Determination of the Volume and Surface Area of Tetrahymena pyriformis,and their Relationship to Endocytosis*

Methods are described for the determination of the mean cellular volume and surface area of Tetrahymena pyriformis GL by direct microscopic measurement or by Coulter counter. The results are compared and discussed. It is suggested that the latter is the more accurate method of estimation. Fed cells showed a bimodal size distribution by Coulter counter determination and had a mean volume of 10,000–11,000 μM³, whereas cells which had been starved for 1 or 2 days showed a unimodal distribution and had mean cellular volumes of ～ 1,600 and 1,200 μ³ respectively. The corresponding mean surface areas were ～ 1,900, 550 and 450 μM² respectively. The discrepancy between the results of the 2 methods of estimation was greater with starved than with fed cells because of the greater asymmetry of the former. Continued cell division during the early part of the starvation period had a considerable reducing effect upon the mean cellular volume, but other unidentified factors were also important in producing the observed diminution in volume. Comparison of the mean surface areas of starved cells with previously recorded rates of membranous utilization in endocytosis upon refeeding indicate that insufficient cell membrane was present to maintain the rates of vacuole formation observed.     

Electrophoretic Characterization of Classical Tetrahymena pyriformis Strains*

The electrophoretic mobility patterns of 8 enzymes have been examined in 43 classical strains of Tetrahymena pyriformis. The strains may be assorted into sets on the basis of a high degree of similarity of their mobility patterns. Strains of similar designation are frequently in different sets, whereas differently labeled strains may be in the same set. It is proposed that new strain designations be made on the basis of phenotypic similarity.     

Temperature Effects on Maturity Periods in Tetrahymena pyriformis Syngen 1*

Cells of Tetrahymena pyriformis syngen 1 grown at 30 C after conjugation achieve sexual maturity more quickly than do cells grown at 19 C, whether time is measured in numbers of cell divisions or in terms of absolute time. This result is achieved regardless of the temperature at which conjugation and nuclear reorganization occur. These observations differ from those of other workers investigating Paramecium, and suggest that the long term “chronometer” is more tightly coupled to cell division in Paramecium multimicronucleatum and Paramecium caudatum than in Tetrahymena pyriformis.     

Isozymic Characterization of Three Mating Groups of the Tetrahymena pyriformis complex*

SYNOPSIS. Strains of 3 unnamed mating groups of the Tetrahymena pyriformis complex have been subjected to starch gel electrophoresis followed by staining the gels for the enzymes isocitrate dehydrogenase (NADP), tyrosine aminotransferase, and tetrazolium oxidase (superoxide dismutase). With respect to the electrophoretic mobilities of these enzyme systems, the mating groups referred to here as 5, 13 and 14 are very similar to Tetrahymena americanis (syngen 2), the most common North American species of the complex. Cultures in our collection labeled Tetrahymena cosmopolitans (formerly syngen 4) are either amicronucleate, with unique isozyme patterns, or micronucleate cells which mate with and have isozyme patterns similar to Tetrahymena canadensis (syngen 7). Immature progeny have been derived from crosses between the latter strains and T. canadensis recently collected in Colorado. The amicronucleate strains are now placed in the Tetrahymena sp. category, and we conclude that strains identifiable as T. cosmopolitanis are no longer available. The reliability of isozymes as characters in ciliate taxonomy was evaluated by comparing the present results for 3 enzymes in 15 groups of strains (syngens and phenosets) that had been compared in an earlier study. These enzyme systems gave correlation coefficients (r) of 0.75 or higher in the separate studies, and can be considered useful diagnostic traits. Other enzymes that were present at threshold levels of detectability or varied highly in concentration from species to species are too unreliable to be of diagnostic value. Some of the strains in the complex are so evolutionarily divergent at the molecular level that we have difficulty finding growth and electrophoretic conditions under which orthologous enzyme activities can be detected simultaneously for all the strains being compared.     

