The Contractile Vacuole of Amoeba proteus. III. Vacuolar Response to Phagocytosis1

The reaction of the contractile vacuole of Amoeba proteus to single and multiple phagocytosis under controlled conditions has been studied. Fluid intake into the cytoplasm from the phagosomes induces secretion by the contractile vacuole of equivalent excess volumes:. Vacuolar response is rapid (200 sec) and may be initiated by increases of protoplasmic hydration of as little as 1%. Cytoplasmic uptake of fluid from the phagosome can occur against an osmotic gradient; thus some form of active transport is implied.     

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS : II. On the Behavior and Possible Nature of the DNA-Containing Elements

Nucleic acid-containing particles in the cytoplasm of Amoeba proteus (cf. reference 1) were counted after acridine orange staining. The number of particles per ameba was found to be correlated with cell age and size. Fresh daughters had a mean particle number of 5400, whereas predivision amebae contained around 11,000 particles. Amebae from two other strains contained similar particles. The particles were found to be clustered in fasted cells and redispersed after feeding. A marked increase in the particle population was noted in anucleate fragments. These results, together with those previously presented, suggest that the particles multiply intracellularly. Their nature and their relationship to previous work on nucleic acid labeling in Amoeba are discussed.     

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS : I. On the Particulate Nature of the DNA-Containing Elements

The incorporation of tritiated thymidine in Amoeba proteus was reinvestigated in order to see if it could be associated with microscopically detectable structures. Staining experiments with basic dyes, including the fluorochrome acridine orange, revealed the presence of large numbers of 0.3 to 0.5 µ particles in the cytoplasm of all cells studied. The effect of nuclease digestion on the dye affinity of the particles suggests that they contain DNA as well as RNA. Centrifugation of living cells at 10,000 g leads to the sedimentation of the particles in the centrifugal third of the ameba near the nucleus. Analysis of centrifuged cells which had been incubated with H₃-thymidine showed a very high degree of correlation between the location of the nucleic acid-containing granules and that of acid-insoluble, deoxyribonuclease-sensitive labeled molecules and leads to the conclusion that cytoplasmic DNA synthesis in Amoeba proteus occurs in association with these particles.     

THE CYTONUCLEOPROTEINS OF AMEBAE : I. Some Chemical Properties and Intracellular Distribution

Autoradiographs of whole Amoeba proteus host cells fixed after the implantation of single nuclei from A. proteus donors labeled with any one of 8 different radioactive amino acids showed that the label had become highly concentrated in the host cell nucleus as well as in the donor nucleus and that the cytoplasmic activity was relatively low. When these amebae were sectioned, the radioactivity was found to be homogeneously distributed throughout the nuclei. The effect of unlabeled amino acid "chaser," the solubility of the labeled material, and the long-term behavior of the labeled material gave evidence that the radioactivity was in protein. At equilibrium, the host cell nucleus contained approximately 30 per cent of the radioactivity distributed between the two nuclei. This unequal nuclear distribution is attributed to the presence of two classes of nuclear proteins: a non-migratory one that does not leave the nucleus during interphase, and a migratory one, called cytonucleoprotein, that shuttles between nucleus and cytoplasm in a non-random manner. It is estimated that between 12 per cent and 44 per cent of the cytonucleoproteins are present in the cytoplasm of a binucleate cell at any one moment. Nuclei of Chaos chaos host cells also concentrated label acquired from implanted radioactive A. proteus nuclei.     

Effect of Chloramphenicol on Bacterial Endosymbiotes in a Strain of Amoeba proteus*

SYNOPSIS. The effect of chloramphenicol (CAP) on the bacterial endosymbiotes of a strain of Amoeba proteus was studied by growing the symbiotic amebae in media containing 0.5–1.6 mg/ml CAP for up to 4 weeks. Treatments with CAP caused such ultrastructural changes as expansion of the nuclear zone and deformation of symbiotes. The CAP treatment also damaged the mitochondria, e.g. disappearance of central and protrusion of peripheral cristae. Number of bacteria per ameba decreased to < 10% of control in CAP-containing media, but no viable amebae became completely free of symbiotes. The resuts supported previous studies that amebae were dependent on endosymbiotes.     

