Tyr-X-X-hydrophobic residue motif associated with transmembrane proteins incorporated into CCVs. When transiently expressed in tobacco (Nicotiana tabacum) suspension-culture protoplasts, a truncated form lacking transmembrane and cytoplasmic domains was secreted. These results, coupled with previous studies of ligand-binding specificity and pH dependence, strongly support our hypothesis that BP-80 is a vacuolar sorting receptor that trafficks in CCVs between Golgi and a newly described prevacuolar compartment.     

Multistage disruption of protoplasts by dikegulac

Dikegulac (2,3:4,6 di-o-isopropylidine-2-keto-I-gulonate) is a growth regulator used to differentially kill terminal apices, and it analogously inhibits basic metabolic functions in dividing cells, but not stationary cells, in suspension culture. This report demonstrates an analogous situation in isolated tobacco protoplasts. At the lowest concentrations, dikegulac partially suppresses division of the protoplasts. Higher concentrations are required to produce visual cytoplasmic damage to the protoplasts, which probably first occurs at the level of the plasmalemma, as the vacuoles can be released intact. Later, tonoplast disruption occurs.Abbreviation FDA fluorescein diacetate     

Fluid phase and receptor-mediated endocytosis in Paramecium primaurelia by fluorescence confocal laser scanning microscopy

In ciliated protozoa, most nutrients are internalized via phagocytosis by food vacuole formation at the posterior end of the buccal cavity. The uptake of small-sized molecules and external fluid through the plasma membrane is a localized process. That is because most of the cell surface is internally covered by an alveolar system and a fibrous epiplasm, so that only defined areas of the cell surface are potential substance uptake sites. The purpose of this study is to analyze, by fluorescence confocal laser scanning microscopy, the relationship between WGA (Triticum vulgaris agglutinin) and dextran internalization in Paramecium primaurelia cells blocked in the phagocytic process, so that markers could not be internalized via food vacuole formation. WGA, which binds to surface constituents of fixed and living cells, was used as a marker for membrane transport and dextran as a marker for fluid phase endocytosis. After 3 min incubation, WGA-FITC is found on plasma membrane and cilia, and successively within small cytoplasmic vesicles. After a 10-15 min chase in unlabeled medium, the marked vesicles decrease in number, increase in size and fuse with food vacuoles. This fusion was evidenced by labeling food vacuoles with BSA-Texas red. Dextran enters the cell via endocytic vesicles which first localize in the cortical region, under the plasma membrane, and then migrate in the cytoplasm and fuse with other endocytic vesicles and food vacuoles. When cells are fed with WGA-FITC and dextran-Texas red at the same time, two differently labeled vesicle populations are found. Cytosol acidification and incubation in sucrose medium or in chlorpromazine showed that WGA is internalized via clathrin vesicles, whereas fluid phase endocytosis is a clathrin-independent process.     

Isolation of plasma membrane fractions from green wheat leaves by free-flow electrophoresis or two-phase partition alone or in combination

Plasma membrane vesicles were isolated from shoots of light-grown wheat seedlings by preparative free-flow electrophoresis, aqueous polymer two-phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K⁺ Mg²⁺ATPase and of glucan synthase II, and concanavalin A-peroxidase binding and membrane asymmetry visualized by electron microscopy. The K⁺Mg²⁺ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two-phase partition were latent and trypsin resistant. The vesicles bound concanavalin A-peroxidase strongly and exhibited a cytoplasmic side-in morphology. These fractions of cytoplasmic side-in vesicles were less than 10% contaminated by cytoplasmic side-out vesicles. By free-flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two-phase partition. The phosphotungstic-reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K⁺Mg²⁺-ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side-out. The electrophoretic mobilities of cytoplasmic side-out (non-latent glucan synthase II activity) and cytoplasmic side-in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side-in. The cytoplasmic side-in and cytoplasmic side-out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free-flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two-phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.     

Nature and Development of Membrane Systems in Food Vacuoles of Cellular Slime Molds Predatory upon Bacteria.

Hohl, Hans R. (University of Hawaii, Honolulu). Nature and development of membrane systems in food vacuoles of cellular slime molds predatory upon bacteria. J. Bacteriol. 90:755-765. 1965.-During the digestion of bacteria by the myxamoebae of cellular slime molds, systems of concentric lamellae begin to appear within the food vacuoles. Each constituent lamella is a unit membrane of 75 to 85 A thickness. A study of these lamellae in Dictyostelium discoideum and Polysphondylium pallidum reveals that most of them do not represent original membranes of the ingested bacteria but are formed mainly in two ways. (i) After swelling and partial digestion of the bacteria, the first membranes appear adjacent to pre-existing membranes, e.g., the membrane lining the food vacuole and the cytoplasmic membrane surrounding the bacterium. Progressive addition of lamellae leads to the formation of the systems of concentric lamellae. (ii) After digestion of the bacteria has proceeded to a high degree, the concentric lamellae are formed spontaneously from clouds of amorphous material through condensation and orientation of precursor material. The study shows that, in biological systems, unit membranes may be formed from amorphous material through template action of pre-existing membranes, and does not necessarily involve fusion of membrane-bound vesicles.     

Giardia muris: ultrastructural analysis of in vitro excystation

Giardia muris cysts were examined by transmission electron microscopy before treatment, after induction, and at timed intervals during the incubation phase of in vitro excystation. Untreated G. muris cysts had a thick cyst wall composed of a fibrous outer wall and a thin, electron-dense inner membrane which extended from the trophozoite plasma membrane. The cytoplasm was devoid of endoplasmic reticulum, Golgi bodies,and mitochondria. Numerous large vacuoles were present within the ectoplasm just beneath the plasma membrane in untreated cysts. Following induction these cysts lacked ectoplasmic vacuoles. Concurrently, numerous membrane bound vesicles were seen in the peritrophic space closely adhering to the surface of the trophozoite. These vesicles appear to be of cytoplasmic origin. The cytoplasm of fully excysted trophozoites lacked ectoplasmic vacuoles but displayed well-developed ribbons of microtubular bodies, probably precursors of ventral disk, lateral flange, and median bodies and also contained extensive granular endoplasmic reticulum. No more than two nuclei were observed within each organism. The earliest excysted organisms were observed 0-5 min after incubation had begun and most organisms had excysted within 10 min. Cytokinesis occurred only after excystation was complete.