Food Vacuole Formation Research Articles

Growth requirements of a new food-vacuole-less mutant of Tetrahymena

The growth requirements of the ciliate Tetrahymena thermophila have been known for a long time. In this study we have investigated the additional requirements induced in a cell line defective in phagocytosis. The mutant cell line of T. thermophila, II 8 G, derived from the wildtype, T. thermophila, CU 399, has apparently no oral structures and forms no food vacuoles at 37°C, but has normal oral structures and the ability to form food vacuoles at normal rates at 28°C. Taking advantage of a synthetic nutrient medium, we observed that the mutant cell line shows increased requirement for Fe(III), Cu(++) and folic acid under restrictive conditions. Phagocytosis obviously plays a role in utilization of low concentrations of the three substances, whereas all other required substances can enter fast enough to support short generation times.

Effects of Glycogen on Food Vacuole Formation in Parameium caudatum

ABSTRACTParamecium caudatum syngen 3 could not reproduce in the defined medium (DM) which had been developed for P. octaurelia stock 299, but we succeeded in culturing it in DM supplemented with glycogen. The number of food vacuoles which formed in 5 and 10 min at 25°C in the DM alone was greater in comparison with the DM supplemented with glycogen. These results showed that the high molecular weight substance which needed to be added to a defined medium for the cultivation of Paramecium did not always support cell reproduction by stimulating food vacuole formation.

Cell Pairing during Mating in Tetrahymena: I. Does Phagocytosis or a Cell Surface Receptor Participate in Con A Block?1

ABSTRACTThe lectin, Concanavalin A (Con A), inhibits cell pairing during mating in Tetrahymena and binds to the surface of pairing cells via receptors concentrated around the conjugation junction. Concanavalin A is also ingested in large amounts into food vacuoles. To dispel the possibility that Con A inhibits pairing via uptake into food vacuoles or through induction of food vacuole formation and to strengthen the idea that pairing is blocked through binding of Con A to cell surface receptors, we have conducted three types of experiments: 1) attempts to inhibit pairing by feeding with nutrients and with tantalum, a non‐nutritive reagent; 2) a temporal analysis of the presence of food vacuoles in mating cells fed with tantalum; and 3) analysis of the restoration of pairing following the addition of α‐methyl mannoside to cells previously treated with inhibitory concentrations of Con A. The results of these studies support the idea that Con A inhibits pairing by binding to receptors located on the cell surface and not by induction of or uptake into food vacuoles. We also present evidence that cells grown in an enriched proteose peptone medium are able to pair and undergo morphogenesis more readily than cells grown in 2% proteose peptone.

Exocytosis, endocytosis and membrane recycling in Tetrahymena thermophila

ABSTRACT Mutant and wild-type cell lines of Tetrahymena thermophila were used to investigate a possible connection between acid hydrolase secretion and the major processes through which membranes are recycled in this ciliated protozoon. These processes consist of food vacuole formation (endocytosis), and food vacuole egestion and muco-cyst release (both exocytosis). We have found that a mutant (MS-1, sec−) blocked in hydrolase secretion is not blocked in either food vacuole formation or egestion and that it has normal mucocyst exocytosis. Another line of experiments with wild-type cells showed also that hydrolase secretion and endocytosis are independent of each other. Thus, sucrose (0·1 M) did not interfere with hydrolase secretion, but blocked food vacuole formation. Furthermore, release of acid hydrolases was selectively stimulated by dibucaine without any effect on food vacuole egestion. Finally, exocytosis of mucocysts could occur without simultaneous release of acid hydrolases, as when cells were exposed to 0’15M-NaCl, which evokes a massive secretory response of mucocysts. Our results demonstrate that formation and egestion of food vacuoles and exocytosis of mucocysts are unrelated to secretion of acid hydrolases. Furthermore, they suggest that secretion of acid hydrolases is not a secondary effect of membrane recycling through these processes.

The somatic function of the micronucleus in asexual reproduction of Paramecium: thermosensitivity of amicronucleates

It has been established that following removal of the micronucleus in Paramecium tetraurelia, the amicronucleate cell line enters a depression period, characterized by slow growth rate and oral abnormalities, at normal growth temperature (27°C). Such cell lines gradually recover in growth rate and stomatogenesis. In the present study, 4 recovered amicronucleate cell lines were challenged with high temperatures (35°C, 36°C, and 36.5°C). They exhibited growth rate reduction and abortive cytokinesis at 35°C and 36°C, and died at 36.5°C. In addition, they demonstrated oral defects similar to those observed in the depression period: disruption of the regular oral membranellar pattern, reduction in the length of the oral apparatus, and impaired phagocytosis (food vacuole formation). These high temperature‐induced abnormalities were largely restricted to amicronucleates, and were rare or seen to a much lesser extent in sister micronucleate cell lines. This study demonstrates the participation of the micronucleus in conferring thermotolerance on the cells. It is hypothesized that the micronucleus specifies heat‐shock proteins to maintain the integrity of oral and somatic cytoskeletal elements at high temperature.

