Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Pattern of DNA synthesis in nuclei of feeding and starving Amoeba proteus : A cytofluorometric study

The DNA content in isolated nuclei of Amoeba proteus was determined for each of the three groups of synchronized amoebae over different intervals after division. Several nuclei of each amoeba group were fixed 1 h after division, before the amoebae were fed. About h after division, some amoebae in each group were given food (Tetrahymena pyriformis), while the rest were left starving. Samples of the nuclei of fed and starved amoebae were fixed 24 h and (in different groups) 42–55 h after division. In each group from 22 to 48% of the fed amoebae had divided prior to the last nuclei fixation. Starved amoebae did not undergo division. In all three amoeba groups the nuclear DNA content of fed cells by the end of interphase had increased to 280–300% the value for 1 h amoebae. The nuclear DNA content of starved amoebae of all three groups was also increased, and in two groups it exceeded the initial level more than two-fold. However, in all three groups, it was lower than that of fed amoebae. In all the groups the nuclear DNA content in fed amoebae grew after 24 h, i.e. during the second half of interphase, the increase accounting for from 11 to 48% of the total increase. The hypothesis is put forward that the increase in the nuclear DNA content during the cell cycle of Amoeba proteus is the result of two processes: (1) one-time replication of the DNA of the whole genome; and (2) repeated replication of some part of the DNA. In amoebae the relation of the pattern of nuclear DNA synthesis to the diet is considered.     

Ultrastructure of the Giant Amoeba Pelomyxa palustris*

SYNOPSIS. The ultrastructure of the herbivorous amoeba Pelomyxapalustris was studied. Nuclear division is not understood in this amoeba, and evidence for the method of nuclear division was sought. This species typically has many spheroidal nuclei which are similar within a given cell. However, some amoebae from our collections differed from this common type in both the number and structure of their nuclei. This suggested stages associated with nuclear division. One current hypothesis of nuclear division in this organism is that of nuclear budding. Our evidence is more in accord with this method than with mitosis. The cytoplasm contained no mitochondria, Golgi bodies, contractile vacuoles or crystals. Most amoebae had 2 types of bacteria (bacteroids or endosymbionts) in their cytoplasm; a separate vesicle enclosed each of these. Characteristically, only 1 type of bacterium (B_n) surrounded the nucleus. Another type (B) was found elsewhere in the cytoplasm. Also in the cytoplasm were the following: food vacuoles enclosing various algae, relatively clear vacuoles and vesicles, glycogen, various electron-opaque particles, and occasional microtubules. The plasmalemma was smooth, lacking the external fringe which characterizes other large fresh-water amoebae.     

Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.     

Developmental features of calcium oxalate crystal sand deposition inBeta vulgaris L. Leaves

Summary Sugar beet (Beta vulgaris L.) leaf has a layer of cells extended laterally between the palisade parenchyma and spongy mesophyll that develop numerous small crystals (crystal sand) within their vacuoles. Solubility studies and histochemical staining indicate the crystals are calcium oxalate. The crystals are deposited within the vacuoles early during leaf development, and at maturity the cells are roughly spherical in shape and 2 to 3 times larger than other mesophyll cells. Crystal deposition is preceeded by formation of membrane vesicles within the vacuole. The membranes are synthesizedde novo in the vacuole and have a typical trilaminate structure as viewed with the TEM. The membranes are formed within paracrystalline aggregates of tubular particles (6–8nm outer diameter) as membrane sheets, but are later organized into chambers or vesicles. Calcium oxalate is then precipitated within the membrane chambers. The tubular particles involved in membrane synthesis are usually present in the vacuoles of mature crystal cells, but in very small amounts.     

