Leif Rasmussen

Dual capacity for nutrient uptake in Tetrahymena. IV. Growth without food vacuoles and its implications.

Abstract A mutant of Tetrahymena pyriformis , syngen 1, having heat-sensitive development of its oral apparatus, can be grown without food vacuole formation indefinitely by supplementing standard growth media with high concentrations of folinic acid, Fe and Cu. Study of a revertant led to the inference that these requirements ensue from lack of food vacuole formation. These results along with those in preceding papers of this series, confirm the dispensability of the food vacuole and reveal a remarkable versatility of the system on the cell surface for uptake of small dissolved molecules. Thus T. pyriformis possesses two efficient and separate nutrient uptake systems, each capable of being nearly self-sufficient (if not completely so). Important implications of these results are discussed. The excellent growth of the mutant without food vacuoles makes available useful cells for a variety of experiments. Our results provide hints for supplementation of growth media for experimentation in which the cells must be under stress. The results are also relevant to the use of Tetrahymena in the bioassay of vitamins and nutritive quality of proteins.

Particulate material as a prerequisite for rapid cell multiplication in Tetrahymena cultures

1. n1. Tetrahymena pyriformis GL, cultivated axenically in 2 % proteose peptone in slanted test tubes at 28°C, had a population doubling time of more than 40 h when the medium was sterilized by asbestos pad or membrane filtration. Under these conditions the cultures could not be propagated successfully. n n2. n2. The doubling time was reduced to 5 h or less when the medium was sterilized in the autoclave instead of by filtration. n n3. n3. Various additives improved the growth potential of the sterile-filtered proteose peptone. These included both insoluble materials such as talcum, quartz, clay and calcium carbonate, and water-soluble compounds which through hydrolytic decomposition readily produce insoluble particles in aqueous solution, as, for example, ferrous ammonium sulphate, ferrous sulphate and potassium aluminium sulphate. Other water-soluble compounds such as amino acids, purines, pyrimidines, vitamins and glucose, added either singly or in combination to the sterile-filtered broth, did not significantly improve the rate of cell multiplication. n n4. n4. The average number of food vacuoles present in the cells of a culture was observed to be correlated with the growth potential of the medium. n n5. n5. On the basis of these observations it is suggested that formation of food vacuoles in Tetrahymena induced by the presence of particulate material is a condition for rapid multiplication of the cells.

Nutrient uptake in Tetrahymena pyriformis

Several morphological structures have been implicated in nutrient uptake in the ciliate protozoon,Tetrahymena pyriformis: food vacuoles, various types of vesicles and the plasma membrane. It is the object of this report to discuss the roles of these organelles in food uptake. Measurements of multiplication rates under conditions where food vacuole formation could be controlled experimentally suggested that the food vacuoles (about 5 μm in diameter) were essential for rapid cell multiplication in various standard growth media. If, however, concentrations of certain specific nutrients (different for different strains ofT. pyriformis) were high, then the cells could multiply rapidly even when food vacuoles were absent. Furthermore, multiplication rates of cells supplied with particulate or dissolved egg albumin as the amino acid source, suggested that the food vacuoles took up particulate egg albumin well, but dissolved egg albumin poorly. The role in food uptake of vesicles with a diameter of less than 1 μm remains largely unknown. Our present knowledge of them is not yet sufficiently detailed to permit estimations of the rates with which they are formed or of their total number per cell. The plasma membrane has carrier-mediated uptake sites for a number of nutrients such as amino acids and nucleosides. It is likely that this type of uptake mechanism plays a quantitatively important role inT. pyriformis whenever such compounds are present in the extracellular fluid.

Survival of Tetrahymena thermophila at low initial cell densities. Effects of lipids and long-chain alcohols.

ABSTRACT. Cells of the ciliate Tetrahymena thermophila failed to establish cultures in lipid‐free standard synthetic nutrient medium if the initial population density was 250 cells per ml or less. These cells died within 10 h, but were saved and formed dense cultures if their medium was supplemented with 10 μg per ml of either certain phospholipids, 1,2‐di‐, 1‐monoglycerides, fatty acids, long‐chain alcohols, or sterols. Cell multiplication was followed in cultures in which the standard synthetic medium was supplemented with a selection of the compounds listed above. It was observed that the cells in the supplemented cultures in their exponential phases of growth had about the same average doubling times as control cells starting multiplication at 10‐fold higher initial cell densities in lipid‐free medium. These cells have been grown for decades in lipid‐free synthetic nutrient media at short (ca. two‐three h) doubling times. Therefore lipids have been considered nutritionally non‐essential for growth and multiplication of these cells. We propose that those compounds that rescue the cells at low cell densities act as “proliferation signals,”sensu lato. This effect of lipids and long‐chain alcohols has so far remained unnoticed.