Scanning Electron Microscope Observations of Amoeba proteus During Phagocytosis*

SYNOPSIS. Phagocytosing Amoeba proteus at different stages of forming foodcups have been observed by scanning electron microscopy. A nonphagocytosing ameba is characterized by dorsal and lateral ridges running longitudinally over the posterior half of the cell and its attachment to the substrate over small areas. When stimulated by prey organisms, the ameba loses polarity and ridges, and adheres to the substrate more firmly over a wider area of contact. Then it forms broad pseudopods to surround its prey and this results in the formation of foodcups. The surface of all amebae is covered with small projections, and membranous blebs are often seen on the surface of phagocytosing organisms.     

Taxonomic serology of Amoeba proteus

The serological distances among six and eight strains of Amoeba proteus were determined. Quantitative immunological precipitation methods have the sensitivity and accuracy to detect the small differences in taxonomic relationships within this species. The data suggest that the _ShP strain might not belong to the proteus species and support the hypothesis that Amoeba indica is a strain of A. proteus. The invasion of a strain of A. proteus by infectious rod-shaped bacteria caused a diversion in taxonomic relationships with the original strain. Present serological findings are compared to previous data.     

Phosphatase activity in <Emphasis Type="Italic">Amoeba proteus</Emphasis> at pH 9.0

In the free-living ameba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called “fast,” “intermediate,” and “slow” phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH values. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be either acid phosphatase, or protein tyrosine phosphatase. Based on data of inhibitor analysis, broad substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, and another localization in the ameba cell than of the fast and intermediate phosphatases, it is concluded that only the slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

CYTOPLASMIC FILAMENTS OF AMOEBA PROTEUS : I. The Role of Filaments in Consistency Changes and Movement

The role of filaments in consistency changes and movement in a motile cytoplasmic extract of Amoeba proteus was investigated by correlating light and electron microscopic observations with viscosity measurements. The extract is prepared by the method of Thompson and Wolpert (1963). At 0°C, this extract is nonmotile and similar in structure to ameba cytoplasm, consisting of groundplasm, vesicles, mitochondria, and a few 160 A filaments. The extract undergoes striking ATP-stimulated streaming when warmed to 22°C. Two phases of movement are distinguished. During the first phase, the apparent viscosity usually increases and numerous 50–70 A filaments appear in samples of the extract prepared for electron microscopy, suggesting that the increase in viscosity in caused, at least in part, by the formation of these thin filaments. During this initial phase of ATP-stimulated movement, these thin filaments are not detectable by phase-contrast or polarization microscopy, but later, in the second phase of movement, 70 A filaments aggregate to form birefringent microscopic fibrils. A preparation of pure groundplasm with no 160 A filaments or membranous organelles exhibits little or no ATP-stimulated movement, but 50–70 A filaments form and aggregate into birefringent fibrils. This observation and the structural relationship of the 70 A and the 160 A filaments in the motile extract suggest that both types of filaments may be required for movement. These two types of filaments, 50–70 A and 160 A, are also present in the cytoplasm of intact amebas. Fixed cells could not be used to study the distribution of these filaments during natural ameboid movement because of difficulties in preserving the normal structure of the ameba during preparation for electron microscopy.     