Peptide stimulation of phagocytosis in Amoeba proteus

The tripeptide n-formylmethionylleucylphenylalanine (NFMLP) at low levels (10−9 M) localized in a glass micropipette elicits a positive chemotactic response in Amoeba proteus. Peptide in solution at higher concentrations (10−5 M) stimulates food vacuole formation. Vacuoles induced by NFMLP result from the fusion of pseudopods which, in turn, entrap a portion of the external medium. Food vacuole formation stimulated by NFMLP is dependent on the concentration of calcium in the external medium. The binding of NFMLP to the amoeba surface may bring about a movement of calcium into the cell which can be inhibited with calcium-channel blockers.

Filamentous actin in paramecium cells: functional and structural changes correlated with phalloidin affinity labeling in vivo.

Rhodaminylated (R)-phalloidin microinjected into Paramecium tetraurelia cells at a final concentration of greater than or equal to 20 micrograms/ml produces considerable functional and structural changes. F-actin bundles (with 20 micrograms/ml phalloidin within 15 min) are formed, which subsequently (greater than 30 min) are sequestered into autophagic vacuoles; simultaneously, the originally intense fluorescence of a narrow cortical layer becomes more and more diminished. When such microinjected cells are processed for electron microscopy, they display concomitant ultrastructural alterations, namely, the formation of transcellular bundles of 5-7 nm-thick filaments, which subsequently appear in autophagosomes, as well as a considerable reduction of filamentous materials in the cortex. This, in turn, entails a considerable restructuring of the cortex, enabling free access of various structural components to the cortex. Higher doses of R-phalloidin abolish cytoplasmic streaming (e.g., 50 micrograms/ml after 20-30 min); although the cells may survive, new secretory organelles (trichocysts) are no longer docked to the cell membrane. In contrast, exocytosis of docked trichocysts (as well as subsequent membrane resealing and retrieval) is not impaired under any conditions. Cortical F-actin may account for the cytoplasmic streaming that may normally guarantee the delivery of new trichocysts to free docking sites at the cell membrane. When docking is inhibited by high R-phalloidin doses, excess free trichocysts are sequestered into autophagosomes (crinophagy). One of the most sensitive cell functions is food vacuole formation (assayed by prelabeling with India ink), which correlates with the presence of R-phalloidin labeling in the cytostomal region and around food vacuoles. The main conclusions from this work are that filamentous actin may be involved in structuring of the cortex and in cytoplasmic streaming, and may therefore influence the formation, and possibly the transcellular transport (cyclosis), of food vacuoles, as well as the docking of trichocysts, whereas it does not play a role in exocytosis per se or in the steps immediately following.

A new principle of cell cultivation: No air-liquid interface but gas exchange across a synthetic membrane

A completely liquid-filled growth chamber for axenic cultures ofTetrahymena pyriformis is described; gas exchange is ensured across a synthetic membrane. The chamber may be incorporated into a continuous flow system with inoculation and removal of cell samples under sterile conditions. Initially, the generation time of the cells was slightly prolonged, about 10%, but after some cell doublings decreased to 5%. The capacity of the cells to form food vacuoles (endocytosis) was unaltered during growth in the chamber. The synthetic membrane was highly permeable to O2 and CO2; however, cells grown in the chamber contained small refractive granules. The culture chamber permits the culture volume to be varied and it may be used for other protozoa, bacteria, and even tissue culture cells.

Feeding Behavior in the Ciliate Pseudomicrothorax dubius is a Series of Morphologically and Physiologically Distinct Events1

Based upon light and electron microscopical observations, the feeding behavior of the ciliate Pseudomicrothorax dubius, when fed the cyanobacterium Oscilatoria formosa, is resolved into two principal phases, contact swimming and phagocytosis, the latter being separable into two steps, attachment aad ingestion. Following collision with an O. formosa filament. cells swim along the filament with their ventral cilia in contact with it during the contact swimming phase. Phagocytosis commences with the attachment of the cytostome to the filament, which initiates lysosomal streaming in the cytostomal‐cytopharyngeal region. The filament then enters the cytopharynx concomitant with food vacuole formation during the ingestion step. Treatment of cells with trypsin or modification of the extracellular ionic medium inhibits the attachment step of phagocytosis but does not affect contact swimming. Behavior of cells when fed different cyanobacterial species as well as artificial food substrates is also examined. Contact swimming is a form of contact guidance since the shape of the food substrate determines the direction of cell movement. Additionally, a chemical factor may be present in or on the cyanobacteria and play a role in contact swimming. Evidence is presented that suggests that during the attachment step, two phenomena are involved: direct adhesion between cell surfaces and adhesion due to material liberated by exocytosis.

