Temporal movement of digestive vacuoles in fedTetrahymena pyriformis GL-9

Summary Studies using cells ofTetrahymena pyriformis GL-9 (which had been grown in and maintained in proteose-peptone/yeast extract medium and then presented first with 0.48 m diameter latex particles for 70 minutes and then with excess carmine particles, or with the particles added in reverse sequence) have shown that there is temporal sequencing in the movement of digestive vacuoles. Under the conditions employed the digestive vacuoles are egested from the cells in approximately the order in which they were formed.     

Periodicity of endocytosis inTetrahymena pyriformis

Summary Starved cells ofTetrahymena pyriformis show marked periodicity in the rates of uptake of digestive vacuoles when presented with a mixture of peptone and polystyrene latex particles. Rates of egestion also show some periodicity. The results show that the supply of membrane is not the sole factor (and possibly not even a main factor) restricting vacuolar uptake. The rates of egestion also do not appear to be greatly influenced by the amounts of digestive vacuole membrane to be reincorporated into the cell membrane. It is suggested that energy and lysosomal supplies may be more important than membrane supply for uptake and that reduction in the supply of uncombined lysosomes may in some way cause cessation of vacuole formation.Fed cells ofTetrahymena also show variations in the rate of digestive vacuolar uptake with time. The maximum rate of uptake is about three times as great as that found in starved cells.     

Studies on Feeding and Digestion in Protozoa. VII. Ingestion of Polystyrene Latex Particles and its Early Effect on Acid Phosphatase in Paramecium multimicronucleatum and Tetrahymena pyriformis

SYNOPSIS. Food vacuole-free P. multimicronucleatum and T. pyriformis readily ingest non-nutritive Dow polystyrene latex particles (PLP) and form vacuoles containing PLP at a rate comparable to the formation of vacuoles containing bacteria. The particles aggregate within the vacuoles and are egested as balls of the size of the vacuoles. PLP containing vacuoles rapidly acquire acid phosphatase activity, which is demonstrated by histochemical (alpha-naphthyl phosphatehexazonium salt or lead phosphate) methods as a peripheric staining. The total activity of the cell does not significantly change as a consequence of PLP uptake as suggested by the histochemical preparations and confirmed in T. pyriformis by measuring the splitting of p-nitrophenyl phosphate at pH 5. Accordingly, no selection between nutritive and non-nutritive particles could be revealed. The vacuole formation is induced by the mechanical action of the particles. The appearance of acid phosphatase activity in the vacuole seems to be dependent on the vacuole formation and not on its content. This early appearance of activity is due to a redistribution of the preexistent activity.     

Food ingestion and egestion in mating reactive populations of Paramecium primaurelia.

The study of food ingestion and egestion carried out on Paramecium primaurelia mating reactive cells shows that, after their transfer into a medium with suspended particles, the complementary mating type cells exhibit very significant differences in the food vacuole formation and egestion rate. Under the same external environmental conditions, the mating type II cells form and egest a higher number of food vacuoles when compared with mating type I cells. The higher rate of food vacuole formation shown by the mating type II cells is related to their faster growth rate.     

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.     

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.     

The effect of puromycin and cycloheximide on vacuole formation and exocytosis in Tetrahymena pyriformis GL-9

It has been shown that both puromycin and cycloheximide, at concentrations of 434 and 100 g/ml respectively, produce a marked inhibition of vacuole formation and exocytosis in Tetrahymena pyriformis GL-9. These effects were analysed in a quantitative manner. At the same time as these inhibitions occurred the incorporation of 1-C¹⁴ leucine into trichloroacetic acid precipitable material was inhibited by 90% and 100% respectively over a 40 min period. This inhibition of protein synthesis by cycloheximide occurred almost immediately, whereas the inhibition of vacuole formation and egestion was delayed. The results suggested that the latter processes were dependent upon a continuing supply of proteinaceous material, of which there was only a small store within the cell. Cycloheximide inhibited exocytosis completely under the conditions employed (with 100% inhibition of protein synthesis) whereas puromycin (with a 90% inhibition of protein synthesis) only inhibited it by about 50%. This suggested that the amount of newly synthesized protein required for the exocytic egestion process was very small in relation to the total cell requirement for protein synthesis. The entry of both inhibitors into the cell was by means other than vacuole formation. Puromycin appeared to have some effect on vacuole formation which was unconnected with protein synthesis. Microscopic observations of living cells indicated that oral apparatus function and endocytic vacuole formation were probably both affected by the inhibitors. Chloramphenicol, at 200 g/ml, had little effect on vacuole formation by starved cells with an exposure of an hour. The uptake of 1-C¹⁴ leucine from the growth medium was found to be a selective process, giving a concentration of about 2000 times into the cells over a 1 hr period. The results are discussed.     