Maintenance and regeneration of cytoplasmic organelles in hybrid amebae formed by nuclear transplantation

The fine structure of cytoplasmic organelles was studied in hybrid amebae that were formed by transplanting the nucleus from one kind of ameba into the cytoplasm of a different type of ameba. Four different species or strains of amebae were utilized; Amoeba proteus, A. discoides, A. dubia, and A. amazonas. The fine structure of A. discoides was indistinguishable from that of A. proteus, but A. dubia and A. amazonas had distinctive features. To determine if the fine structure of cytoplasmic organelles in hybrid cells is like that of the nuclear parent or the cytoplasmic parent, nuclei were transferred between A. proteus and A. amazonas, which differ in their cytoplasmic ultrastructure, especially in the morphology of mitochondria. The cytoplasm of these hybrids resembled most closely that of the cytoplasmic parent. The degree to which different heterologous nuclei are capable of maintaining normal cytoplasmic organization was then studied by replacing an A. proteus nucleus with a nucleus from one of the other kinds of amebae. An A. discoides nucleus maintained A. proteus cytoplasm for several weeks. A nucleus from A. dubia or A. amazonas was able to maintain A. proteus cytoplasm for a few days, but then the cytoplasm began to show ultrastructural changes characteristic of enucleated amebae. The ability of a heterologous nucleus to promote the regeneration of Golgi bodies and endoplasmic reticulum after their decline in the absence of the nucleus was tested by transplanting a heterologous nucleus into A. proteus cytoplasm that had been enucleate for 5 days. A. discoides was the most effective heterologous nucleus donor but was less successful than normal A. proteus. Insertion of a nucleus from either A. amazonas or A. dubia resulted in “reactivation” of the cytoplasm with extension of pseudopods and resumption of motility but little or no regeneration of cell organelles. Thus, the ability of a heterologous nucleus both to maintain and to regenerate normal features of cytoplasmic organelles varied with the type of heterologous nucleus and was related to the degree of morphological resemblance between the donor and recipient cells. The role of the nucleus in motility and its role in regeneration of cytoplasmic organelles were dissociated from one another in the case of some hybrid renucleates.     

ELECTRON MICROSCOPY OF THE NUCLEAR MEMBRANE OF AMOEBA PROTEUS

An electron microscope study of the nuclear membrane of Amoeba proteus by thin sectioning techniques has revealed an ultrastructure in the outer layer of the membrane that is homologous to the pores and annuli observed in the nuclear membranes of many other cell types studied by these techniques. An inner honeycombed layer apparently unique to Amoeba proteus is also described.     

Locomotion ofAmoeba proteus after standardizing its body shape

Summary Amoeba proteus obliged to follow dark stripes in the form of Y may be studied in three repeatable simple configurations: 1. tail + 1 advancing front, 2. tail + 2 advancing pseudopodia, 3. tail +1 advancing pseudopodium + 1 contracting pseudopodium. Formation of two advancing pseudopodia and the later conversion of one of them into a contracting pseudopodium affect the rate of movement of all the other body parts in the manner predictible by the hydrodynamic concept of the endoplasmic flow in amoeba. An active front stops and begins to retreat when arriving to a constant distance from the posterior body end. The locomotion is disfavoured if new pseudopodia deviate from the former body axis at the angle wider than 35°.Study supported by Research Project II. 1 of the Polish Academy of Science.     

I. THE RELATION BETWEEN ATTACHMENT TO THE SUBSTRATUM AND INGESTION BY AMEBA IN STRYCHNINE SULFATE SOLUTION AND CONDITIONED MEDIA : II. BIOLOGICAL ASSAY OF CONDITIONED MEDIA