Cortical microtubular structures of the ciliate Lepidotrachelophyllum fornicis Nicholls &Lynn, 1984 and phytogeny of the litostomate ciliates

The somatic and oral cortical ultrastructure of Lepidotrachelophyllum fornicis Nicholls &Lynn, 1984 are described. The somatic kinetids are monokinetids whose fibrillar associates include the following: (i) a kinetodesmal fibril which originates near triplets 6, 7, and 8 and extends laterally and to the right at an angle of about 45° to the longitudinal axis of the kinety; (ii) a postciliary ribbon which originates in dense material near triplet 9 and extends posteriorly and to the right; (iii) a primary transverse ribbon which originates near triplets 3, 4, and 5 and extends to the left and anteriorly; (iv) a secondary transverse ribbon which originates near triplet 5 and curves to extend to the lateral left; and (v) a ribbon of rootlet microtubules which originates on the left at the base of the kinetosome. The oral kinetids are dikinetids. The ciliated posterior kinetosome has the following: (i) a postciliary ribbon which extends posteriorly and to the right; and (ii) a transverse ribbon which extends only a short distance anteriorly. The nonciliated anterior kinetosome has the following: (i) a single postciliary microtubule; (ii) a large transverse ribbon which extends into the oral cone; and (iii) near its base a hexagonal nematodesma. The cytopharyngeal apparatus or rhabdos is described and a schematic figure is presented. The cellular mechanisms underlying body shape changes and food vacuole formation and the phylogeny of the class Litostomatea are discussed.

Utilization of iron complexes in an animal cell.

The ciliate protozoan Tetrahymena thermophila was grown in synthetic nutrient medium in the absence of the iron chelator citrate. Utilization and toxicity of various iron compounds or complexes in iron-starved cells were assessed from the number of cell doublings obtained within a standard time. The compounds tested included complexes formed between ortho-phosphates and two forms of ferric hydroxides, native and cationized ferritin, and tris-acetylacetonato Fe(III). The ferric hydroxo ortho-phosphate particles are toxic and can be removed from the medium by Millipore filtration. Uptake of ferritin and tris-acetylacetonato-Fe(III) is independent of food vacuole formation and seems to occur by micropinocytosis and by plasma membrane translocation, respectively.

Changes in Oral Apparatus Structure Accompanying Vacuolar Formation in the Macrostomal Form of Tetrahymena vorax. A Model for the Formation of Food Vacuoles in Tetrahymena1

ABSTRACTThe large cytopharyngeal pouch of the macrostomal form of Tetrahymena vorax, following the addition of calcium, can form a sealed, empty vacuole. The open cytostomal region of this cell, which averages about 16 μ in diameter, is closed by an upward (ventral) movement of the right and posterior ribbed walls, both of which project into the cytostomal cavity. At the same time, the anterior and left walls of the cytostome‐cytopharyngeal complex move to the right, forming a diagonally (right to left) placed furrow in the floor of the buccal cavity as these walls meet. As a result of the movement, the edges of the single membrane‐bounded cytopharyngeal pouch are brought together and fuse, producing the closed vacuole. Elements of the cytoskeleton appear to participate in the closure process. Three major groups of ribbed wall microtubules support the open cytostome. The anterior ribbed wall microtubules pass laterally along the anterior (dorsal) portion of the cytopharyngeal pouch to the left where they end in the specialized cytoplasm. Middle oral rib microtubules terminate at the right and posterior margin of the cytopharynx while microtubules from the most posterior region of the ribbed wall pass to the left terminating in the specialized cytoplasm. The fine filamentous reticulum, a striated reticulum that borders the right, posterior, and anterior margins of the cytostome‐cytopharyngeal complex, is in an ideal position to participate in these movements. It is anchored anteriorly high up in the buccal cavity to the cross‐connective between the third membranelle and the undulating membrane complex. It courses beneath the right and posterior ribbed walls and runs laterally along the anterior margin of the cytopharynx to the left side. Contraction or pulling of this reticulum would act to bring the microtubule‐reinforced walls of the cytopharynx together permitting fusion of the cytopharyngeal pouch membranes to form a sealed vacuole.