Symbiotic Chlorella vulgaris of the ciliate Paramecium bursaria plays an important role in maintaining perialgal vacuole membrane functions

Treatment of symbiotic alga-bearing Paramecium bursaria cells with a protein synthesis inhibitor, cycloheximide, induces synchronous swelling of all perialgal vacuoles at about 24h after treatment under a constant light condition. Subsequently, the vacuoles detach from the host cell cortex. The algae in the vacuoles are digested by the host's lysosomal fusion to the vacuoles. To elucidate the timing of algal degeneration, P. bursaria cells were treated with cycloheximide under a constant light condition. Then the cells were observed using transmission electron microscopy. Results show that algal chloroplasts and nuclei degenerated within 9h after treatment, but before the synchronous swelling of the perialgal vacuole and appearance of acid phosphatase activity in the perialgal vacuole by lysosomal fusion. Treatment with cycloheximide under a constant dark condition and treatment with chloramphenicol under a constant light condition induced neither synchronous swelling of the vacuoles nor digestion of the algae inside the vacuoles. These results demonstrate that algal proteins synthesized during photosynthesis are necessary to maintain chloroplastic and nuclear structures, and that inhibition of protein synthesis induces rapid lysis of these organelles, after which synchronous swelling of the perialgal vacuole and fusion occur with the host lysosomes.     

Membrane markers of endoplasmic reticulum preserved in autophagic vacuolar membranes isolated from leupeptin-administered rat liver

We isolated membranes from leupeptin-induced autophagic vacuoles and compared them with lysosomal membranes purified from dextran-administered rats. In protein composition, autophagic vacuole membranes prepared from long term-starved (36 h) rats bear marked resemblance to lysosomal membranes, whereas vacuole membranes prepared from short term-starved (12 h) animals differ significantly from lysosomal membranes. Immunoblotting analyses showed that only autophagic vacuole membranes from short term-starved rats possess endoplasmic reticulum markers such as cytochrome P450 and NADPH-cytochrome c reductase. None of the membranes contain sialyltransferase, a Golgi membrane marker. In experiments in which rats were starved after feeding to induce autophagy, the appearance of the endoplasmic reticulum markers occurred during 6-12 h of starvation, concomitantly with increases in vacuolar proteins and sequestered cytosolic aldolase. The endoplasmic reticulum membrane markers and sequestered aldolase declined gradually after 20-36 h of starvation, suggesting that prolonged starvation causes no further increase in the formation of autophagic vacuoles but an increase in the population of matured autophagic vacuoles. Thus, the prominent markers of endoplasmic reticulum from which autophagosomes originate are well preserved in autophagic vacuole membranes, and retention of these markers is highly dependent on the formation and subsequent maturation process of autophagic vacuoles.     

Fate of the nucleolar vacuole during resumption of cell cycle in pea cotyledonary buds

Summary Meristematic cells of pea cotyledonary buds blocked in G_0–1 state contain a small nucleolus with a large central clear area surrounded by a fibrillar rim. The nucleolar structure varies according to the cell cycle from the G_0–1-blocked state until the first mitoses occurring between 24 and 27h after removal of the main stem. In order to better identify and understand the role of the central area in the nucleolar function, its content was investigated by cytochemical and terminal deoxynucleotidyl transferase-immunogold methods. The central area showed the characteristics of a vacuole commonly constituted of the condensed chromatin, ribonucleoprotein granules, and lack of argyrophilic proteins. 3 h after decapitation, a thickening of the fibrillar rim occurred, accompanied by an increase of granules in the vacuole. After 6h, the unique vacuole broke up into two to four small vacuoles in which the granules are more abundant. After 12 h the nucleolus acquired compact structure with few minute vacuoles dispersed over the fibrillar component. During the whole cell cycle, the condensed chromatin is always observed in the vacuole. Our findings suggest that the appearance of the vacuoles is subsequent to the output of preribosomes from nucleolus. These vacuoles might play a role in condensation and decondensation of the chromatin.     