1. Strychnine sulfate 0.000069 M decreased percentage attachment to the substratum by Amoeba proteus in 0.0029 M NaCl from 77.3 to 1.3, in 0.0029 M KCl from 40.8 to 2.5, in 0.002 M CaCl₂ from 73.3 to 68.0, in 0.002 M MgCl₂ from 85.5 to 83.3. 2. Frequency of ingestion of chilomonads by Amoeba proteus is increased by adding strychnine sulfate to solutions of NaCl, KCl, or CaCl₂. Frequency of ingestion is increased in NaCl solution from 1.3 to 2.3, in KCl from 0.75 to 2.25, and in CaCl₂ from 1.1 to 1.9 chilomonads per minute. Ingestion is not significantly increased by the addition of strychnine to MgCl₂ solution. 3. Frequency of ingestion of food by Amoeba proteus is not closely correlated with attachment to the substratum in NaCl and KCl solutions to which strychnine sulfate is added. 4. Chilomonads adhere to the plasmalemma of Amoeba proteus in solutions of NaCl, KCl, or CaCl₂ containing strychnine, but in MgCl₂ plus strychnine only a few adhere to it. Strychnine appears to make the surface of the amebae and chilomonads sticky in the former but not in the latter. Frequency of ingestion is apparently correlated with adherence of chilomonads to the plasmalemma. 5. Attachment to the substratum and ingestion by Pelomyxa carolinensis is increased by dead Chilomonas, Colpidium, and Paramecium in aqueous solutions, by materials obtained from paramecia by alcoholic-ether extraction, and by solutions in which these organisms have lived. 6. Attachment to the substratum by Pelomyxa carolinensis is not closely correlated with kind or concentration of inorganic salts used in this study. 7. Materials were found in extracts of paramecia which had certain characteristics in common with choline esters. There is no reason to doubt that under certain conditions materials are present in aqueous and alcoholic extracts which are pharmacologically similar to choline and acetylcholine. 8. Aqueous suspensions of paramecia when subcutaneously injected into young mice for 21 days inhibit the gonadotropic luteinizing hormone of the pituitary. Ovaries from injected mice showed no corpora lutea, and the seminal vesicles from injected males were smaller and contained less fluid than those of the controls.     

INTRACELLULAR OXIDATION-REDUCTION STUDIES : I. REDUCTION POTENTIALS OF AM?BA DUBIA BY MICRO INJECTION OF INDICATORS.

Twenty-five oxidation-reduction indicators were injected in oxidized or reduced form into Amoeba dubia and Amœba proteus under controlled conditions of oxygen access. (1) Under anaerobiosis the ameba was able to reduce completely all the reversible oxidation-reduction indicators down to and including indigo disulfonate. (2) Under anaerobiosis the ameba was unable to reoxidize six of the most easily oxidizable indicators. (3) Under aerobiosis the ameba was able to reduce completely all the indicators down to and including 1-naphthol-2-sulfonate indo-2, 6-dichlorophenol. Toluylene blue, methylene blue and indigo tetrasulfonate were sometimes completely and sometimes only partly reduced, depending on the quantity of indicator injected and the duration of observation. (4) The time of reduction varied approximately with the size of the injection. Reduction was more rapid under anaerobiosis than under aerobiosis, more rapid in active than in sluggish cells and was retarded by toxic compounds. (5) Sulfonated compounds were somewhat toxic, as a rule. In interpreting reduction phenomena of micro injection, it is necessary to take into consideration the intensity, capacity and rate factors. It then becomes apparent that the ameba has a high reducing potential lying on the rH scale below the zone of indigo disulfonate. The reducing capacity of the ameba seems to be relatively great in the region of the simple indophenols and of a progressively diminishing magnitude as the zone of the indigos is approached. Material of high reduction potential appears to be generated within the ameba at a measurable rate. These phenomena, observed in the interior of the cell with the aid of indicators, parallel very closely those found in reduction electrode studies on bacterial cultures.     

THE CELL IN STARVATION: PHYSIOLOGICAL AND CHEMICAL OBSERVATIONS

Observations made on Amoeba proteus during total inanition revealed the following changes: Dry weight declined progressively, but at a decreasing rate to about 45 per cent of the initial levels when determined in surviving members of a dying population. Protein fell to about 70 per cent of the initial level. A hexane-alcohol extractable component fell during early starvation then rose to about its initial absolute level in the dying cells. While initially most of this component is probably lipide, it is not certain that other materials are not extracted during cell degeneration. Survival as a function of cell size was studied. No advantage in survival was apparent for any size class. Nucleate cell "halves" likewise showed no survival time differential, unlike a highly significant decrease in the survival of enucleate portions. The maintenance of the initial variance about the mean population weight (after hexane-alcohol extraction) during starvation, likewise supports the idea that survival depends largely on concentration parameters.     