Actin in Tetrahymena paravorax: Ultrastructural Localization of HMM‐Binding Filaments in Glycerinated Cells1,2

ABSTRACTActin has been identified in the ciliated protozoon Tetrahymena paravorax on the basis of the ultrastructural detection of filaments typically decorated with heavy meromyosin (HMM) in glycerinated microstome cells. These filaments are widely distributed in endoplasmic and cortical regions and can form bundles. They are particularly numerous in elongating cells; HMM‐binding filaments run approximately parallel to rib microtubules in the ectoplasm of the right wall of the buccal cavity and seem to extend to the cytopharyngeal region, suggesting some role of actin in maintenance of the crest‐trough pattern of ribbed wall and/or in formation of food vacuoles. Extensive actin bundles are observed below some membranellar areas and are thought to follow the course of the microtubular “deep fiber bundle.” The “fine filamentous reticulum” underlying the oral ribs and the “apical ring” extending beneath kinetosomes of ciliary couplets display filaments that do not bind HMM and are ˜ 14 nm in diameter. No evidence for actin in these structures was obtained in the present study. The “specialized cytoplasm” of the cytostome‐cytopharyngeal region appears as an undecorated reticulum with 20 nm‐spaced nodes. Occasionally HMM‐binding filaments were found inside the macronucleus, just beneath its envelope. Actin is suggested to be involved in cell shaping and in control of the transport of food vacuoles.

Actin microfilaments in paramecium: localization and role in intracellular movements.

Using heavy meromyosin (HMM) or the fragment S1 of myosin as probes for actin microfilaments, we studied their organization in Paramecium both by fluorescence and electron microscopy. In interphasic cells, HMM decorates (a) most prominently the periphery of nascent and young food vacuoles and their route during the early phase of their intracellular transit; (b) a thin meshwork radiating from the gullet throughout the cytoplasm; (c) a small area beneath the pore of contractile vacuoles and beneath the cytoproct when open to release food residues. Most of these HMM-decorated structures are in close contact with microtubular arrays. All HMM decoration disappears in dividing cells and in cytochalasin-treated cells. In vivo, the drug immediately blocks food vacuole formation but does not affect cytokinesis, cyclosis, contractile vacuole pulsation, defecation, or nuclear movements. The data show that, as in the cells of other organisms, actin microfilaments form defined arrays that undergo physiologically controlled cycles of assembly/disassembly. These arrays contribute (at least in the phagocytotic process) to diverse types of movement: constriction, membrane fusion, and migration of food vacuoles. However, aside from their massive concentration along the phagocytotic tractus, actin microfilaments are neither major structural components of Paramecium cytoplasm nor the only cytoskeletal components ensuring motility or contractility processes.

Coated pits with pinocytosis in Tetrahymena.

The possible role in pinocytosis of coated pits at the parasomal sacs of Tetrahymena has been studied using cationized ferritin (CF; pI = 8.5) as a marker of membrane and content. It is shown that CF binds evenly to the surface, including the coated pits, of Tetrahymena in an inorganic salt medium (to avoid formation of food vacuoles) at the normal growth temperature. Moreover, CF is internalized by coated vesicles (shown to be truly free by thin serial-section analysis) and delivered initially (1-5 min of incubation) to cisterna near the cell surface. Later (5-10 min) CF occurs also in autophagic vacuoles, formed as a result of starvation, and eventually (15-90 min) it is present in preformed (old) food vacuoles. These observations indicate that the coated pits at the parasomal sacs of Tetrahymena function in adsorptive pinocytosis in much the same manner as coated pits at the surface of mammalian cells.

The oral apparatus of the ciliate Nyctotherus ovalis Leidy: possible involvement of microtubules in the structural support of a cortical domain and the translocation of vesicles

Microtubule bundles anchored at the kinetosomes of cilia of the adoral zone of membranelles at the oral apparatus of Nyctotherus ovalis seem to represent a fairly rigid framework. No evidence for gliding or any other form of motility of these bundles could be obtained. Food uptake at the base of the funnel-shaped oral tube is accompanied by the appearance of electron-translucent discoidal vesicles between microtubule ribbons and nemadesmata-like microtubules inserting at the kinetosomes of cilia of the paroral membrane. After incubation with peroxidase, label is found in large food vacuoles but not in these discoidal vesicles. Such vesicles are still found after exposure of ciliates to cycloheximide. Colchicine, nocodazole, and low concentrations of vinblastine do not visibly affect the cytopharyngeal microtubules, and the transport of the vesicles appears undisturbed. However, vesicles are absent from microtubule ribbons in ciliates exposed to high concentrations of vinblastine or low temperature, although the microtubules themselves are morphologically unchanged. The presumably exocytotic membrane flow continues after treatment with D 2O, implying that the discoidal vesicles are not transported along the bundles by attachment to ‘treadmilling’ microtubules [35]. Since we observed microfilaments in the cytoplasm of vinblastine-treated ciliates and at the cytopharynx of Triton-lysed cell models these filaments may be involved in vesicle movement and food uptake. However, cytochalasin B has no immediate effect on the distribution of the discoidal vesicles. The appearance of fine filaments in the vicinity of the presumed endocytotic site of the cytopharynx and the attachment of these filaments to microtubules and membranes rather suggests that microfilaments together with the microtubules could cooperate in food vacuole formation. Evidence for a direct participation of microtubules in the transport of the discoidal vesicles was not obtained.