On Food Vacuoles in Tetrahymena pyriformis GL

SYNOPSIS. The following problems concerning food vacuoles were studied by in vivo observations of Tetrahymena: (A) Formation of food vacuoles . The process may be divided into 4 stages. Stage 1—gradual growth of the limiting membrane of the open food vacuole (of short duration). Stage 2—"filling up" of the fully expanded vacuole (of long duration). Stage 3—"closing off" of the vacuole (of brief duration). Stage 4—initial movement of the detached vacuole away from the cy-tostome. The possible role of the oral components (apart from membranellar beating) in the process is discussed. (B) Change of pH in the food vacuole . After ingestion of heat-killed yeast stained with indicator dyes (neutral red, bromcresol purple, bromcresol green, bromphenol blue), the observed color changes indicate that pH is neutral in the forming vacuole as well as in newly formed vacuoles; that a pH value of 6.0–5.5 is reached after ∼ 5 min; and that the lowest pH value between 4.0 and 3.5 is reached after 1 hr. Before egestion the pH again increases. (C) Length of the digestive cycle . A determination of the time required to deplete the cells of labeled vacuoles formed during a short exposure, was attempted. Defecation was observed after 1/2 hr and it was frequent after 2 hr. About 25% and 50% of the labeled vacuoles were removed after 1 hr and 2 hr, respectively; however, labeled vacuoles may still be seen in some cells 6 hr after ingestion. The conclusion is that the digestive cycle lasts ∼ 2 hr and that egestion of undigestible material is a random process.     

Confusing Selective Feeding with Differential Digestion in Bacterivorous Nanoflagellates

Food selectivity and the mechanisms of food selection were analyzed by video microscopy for three species (Spumella, Ochromonas, Cafeteria) of interception-feeding heterotrophic nanoflagellates. The fate of individual prey particles, either live bacteria and/or inert particles, was recorded during the different stages of the particle-flagellate-interaction, which included capture, ingestion, digestion, and egestion. The experiments revealed species-specific differences and new insights into the underlying mechanisms of particle selection by bacterivorous flagellates. When beads and bacteria were offered simultaneously, both particles were ingested unselectively at similar rates. However, the chrysomonads Spumella and Ochromonas egested the inert beads after a vacuole passage time of only 2-3 min, which resulted in an increasing proportion of bacteria in the food vacuoles. Vacuole passage time for starved flagellates was significantly longer compared to that of exponential-phase flagellates for Spumella and Ochromonas. The bicosoecid Cafeteria stored all ingested particles, beads as well as bacteria, in food vacuoles for more then 30 min. Therefore "selective digestion" is one main mechanism responsible for differential processing of prey particles. This selection mechanism may explain some discrepancies of former experiments using inert particles as bacterial surrogates for measuring bacterivory.     

The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce "tails" in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.     

Retention of lead within the digestive vacuole inTetrahymena

Summary The ciliateTetrahymena pyriformis was exposed to lead acetate. Cell proliferation in the presence of 0.1% lead salt (with or without EDTA) equaled, after a variable lag period, that of the control cells. The lead (550 ppm) forms a fluffy precipitate with the organic growth medium; this was in part prevented by addition of EDTA. The cells primarily ingested the fluffy precipitate whereby they became exposed to large amounts of lead. Within the digestive vacuole, the fluffy precipitate became converted into refractile structures (3 m in diameter) which were egested and accumulated at the bottom of the culture flask. The lead content of these defecation balls was higher than that of the fluffy precipitate. In addition to the lead-containing vacuoles, the cells contained small, refractile granules. The apparent, high tolerance ofTetrahymena towards lead is believed to be due in part to the low ionic concentration of lead under the present conditions and in part to a detoxication mechanism consisting of retention of lead within the digestive vacuoles and perhaps of accumulation of lead within the small, refractile granules.     

Lysosomes of root tip cells in corn seedlings

Summary Nine acid hydrolases are present in lysosomes which are found in the mitochondrial fraction of a cell-free extract prepared from root tips of corn seedlings.Light and heavy lysosomes can be distinguished. The latter are sedimentable in a sucrose-medium, the former only in sorbitol-medium. The fraction of heavy lysosomes is in turn composed of at least three populations of lysosomes differing in density and enzyme content.Light lysosomes are membrane-bound particles with diameters from 0.3 to 1.5 . Electron micrographs of frozen-etched tissue and isolated particles provide evidence that light lysosomes are identical with small vacuoles. This type of lysosome is characterized by presence of transaminases in addition to that of hydrolases. Heavy lysosomes are small spheres (diameters from 0.1–0.3 ) with membranes resembling those of vacuoles and of the endoplasmic reticulum. These lysosomes are characterized by high specific activities of two oxydoreductases known to occur also in the membranes of the reticulum.The different types of particles are thought to represent stages of the development of the lysosomal apparatus; according to this hypothesis the large vacuole of parenchymatous cells represents the end product of this process.     