The Stability of Cortical Phenotypes in Continuously Growing Cultures of Tetrahymena pyriformis*

Cortical features were analyzed in successive samples of continuously growing stock cultures of amicronucleate strains GL-C and GL-I, and in micronucleate strain WH-6 (syngen 1, mating type I). Thirteen successive samples of strain GL-C, representing a time span of 111 months, 5 samples of WH-6 (43 months) and 2 samples of GL-I (1 month) were examined. The observed range of commonly expressed ciliary row numbers (corticotypes) was 16–20 rows in strain GL-C, 15–20 in strain GL-I, and 16–20 in strain WH-6. These ranges remained constant through time within each strain. The individual samples each included all or a large part of the total range observed in the strain, but the relative abundances of different corticotypes within this range shifted through time. The shifts appeared random, with no discernible trends. Mean contractile vacuole pore (CVP) position and number of CVP meridians were assayed in the 2 “GL” strains. Mean CVP position was an apparently stable character, with only slight fluctuations through time, while the distribution of number of CVP meridians was somewhat less constant. The CVP parameters of strains GL-C and GL-I were considerably different, and both of these strains were very different from the GL strain which had been studied by Nanney. In fact, these 3 “GL” strains have, among them, virtually the entire gamut of known CVP characteristics. The possible significance of these wide differences among strains presumed to be closely related is considered in the Discussion.     

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.     

Further Electrophoretic Characterization of Strains of Tetrahymena pyriformis*

Mobility patterns of 5 isozymes in strains of Tetrahymena pyriformis were demonstrated using polyacrylamide disc gel electrophoresis. Six stock strains were compared in these patterns to 4 strains representative of each of the previously described 4 major “phenotypic sets.” Stock strains segregated into predicted “phenosets,” and essentially confirmed validity and reproducibility of such a discrimination method. The proposal that new strain designations be assigned on a basis of “phenoset” conformity is reaffirmed.     

Transformation in Tetrahymena pyriformis: Description of an Inducible Phenotype*†

SYNOPSIS. Transformation of Tetrahymena pyriformis to a rapid-swimming (presumably dispersal) form can be induced by washing cells and suspending them in distilled H₂O, Dryl's solution or 10 mm Tris. Transformation is possible with high efficiency in mass cultures of axenically grown cells within ～ 5 h at 30 C. The radically different phenotype produced during transformation is characterized by a more elongate body form, increased numbers of somatic basal bodies and cilia, a long caudal cilium and oral membranelles positioned beneath the cell surface. DNA quantities characteristic of G1, S, and G2 cells are found in these transformed ciliates, suggesting that achievement of a particular stage in the DNA-division cycle is not a prerequisite for transformation. Preliminary observations on cells belonging to syngens 2–12 indicate that they also have a capacity to form a caudal cilium, but that the amicronucleate strain GL-C does not. Possible relevance of the transformed phenotype for taxonomy of Tetrahymena is discussed.     

Tetrahymena pyriformis as a Model System for Membrane Studies*†

SYNOPSIS. Tetrahymena pyriformis is an exceptionally useful subject for studying metabolic interrelationships among intracellular membranes. Its advantages include the striking differences in lipid composition among the cell's various functionally distinct membrane systems, indicating a pronounced lipid specificity at the membrane sites. The magnitude of these differences permits analysis of the mechanisms underlying the specificity. Even more valuable is the unique physical isolation of ciliate surface membranes from the cytoplasm of the cell. In contrast to the almost immediate equilibration of newly made lipids with preexisting lipids found in most cells, Tetrahymena surface membranes have a lipid turnover slow enough to be conveniently analyzed. Finally, the well-studied responses of Tetrahymena to such physiological stresses as heat and starvation may be used to evaluate the effects of environmental factors on membrane formation.     

Sublethal Cytotoxic Effects of Mercuric Chloride on the Ciliate Tetrahymena pyriformis*

SYNOPSIS. The behavior and ultrastructure of Tetrahymena pyriformis was assessed after exposure to dosages of 8 and 16% of the lethal concentration of HgCl² (TLm 96 hr). The lower dosage caused no abnormal changes in cell motility, activity of the water explusion vesicles, or cell shape; the higher dosage caused deleterious changes in these parameters. The higher sublethal HgCl² concentration (0.50 mg/liter) elicited damage of several cell structures. This damage persisted and accumulated with time up to 24 hr. At the lower HgCl² dosage (0.25 mg liter) there were extensive changes after 1-hr exposure involving primarily mitochondria; however, all major changes were repaired after 24 hr of constant exposure to the HgCl², indicating adaptation to the toxicant. Based solely on cytotoxic evidence an attempt is made to apply the findings defining what constitutes a “safe'’concentration of HgCl₂ in the cell's environment.     