INFECTIVE ORGANISMS IN THE CYTOPLASM OF AMOEBA PROTEUS

Evidence from electron and phase microscopy is given which shows that infective organisms are present in the cytoplasm of Amoeba proteus. Vesicles containing living organisms have been observed after repeated washing and starvation of the amebae for a period of 2 weeks. Exposure to γ-radiation in conjunction with starvation, repeated washing, isolation of single amebae, refeeding with contaminant-free Tetrahymena, and clone selection has produced clones with reduced cytoplasmic infection. These findings are discussed in regard to the autoradiographic studies of other investigators on Amoeba proteus. The controversies over whether DNA and RNA are synthesized in the cytoplasm may be resolved by the finding of cytoplasmic infection.     

Variability of the limited lifespan state in ameba

Muggleton & Danielli [6] reported that the normally immortal Amoeba proteus could be switched into a state of limited lifespan by restricting their food intake for several weeks so as to preclude division. Upon return to normal feeding, these cells were reportedly no longer immortal, but were only capable of a limited, unspecified number of divisions before the resulting clone died out. Attempts to duplicate these results with our present ameba stocks have resulted in failure. It appears that the presence of this phenomenon is variable and the life-spanned state may no longer be present in these cultures. An extended period on a maintenance diet which precludes division may sometimes have effects on cell viability for several generations but it no longer results in a general limitation of lifespan. Further work on this phenomenon must involve rigorous proof that the phenomenon is indeed present in the ameba being used.     

Karyotyping of <Emphasis Type="Italic">Amoeba proteus</Emphasis>

In this paper, we have developed a method to prepare spreads of mitotic chromosomes of Amoeba proteus and described the process of Amoeba proteus karyotyping. This protocol allows obtaining spread chromosomes with a characteristic pattern of chromomeres on individual chromosomes. It is shown that, in the metaphase of mitosis, amoebas of strain B (one of the type strains of A. proteus in the Amoebae Cultures Collection of the Institute of Cytology, Russian Academy of Sciences) contain 27 pairs of chromosomes. It is established that the pattern of chromomeres is a chromosome-specific feature. A typical karyogram and image bank of DAPI- and YoYo1-banded individual chromosomes of A. proteus, strain B composed of five different spreads of mitotic cells are presented.     

Ultrastructure of the Contractile Vacuole and Its Periphery in Amoeba proteus: Evolution of Vesicles During the Cycle1

ABSTRACT. The population of organelles in the periphery of the contractile vacuole of Amoeba proteus has been studied throughout the cycle using proper fixation and dehydration procedures on serial semithin and thin sections. Three distinct types of perivacuolar vesicles which arise successively from one another in the course of the cycle have been described. Size relationships have been determined by stereology.     

Pinocytosis and locomotion of amoebae: XII. Dynamics and motive force generation during induced pinocytosis in A. proteus

Summary The mechanism of induced pinocytosis was investigated in Amoeba proteus by light and electron microscopy. The application of nine different inducing substances revealed that pinocytotic channel formation, elongation, vesiculation, shortening and disappearance are the result of the successive or simultaneous action of both traction and pressure forces, which are produced by the contractile activity of a plasma membrane-associated layer of filaments ranging from a few hundred nm to several in thickness. The initial phase of channel formation is caused by traction forces according to the membrane flow concept, whereas channel elongation and vesiculation mainly result from pressure forces in conjunction with the extrusion of small hyaline pseudopodia. Shortening and disappearance of the pinocytotic channels are brought about by local contractions of the cortical filament layer in the basal region of the hyaline pseudopodia. Experiments using latex beads as marker particles together with inducing substances show that a rapid membrane turnover during pinocytosis can be excluded, and that the plasma membrane slides as an entire structure over the underlying cytoplasm.The authors are most grateful to Mrs. J. Ruch for technical assistance