Digestive system membranes: freeze-fracture evidence for differentiation and flow in Paramecium

Freeze-fractured membranes of digestive vacuoles of randomly feeding Paramecium caudatum exhibit dramatic differences in intramembrane particle (IMP) number and distribution on both E- and P-fracture faces. By pulse-feeding latex spheres to cells we have demonstrated that these differences are related to the age of the digestive vacuoles, and that the membranes of such vacuoles undergo a specific sequence of changes during the digestive cycle. Young digestive vacuoles (DV-I; less than or equal to 6 min), nascent vacuoles still connected to the cytopharynx, and discoidal vesicles, from which vacuole membrane is derived, all have a highly particulate E face and a less particulate P face. As early as 3 min after feeding, a second category of digestive vacuoles (DV-II) can be recognized, which are both considerably smaller in diameter and lack particles on their E face. These findings suggest that the endocytic removal of DV-I membrane material associated with the formation of DV-II vacuoles involves a concomitant and selective removal of E-face particles, as essentially no changes are seen in the density of P-face particles on the two types of vacuoles. Beginning at 10 min the first DV-III vacuoles are encountered. These are both larger than the DV-II vacuoles and possess very prominent E-face particles, which resemble those on the E face of the numerous lysosomes bordering the digestive vacuoles. DV-III vacuoles also exhibit a substantial increase in P-face particles. These membrane changes closely parallel, and are probably correlated with, the physiological events occurring within the vacuole lumen: concentration of food, killing of prey, and digestion. Calculations of the amount of membrane removed from DV-I to form DV-II and of the increase in membrane surface area during the transition from DV-II to DV-III indicate that as much as 90% of the initial phagosome (DV-I) membrane can be removed before digestion begins. The enlargment of DV-II must be caused by fusion with adjacent lysosomes which also contribute the new populations of IMPs to the DV- III membrane. The appearance of numerous endocytic structures on older DV-III vacuoles suggests that membrane is retrieved from DV-III before defecation.     

Symbiotic <Emphasis Type="Italic">Chlorella variabilis</Emphasis> strain, 1 N,can influence the digestive process in the host <Emphasis Type="Italic">Paramecium bursaria</Emphasis> during early infection

The association between the ciliate Paramecium bursaria and symbiotic Chlorella spp. is mutually beneficial. However, this relationship is facultative mutualism because both the host and the symbiotic algae can grow by themselves. This association is easily re-established by mixing the two species together. Following algal mixing, some algae become enclosed in the digestive vacuole membrane of the paramecia to which both acidosomes and lysosomes fuse. To establish endosymbiosis, some algae acquire temporal resistance to the host lysosomal enzymes in the digestive vacuoles. We examined whether the algae influence the differentiation of the host digestive process using LysoSensor staining to evaluate the acidification of the digestive vacuoles. Furthermore, to assess lysosomal fusion with the digestive vacuole, Gomori’s staining was conducted. Acidification and lysosomal fusion occurred later in digestive vacuoles containing living algae than in those containing boiled algae or latex spheres. This phenomenon was observed when the living algae were maintained under a constant light condition. These results suggest that the algae release some unknown factor in response to light exposure, and the factor may be associated with the alteration of the host digestive process, indicating that the living algae can influence the host digestive processes during early algal infection.     

Membrane differentiation in the cytoplasmic-tubule system of the intestinal absorptive cells of the lamprey,Lampetra japonica

Summary Specific membrane differentiation occurs in the cytoplasmic-tubule system of the absorptive cells lining the mucosa of the lamprey anterior intestine. The absorptive cells are characterized by the presence of abundant mitochondria and a system of well-developed cytoplasmic tubules (120 nm in diameter). The cytoplasmic tubules open on to the basolateral cell surface and contain numerous lipoprotein particles (50–100 nm diam.) in their lumina. Lipoprotein particles are also observed in the endoplasmic reticulum and the Golgi complex, and they are transfered to the lateral intercellular space and lamina propria by way of the cytoplasmic tubules. Spirally-wound parallel rows of particles are found in the luminal surface of the cytoplasmic tubules. The rows are 17 nm apart and are wound spirally at a pitch of 210 nm. Freeze-fracture images of the tubule membranes also show spiral arrays of particles (9 nm in diameter) on the P-face, and complementary shallow grooves on the E-face. From these observations, it is suggested that the cytoplasmic-tubule system of the intestinal absorptive cells serves as a channel for the transport of synthesized lipoprotein into the interstitium, and is also the site of the ion and water exchange essential for the maintenance of ionic homeostasis.     