Neuronal cell death in transmissible spongiform encephalopathies (prion diseases) revisited: from apoptosis to autophagy

Neuronal autophagy, like apoptosis, is one of the mechanisms of the programmed cell death (PCD). In this review, we summarize the presence of autophagic vacuoles in experimentally induced scrapie, Creutzfeldt–Jakob disease and Gerstmann–Sträussler–Scheinker (GSS) syndrome. Initially, a part of the neuronal cytoplasm was sequestrated by concentric arrays of double membranes; the enclosed cytoplasm appeared relatively normal except that its density was often increased. Next, electron density of the central area dramatically increased. The membranes then proliferated within the cytoplasm in a labyrinth-like manner and the area sequestrated by these membranes enlarged into a more complex structure consisting of vacuoles, electron-dense areas and areas of normally-looking cytoplasm connected by convoluted membranes. Of note, autophagic vacuoles form not only in neuronal perikarya but also in neurites and synapses. Finally, a large area of the cytoplasm was transformed into a collection of autophagic vacuoles of different sizes. On a basis of ultrastructural studies, we suggest that autophagy plays a major role in transmissible spongiform encephalopathies (TSEs) and may even participate in a formation of spongiform change.     

Zur Digestion bei Pseudomicrothorax dubius Mermod Nahrungsvakuolen-Vesikulation (Ciliophora) im Anschluß an die Phagocytose

Zusammenfassung Der Schlinger Pseudomicrothorax dubius ingestiert innerhalb von 1–2 min ein großes Volumen fädiger Blaualgen. Die Nahrung ist unmittelbar nach dieser rapiden Phagocytose in einer einzigen, sehr großen Vakuole eingeschlossen, die fast den ganzen Ciliaten ausfüllt. Im Verlaufe der folgenden Stunde vesikuliert diese große Nahrungsvakuole über Zwischenstufen zu einer Vielzahl von Vakuolen mit 1–2 m Durchmesser. Gleichzeitig erfolgt eine Kondensierung des Vakuoleninhaltes. Erst zu diesem Zeitpunkt setzt die Verdauung der Nahrung ein, wie an Hand von zahlreichen Dictyosomen belegt wird, die nun in unmittelbarer Nähe der Nahrungsvakuolen nachzuweisen sind. Durch die Vesikulation der großen Nahrungsvakuole in kleinere Einheiten sowie durch die Kondensierung der Nahrung wird bewirkt, daß die über Lysosomen in die Nahrungsvakuolen abgegebenen Verdauungsenzyme optimal eingesetzt werden. Nach Beendigung der Verdauung liegen viele leere Vakuolen vor, die durch eine stark gefaltete Kontur gekennzeichnet sind. Diese Vakuolen gehen allem Anschein nach wieder in den Membranhaushalt der Zelle ein.

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On the digestion in Pseudomicrothorax dubius Mermod (Ciliophora) vesiculation of the food vacuole following phagocytosis

Summary The gulper Pseudomicrothorax dubius ingests a large volume of filamentous blue-green algae within 1–2 min. Immediately after this rapid phagocytosis, the food is enclosed in a single, extremely large food vacuole, which fills up the ciliate almost entirely. During the following hour this giant food vacuole vesiculates. Finally numerous small vacuoles are present, 1–2 m in diam. Simultaneously the content of the vacuoles is noticeably condensed. At this time the digestion of the food starts as is indicated by numerous dictyosomes, which now surround the periphery of the food vacuoles. Due to both, the prior vesiculation of the food vacuole and the condensation of the food, the digestive enzymes can act very effectively. After 6–8 hours, when the digestion of the food is finished, numerous empty vacuoles are found. Each is characterized by a highly irregular, convoluted outline. Apparently these vacuoles are eventually recycled to the membrane pool of the cell.