The Conversion of Phenylalanine to Tyrosine in Tetrahymena pyriformis*

SYNOPSIS. Phenylalanine hydroxylase could not be assayed in extracts of Tetrahymena pyriformis strain W in a system by which the enzyme could be assayed in rat liver extracts. Isotopically labelled phenylalanine, however, was converted to tyrosine by growing or washed cells. Growth conditions which allowed limited synthesis of unconjugated tetrahydropteridine severely reduced the ability of the cells to synthesize tyrosine from phenylalanine. The presence of glucose and acetate in the growth medium resulted in elevated free tyrosine pools and an increased capacity of washed cell suspensions to convert phenylalanine to tyrosine. It would appear that the putative phenylalanine hydroxylation system is not subject to the repressive effects of glucose and acetate which apply to the enzymes of tyrosine catabolism. The significance of this distinction is discussed.     

Studies of Membrane Formation in Tetrahymena pyriformis. VIII. On the Origin of Membranes Surrounding Food Vacuoles*†

SYNOPSIS. A procedure was devised for the isolation of purified food vacuoles from Tetrahymena pyriformis fed particles of ferric oxide. Phospholipids extracted from vacuolar membranes were more similar in composition to the lipids of microsomes than to lipids of whole cells, cilia or post-microsomal supernatant. Fractionation of cells grown in the presence of [¹⁴C]palmitic acid or [³²P]inorganic phosphate also revealed similarities in the specific radioactivities of microsomes and vacuolar membranes. The data suggested that vacuolar membranes arise from a pool of cytoplasmic membranes.     

Morphometric Studies of Mitochondria in Tetrahymena pyriformis Exposed to Chloramphenicol or Ethidium Bromide*

Cultures of Tetrahymena pyriformis strain ST were exposed to 300 μg chloramphenicol/ml or 15 μg ethidium bromide/ml for 48 hr. Qualitative assessments of electron micrographs reveal that the abundance of mitochondrial cristae decreases greatly. By equating the spatial characteristics of the organism with those of a prolate spheroid, the distribution and abundance of mitochondria were quantified. Such characterizations reveal that the size of individual mitochondria decreases by 35–60% and that the number of mitochondria/cell increases ～8 fold. The observations are discussed in terms of coordinated mitochondrial and nuclear genetic activities.     

Effects of Dimethyl Sulfoxide on Tetrahymena pyriformis GL. Fine Structural Changes and Their Reversibility*

SYNOPSIS. Fine-structural changes are induced in Tetrahymena by exposure to 7.5% dimethyl sulfoxide (DMSO) in the presence of growth medium. Some of these changes (nucleolar, mitochondrial, peroxisomal) resemble those seen during starvation, in agreement with the previously reported inhibitory effect of DMSO on food-vacuole formation; however, changes such as helical formations of polyribosomes indicate additional internal actions of the reagent. The effects vary to some extent within the same group of cells, suggesting that sensitivity to the reagent may differ with the stage in the cell cycle. The structural changes induced by a 1-hr exposure to DMSO are reversible, but recovery of the cells after removal of the reagent is slower than that seen after starvation. The observations suggest that the recovery is associated with renewed synthesis.     

Cytotoxicity of Synthetic Fuel Products on Tetrahymena pyriformis. I. Phenol*

SYNOPSIS. Phenol is the major organic constituent of coal-conversion scrub water and is therefore a potential environmental contaminant. After Tetrahymena pyriformis strain GL-C, syngen 1 was exposed to phenol, its behavior, cytology, respiration, and growth rates were examined. Concentrations ≥ 75 mg/liter alter cell motility, shape, and contractile vacuole activity. O₂ uptake was abruptly reduced within 3 min of exposure to phenol in concentrations as low as 10 mg/liter. Concomitantly there was an increase in the electron density of the mitochondrial matrix. Recovery to normal rates of O₂ consumption was paralleled by a return to normal matrix density. Alterations of mucocysts, pellicle, and glycogen were also observed. The length of lag phase growth curves increased generally in proportion to concentration of toxicant. Phenol, however, did not affect the rate of cell multiplication during the exponential growth phase. The potential use of this system to examine the effects of other possible organic pollutants is discussed.     