Intracellular digestion and symbiosis in Paramecium bursaria

Electron microscopic cytochemical methods reveal that acid phosphatase activity appears exclusively in vacuoles containing recently ingested bacteria or inert particles such as carmine, Celkate or latex spheres, and not in the vacuoles surrounding established symbionts. Although newly ingested symbiotic algae are digested in large numbers, some remain to reestablish the symbiosis. Since symbiotic algae are able to delay the digestion of heat-killed algae when they coexist in a phagosome, we propose that symbiotic Chlorella actively interfere with an early event in the host digestive process.     

The Induction of Endocytosis in Starved Tetrahymena pyriformis

SYNOPSIS. The formation of digestive vacuoles by starved Tetrahymena pyriformis could be induced by mixtures of latex particles and a variety of potentially digestible solutes. Latex particles themselves had little effect in inducing vacuole formation. Protein, polypeptide, and RNA were highly effective inducers, while glutamate, amino acid mixtures, polysacharides, and glucose were moderately effective. Sodium-β-glycerophosphate had a slight effect and sodium acetate was ineffective. The possible stimulus to endocytosis is discussed. The endocytic response to inducers does not appear to be an all-or-none phenomenon and varies with the concentration of inducer. The stimulatory effect for protein-related inducers seems to be produced by a large number of stimulatory molecules acting upon a single cell and the magnitude of the response appears to be related to molecular size.     

Ultrastructural investigation of spicule formation in the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Ultrastructural examination of original and regenerated branch tips of the gorgonianLeptogorgia virgulata reveals that spicule formation begins with the aggregation of scleroblasts in the mesoglea. Calcite crystal deposition occurs within a Golgi vacuole containing organic matrix. Vacuole size increases while matrix incorporation and subsequent crystal growth continue, filling the vacuole. At approximately this time, the scleroblasts dissociate and wart formation begins. Further spicule growth stretches the cell into a thin envelope. Fusion of vacuole and plasma membrane followed by breach formation during spicule growth, as well as scleroblast atrophy or migration from mature spicules, result in the transition of the spicule from the intracellular to the extracellular environment. The results also reveal aborted spicules and digestive bodies, implying possible relationships among calcification, detoxification, and waste management.Contribution No 436, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

On the gas vacuoles of the halobacteria

Summary The cells of Halobacterium sp., strain 5, contain a large number of highly refractile bodies of the type which Petter (1932) suggested were gas-filled vacuoles. The present studies support Petter's contention, but the evidence for the exact chemical nature of the vacuole content is still indirect. It is not carbon dioxide or oxygen, but might possibly be nitrogen. Strain 5 loses spontaneously and with a high frequency the ability to make the vacuoles.When vacuolated cells are subjected to pressure, the vacuoles disappear, but can recover upon aeration. Oxygen and the organic constituents of the growth medium stimulate the recovery, whereas 2.4-dinitrophenol inhibits it. A procedure is described for the isolation of the vacuoles. The vacuoles are bounded by a membrane which reveals itself in electron micrographs of thin sections as a 1-layered structure about 30 Å thick.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.     

The content and chain length of polyphosphates from vacuoles of Saccharomyces cerevisiae VKM Y-1173

The content of inorganic polyphosphates (polyP) in vacuoles of the yeast Saccharomyces cerevisiae is 15% of the total cellular polyP. Over 80% of the vacuole polyP are in an acid-soluble fraction. It was first established by ³¹P-NMR spectroscopy that a polymeric degree (n) of two subfractions obtained by precipitation with Ba²⁺ in succession at pH 4.5 and 8.2 was approximately 20 ± 5 and 5 ± 2 residues of ortho-phosphoric acid, respectively. Under a deficit of phosphate (P_i) in the cultivation medium, the polyP content in vacuoles decreased 7-fold with the same drastic reduction of their content in the cell. Unlike intact yeast cells, where polyP overcompensation is observed after their transfer from phosphate-free to phosphate-containing medium, the vacuoles do not show this effect. The data indicate the occurrence of special regulatory mechanisms of polyP synthesis in vacuoles differing from those in the whole cell.