Thermotropic changes occur at fixed temperatures in Tetrahymena in spite of variations in culture temperature

A number of experimental observations indicate that when Tetrahymena thermophila are grown in defined medium, a thermotropic change occurs in the membranes at about 25°C. Thus, the curve showing food vacuole formation as a function of temperature is biphasic, with a shoulder or minimum at 24–25°C. Similarly, when the maximum swimming velocity or the growth rate is measured as a function of temperature, Arrhenius plots show clear breaks at 23.2 and 23.9°C, respectively. When isolated pellicles are examined using the fluorescence of polarisation technique, a break is seen in the curves of ln P against 1/T at 24.6°C, demonstrating that a phase change occurs at this temperature. Furthermore, this same phase change was demonstrated in the pellicles of a fatty-acid-requiring mutant of T. thermophila, RH179E1, at 25.2°C. A review of the literature revealed that in cells cultured in peptone media, there is evidence of thermotropic changes in many Tetrahymena membranes around 25°C, but that in addition, changes also occur around 16°C. The experiments on the temperature variation of swimming and growth were therefore repeated in peptone media, and it was confirmed that, in contrast to defined medium, two break points are always seen in Arrhenius plots. In particular, it was noted that these break points are practically invariable, having values of 25.8 and 15.6°C, a fact that is of considerable theoretical interest. The experiments described here and in the literature demonstrate that a wide range of biological functions are apparently correlated with thermotropic changes in the membranes occurring at 25°C and 16°C.

Characterization of the parapharyngeal mass of the ciliateHomalozoon vermiculare

The structure and elemental composition of the granules which make up the parapharyngeal mass of the ciliateHomalozoon vermiculare are analyzed. The mass is made up of two kinds of granules. One is not membrane-bounded and is composed of paraglycogen. The second type is comprised of concentric lamellae. It is rich in magnesium, phosphorus, potassium, and calcium. These results do not support the contention that the granules are directly involved in the process of food vacuole formation. The role of the parapharyngeal mass remains obscure.

Effects of nickel on the rates of endocytosis, motility, and proliferation inTetrahymena and determinations on the cell content of the metal

At concentrations above 1 mM, nickel has a dose-dependent effect on the rate of food vacuole formation in cells in the growth medium, proteose peptone (PP); total inhibition of endocytosis occurs within 10 minutes in 6mM nickel. However, only a 10 times lower concentration of nickel is tolerated by starved cells in an inorganic salt medium, a difference which may be ascribed to the high binding property of nickel to organic material. In the PP medium, nickel affects cell motility by increasing the rate of movement at a concentration of 1 mM, and by causing immobilization after 30 minutes in 6mM nickel; a spontaneous, partial recovery of cell motility is seen after 3 hours in 6 mM nickel. The effects of nickel on endocytosis and cell motility are reversible after removal of nickel. Cell proliferation continues at a reduced rate in 1 mM nickel, while only 1 1/2 cell doublings are achieved in 3 mM nickel during a 72-hour exposure, and no proliferation occurs in 6mM nickel, where an increasing cell mortality is observed after 12 hours. The cell content of nickel relates initially to the external concentration of the metal; however, cells in 1 mM nickel are capable of maintaining a constant content of the metal, whereas in 3 mM, the rate of accumulation is reduced after 3 hours, and cells in 6mM nickel accumulate the metal at a constant rate. All nickel-treated cells contain small refractive granules, previously proposed as representing an ion-regulating system, and the apparent adaption ofTetrahymena to the effects of nickel may be ascribed to such a regulation of the intracellular concentration of the metal.

Wild-type and food-vacuole-less Tetrahymena thermophila: Model for iron uptake and utilization in animal cells

Wild-type Tetrahymena thermophila and a conditional lethal mutant defective in food-vacuole formation constitute a useful model system for studies of iron uptake and utilization in animal cells.