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Für umsichtige und sorgfältige technische Assistenz danke ich Frl. A. Rüskens. Die Deutsche Forschungsgemeinschaft unterstützte diese Untersuchung durch die Sachbeihilfe Ha 818/7     

Freeze-fracture study of Blastocystis hominis

The ultrastructure of Blastocystis hominis was investigated by the freeze-fracture method. Freeze-fracture replicas of the membranes of B. hominis and its organelles were studied with special regard to the density and distribution of the intramembranous particles (IMP's). On all membrane replicas, the concentration of IMP's on the protoplasmic face (P face) invariably was greater than on the exoplasmic face (E face). On the P face, IMP's were heterogeneously distributed in dense aggregates, alternating with particle-free, smooth surface areas. Occasionally, small depressions and protrusions were observed in these areas. On the membrane of the central vacuole, invaginations into the vacuole were frequently observed within the smooth surface regions. Since most of the granules in the central vacuoles had no IMP's, it seems likely that the intervacuolar granules were formed from these invaginations of the vacuole membrane. The width of the intermembrane space between the inner and outer membranes of the nuclear envelope was uneven, with regions of relative narrowness interspersed with regions of expansion. Nuclear pores were localized within the narrow portions of this space. A nucleus, apparently in the process of dividing, was observed enclosed within an intact outer membrane. Division of the outer membrane would then result in the formation of two discrete nuclei.     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Freeze-Fracture Study of Blastocystis hominis1

The ultrastructure of Blastocystis hominis was investigated by the freeze-fracture method. Freeze-fracture replicas of the membranes of B. hominis and its organelles were studied with special regard to the density and distribution of the intramembranous particles (IMF's). On all membrane replicas, the concentration of IMF's on the protoplasmic face (P face) invariably was greater than on the exoplasmic face (E face). On the P face, IMP's were heterogeneously distributed in dense aggregates, alternating with particle-free, smooth surface areas. Occasionally, small depressions and protrusions were observed in these areas. On the membrane of the central vacuole, invaginations into the vacuole were frequently observed within the smooth surface regions. Since most of the granules in the central vacuoles had no IMF's, it seems likely that the intervacuolar granules were formed from these invaginations of the vacuole membrane. The width of the intermembrane space between the inner and outer membranes of the nuclear envelope was uneven, with regions of relative narrowness interspersed with regions of expansion. Nuclear pores were localized within the narrow portions of this space. A nucleus, apparently in the process of dividing, was observed enclosed within an intact outer membrane. Division of the outer membrane would then result in the formation of two discrete nuclei.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

Lysosomal Activities of the Vacuole in Damaged and Recovering Plant Cells

ON the evidence from cytochemical studies it seems that a wide range of lysosomal enzymes is present in the vacuole of plant cells^1,2. Cytological studies have also suggested that biochemically identified lysosomal particles are equivalent to small vacuoles and because membranous material has been seen within these vacuoles it was suggested that the plant cell vacuole is functionally equivalent to the secondary lysosome of the animal cell³. In this role it would be engaged in the. breakdown of cytoplasmic constituents and, using light microscope cinematography, it has been shown that after certain experimental environmental changes, structures appeared and then disappeared in the vacuoles of yeast cells⁴. Generally, electron micrographs of plant cells have remnants of disorganized membranes within their vacuoles, which have consequently come to be regarded as repositories of waste materials.     

Theonellamide F, a Bicyclic Peptide Marine Toxin, Induces Formation of Vacuoles in 3Y1 Rat Embryonic Fibroblast

The effect of a marine bicyclic peptide, theonellamide F, on vacuole formation in exponentially growing 3Y1 rat embryonic fibroblasts was studied in comparison with the effect of monensin. Many abnormally large vacuoles appeared around the nucleus in the cells treated with 6 μM theonellamide F within 24 hours. Following prolonged treatment with this agent, the number of enlarged vacuoles increased. Such vacuoles accumulated many granules that showed Brownian movement. The effect of theonellamide F on the cells was more drastic in an amino-acid-deficient medium, in which all cells died within 1 hour at a 3-μM concentration of the agent. Theonellamide F probably affects cellular autophagy, inhibiting the degradation of the organelles and turnover of proteins. Monensin, a well-known Na⁺ ionophore that disintegrates the Golgi apparatus, induced similar types of vacuole formation, although the vacuoles were localized in a region slightly distant from the nucleus. Monensin readily affected cell morphology, resulting in cell death. We propose that theonellamide F, like monensin, is a useful agent for investigating membrane structures in cells. Received November 25, 1998; accepted February 10, 1999     

Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga

We have previously demonstrated that isolates of the Burkholderia cepacia complex can survive intracellularly in murine macrophages and in free-living Acanthamoeba. In this work, we show that the clinical isolates B. vietnamiensis strain CEP040 and B. cenocepacia H111 survived but did not replicate within vacuoles of A. polyphaga. B. cepacia-containing vacuoles accumulated the fluid phase marker Lysosensor Blue and displayed strong blue fluorescence, indicating that they had low pH. In contrast, the majority of intracellular bacteria within amoebae treated with the V-ATPse inhibitor bafilomycin A1 localized in vacuoles that did not fluoresce with Lysosensor Blue. Experiments using bacteria fluorescently labelled with chloromethylfluorescein diacetate demonstrated that intracellular bacteria remained viable for at least 24 h. In contrast, Escherichia coli did not survive within amoebae after 2 h post infection. Furthermore, intracellular B. vietnamiensis CEP040 retained green fluorescent protein within the bacterial cytoplasm, while this protein rapidly escaped from the cytosol of phagocytized heat-killed bacteria into the vacuolar lumen. Transmission electron microscopy analysis confirmed that intracellular Burkholderia cells were structurally intact. In addition, both Legionella pneumophila- and B. vietnamiensis-containing vacuoles did not accumulate cationized ferritin, a compound that localizes within the lysosome. Thus, our observations support the notion that B. cepacia complex isolates can use amoebae as a reservoir in the environment by surviving without intracellular replication within an acidic vacuole that is distinct from the lysosomal compartment.     

Morphological study of cysts of Pelomyxa palustris Greeff, 1874

Pelomyxa palustris Greeff, 1874, is the only representative of pelomyxoid amoebae with rest cysts in its life cycle. The morphology of the P. palustris cysts was studied using light and electron microscopy. The encystation of P. palustris under the climatic conditions of northwestern Russia occurs in August through September. The rest of the cysts have complex, trilaminar walls. The most developed are the two inner layers, i.e., the electron-dense structureless endocyst and the laminated mesocyst; the thickness of each layer can reach 0.6–0.7 μm. The thickness of the superficial electron-dense ectocyst lamina usually does not exceed 0.1–0.2 μm. The encysted cell of P. palustris has a unique structure. About 60% of its cell volume is occupied by a giant central vacuole filled with prokaryotic cytobionts. This vacuole has also been found to contain vacuoles and vesicles of different natures, restricted by vacuole membranes, autophagosomes, and lipid droplets. The amoeba cytoplasm occupies the space between the endocyst inner surface and the central vacuole. It contains no inclusions, prokaryotic cytobionts and most of cell organelles. In the cytoplasm there are 4 large nuclei filled with relatively homogeneous karyoplasm. The nuclear envelope forms numerous long tubular outgrowths, piercing the cytoplasm and underlining the central vacuole membrane. In this state the encysted pelomyxoids survive until the beginning of excystation. The excystation of P. palustris in the studied region occurs in spring, during the second half of April through the beginning of May. The cysts undergo complex morphofunctional changes due to reorganization of the wall and formation of young multinucleate amoebae. Out of the three initial lamina of the wall, only one persists until the moment of encystation. The central vacuole is destroyed and its content penetrates into the cytoplasm. Pelomyxoid nuclei divide twice. Prokaryotic cytobionts are localized in the cytoplasm and in the perinuclear area. Young multinuclear P. palustris individuals exiting cysts do not differ from the adult forms by their organization.     