Timing and Regulation of Nuclear and Cortical Events in the Cell Cycle of Tetrahymena pyriformis*

SYNOPSIS. Using continuous flow cultures based on the chemostat principle, we varied the cell generation times of the ciliate Tetrahymena pyriformis strain GL, from 4.9 to 22.2 hr and studied various parameters of the cell cycle at 28 C. These included: the duration of the periods required for oral morphogenesis, macronuclear division, cell division, G₁ S, and G₂. The size of individual cells was also measured. Independent of the growth rate, the period of oral morphogenesis occurred during the last 90 min of the cell cycle. In all cases macronuclear and cell divisions took place during the last part of these 90 min, and the final macronuclear separation occurred just before final cell separation. The S-period increased slightly, while the G₁ and G₂ both increased in roughly the same relative proportion to the increasing generation times. Slowly growing cells (generation time 20.5 hr) were shorter but broader and somewhat larger in volume than quickly growing cells (generation time 4.9 hr).     

Isolation and Identification of Specific Cortical Proteins in Tetrahymena pyriformis strain GL*†

SYNOPSIS. Pellicles of the ciliate Tetrahymena pyriformis strain GL (phenoset A) were isolated by a new procedure. Oral apparatuses were also purified by a modification of a previous method. Both preparations were characterized by electron microscopy. Proteins of the isolates were separated by analytical SDS polyacrylamide gel electrophoresis. The isolated pellicles, which included oral apparatuses, contained only 6 major proteins (gel bands), designated A through F. Bands A, B, and C, were found in the pellicle fraction, but not in the oral apparatus fraction. Therefore, these proteins are believed to be present in the somatic cortex of Tetrahymena. Bands D and E were greatly enriched in the oral apparatus fraction; these proteins are therefore believed to be present primarily in the oral apparatus. Band F, identified as tubulin, was present in both preparations. Molecular weight determinations and some selective solubilization experiments are also presented.     

Efficiency of Filter Feeding in Two Species of Tetrahymena*

SYNOPSIS. Rates of removal of suspended India ink particles from the surrounding medium by 2 ciliates, Tetrahymena pyriformis strain GL and Tetrahymena vorax strain V₂S, have been measured. It is evident from the results that the food vacuoles concentrate the suspended particles, clearing a volume of the surrounding suspension fluid 500 × greater than the total volume of food vacuoles made during the same period of time.     

The Fate of RNA Degradation Products in Starved Cultures of Tetrahymena pyriformis W.*

The RNA content of a population of Tetrahymena pyriformis W was followed during the growth phases of the culture. The cellular RNA levels were found to reach a maximum in early log phase and to decrease throughout the remainder of the log and deceleration phases. There was a 25% decrease in RNA amount when cells in late stationary phase were compared to those in deceleration. This loss of RNA was mimicked when cells from the deceleration phase were suspended in a non-nutrient buffered medium. Procedures were established to determine RNA content and the intra- and extracellular distribution of RNA degradation products, namely purine and pyrimidine bases and orthophosphate. Balance sheets are presented to show that the decrease in RNA levels was accompanied by an equivalent increase in purine and pyrimidine bases and phosphorus derivatives. The validity of the procedures employed was demonstrated. The influence of magnesium, cholesterol and glucose on the cells suspended in a non-nutrient buffer was examined. Each was found to affect the ultimate distribution of RNA products in a characteristic fashion suggesting that each compound acts by a different mode of action.     

Influence of Sphingolipid Bases,Fatty Acids and Tween 80 on the Folate Requirement of Tetrahymena pyriformis*

SYNOPSIS The fatty acids lauric, myristic, and oleic, as well as long-chain bases (LCBs) obtained from sphingolipids, and Tween 80 “spare” the requirement for folate by Tetrahymena pyriformis W. Since LCBs are metabolized by the ciliate to ethanolamine phosphate and fatty aldehydes which can be converted to either fatty acids or fatty alcohols, the latter compounds are used as precursors of phospho- and phosphonolipids and ether phospholipids. It is suggested that lipid biosynthesis is a rate-limiting step in growth of the ciliate as is the folate concentration. Removal of one restraint on growth rate mimics the effect of increased folate concentration. Alternatively, if the enzymes responsible for lipid synthesis are repressible, the presence of exogenous fatty acids would make available more formylmethionyl-tRNA for the initiation of synthesis of other proteins.