The Supramolecular Organization of Red Algal Vacuole Membrane Visualized by Freeze-fracture

The supramolecular organization of the vacuole membrane (orof the membranes of mucilage sacs) in 27 species of red algaeis studied in replicas of rapidly frozen and fractured cells.Intramembranous particle complexes composed of four particles('tetrads' with average diameters between 8·5 and 14·5have been observed in the protoplasmic fracture (PF) face butmost clearly and more frequently in the exoplasmic fracture(EF) face of the vacuole membrane of all red algae investigated.The tetrads lie individually within the vacuole membrane orform clusters in several species and are randomly distributed.In the species Ceramium diaphanum var. strictum and Laurenciaobtusa the intramembranous particle complexes ('tetrads') havebeen observed both in the EF and PF faces of the vacuole membrane;the 'membrane tetrads' at least as regards these two speciesseem to span both the outer and inner leaflets of the vacuolemembrane ('transmembrane particles'). The occurrence of particletetrads in the plasma membrane is probably due to exocytosiseither of the Golgi vesicles or of the mucilage sacs. Tetradfrequency in the EF face of the vacuole membranes of the investigatedred algae varies between 2 and 87 µm^-2, while that ofsingle particles varies between 102 and 695 µm^-2. ThePF face of the vacuole membrane is characterized by a higherparticle density than the EF face. The particle densities ofthe PF and EF faces of the plasma membrane for a given speciesare higher than those of the corresponding fracture faces ofthe vacuole membrane. Some members of Bangiophycidae bear smallerprotein particles (diameter between 8·5 and 10·5nm) in comparison with those of Florideophycidae (diameter between10·5 and 14·5 nm). It is suggested, based uponthe particle tetrads lying in depressions of the vacuole membraneand the origin of vacuoles (mucilage sacs) from ER, that theparticle tetrads originate from the ER or the Golgi complex.Since vacuoles (mucilage sacs) in red algae, along with theGolgi complex, are involved in the synthesis and export of cellsurface polysaccharides, it could be assumed that the 'membrane-tetrads'within the vacuole membrane represent a membrane-bound multienzymecomplex, participating in the synthesis of amorphous extracellularmatrix polysaccharides.Copyright 1993, 1999 Academic Press Red algae, freeze-fracture, vacuole membrane, mucilage sacs, membrane tetrads, supramolecular organization     

Sustained Food Vacuole Formation by Axenic Paramecium tetraurelia and the Inhibition of Membrane Recycling by Alcian Blue

It is believed that the uptake mechanism of some nutrients by Paramecium tetraurelia primarily involves transport through the cell surface, whereas the uptake of other compounds appears to be restricted to bulk transport during food vacuole (phagosome) formation. In this study, we established that, in axenically grown cells, food vacuole formation occurred at continuous rates over long periods. This information allows quantitation of the volume of media taken up by bulk transport. India ink and latex beads were shown to be inert food vacuole markers and carmine was found to have an initial stimulatory effect on phagosome formation rates. Cultures grown for 3.5 h or longer with the glycocalyx stain Alcian Blue, contained only three phagosomes/cell, whereas cells cultured with the other markers contained 15 phagosomes/cell. Electron microscopy of fecal material that accumulated at the bottom of Alcian Blue-grown cells demonstrated the presence of membranes, suggesting that the vacuolar membrane was eliminated during defecation. Neither cell lysis nor the formation of autophagous vacuoles was detected in Alcian Blue-grown cells, indicating that the stain was not cytotoxic at the concentrations used. Thus it appeared that the binding of Alcian Blue to the digestive vacuole membrane resulted in a loss of the vacuole membranes from the cell which reduced the amount of membranes retrieved and recycled and hence eventually reduced the rate of phagosome formation. Alcian Blue-treated cultures exhibited decreased rate of growth and final density, which is consistent with a decrease in bulk transport of nutrients resulting from reduced membranes of digestive cycle organelles available in the cell.