Dual capacity for nutrient uptake in tetrahymena importance of the oral uptake system for Fe and Cu uptake

We have reported that a mutant ofTetrahymena pyriformis with heat-sensitive development of the oral apparatus can be grown indefinitely without food vacuoles if the medium is supplemented with folinic acid and a mixture of trace metal salts. We report here that the trace metal mixture can be replaced completely and specifically by salts of iron and copper. Fe(II) and Fe(III) are interchangeable. Addition of citrate has proven useful to reduce precipitate formation and improve the reproducibility of growth of the mutant cell. Thus it appears to serve as an Fe buffer.From the increased concentrations of Fe and Cu required to permit good growth of the mutant strain at 37°C, we conclude that the oral uptake system plays a much more important role in the case of these two metals than the surface uptake system. The oral uptake system may facilitate Fe uptake in at least two ways: a) by a mechanical concentration of precipitates affected by the ciliary membranelles surrounding the oral cavity and, b) by lowering of the pH of the food vacuole and thereby releasing Fe from precipitates and from complexes which cannot be transported across the membrane as such. The second factor may also be important in Cu uptake.A specific effect of Mg and Fe uptake or retention in the mutant strain growing without food vacuoles has been detected. The significance of this effect remains unclear.Practical implications of the findings are discussed.

On the role of food vacuole formation in the uptake of dissolved nutrients by Tetrahymena

Abstract 1. 1. In sterile filtered 2 % proteose peptone medium, growth of Tetrahymena pyriformis was slow (doubling time: 100 h at 18 °C) and the mean number of food vacuoles per cell was low (less than 1). 2. 2. Addition of indigestible polystyrene beads (approx. 1 μm in diameter) or ferric hydroxide particles to the sterile filtered medium stimulated considerably both formation of food vacuoles and growth rates (doubling time: about 10 h). 3. 3. Addition of nucleosides at high concentration (6 mM) and glucose to the sterile filtered medium also increased growth rates (doubling time: 20 h), but in this case the number of food vacuoles per cell remained low. 4. 4. The organism T. pyriformis is unable to synthesize purines and pyrimidines de novo. The above findings show that in order to multiply rapidly, it must either have these compounds (or a source of them, e.g. the corresponding nucleosides) supplied at relatively high external concentrations, or be able to obtain them from lower external concentrations through formation of food vacuoles. The same appears to hold for other groups of nutrients, e.g. the amino acids.

Chapter 3 Recording of Clonal Growth of Mammalian Cells through Many Generations

Publisher Summary This chapter discusses experimental studies focusing on the recording of clonal growth of mammalian cells through many generations. The cultures were followed over a period of 6–7 days by exposures made every hour. Because cell division occurred at an unknown time between two photographic recordings spaced an hour apart, a method was needed for assigning the division time to these cells in an accurate manner. Assigning of the division time made use of several morphological criteria. When one frame showed one cell in the clone and the next frame showed two cells in its place, it was obvious that cell division had occurred between the times of the two recordings. The average duration of the four successive cell generations was calculated. Correlations among generation times were tested. The frequency distribution of the generation times obtained in the study was analyzed. The mean generation time was found to be 21.5 ± 3.3 hours and included minimum and maximum values of 14 and 37 hours, respectively. The method described in the chapter can be used for several different types of experiments.

Na+-independent uptake of nutrients inTetrahymena

SummaryCell multiplication rates in cultures ofTetrahymena pyriformis andT. thermophila were independent of the external Na+ concentrations at the levels of 0.5, 10 and 22 mM. The Nai/Na0 and Cli/Cl0 ratios were determined at the low and high Na+ concentrations, and assuming that Cl− is distributed passively, the electrochemical Na+ gradients for the two situations were calculated. The energy in these gradients allows a net co-transport of nutrients only in the high Na+ medium. Since the doubling times are independent of the Na+ concentrations we conclude thatTetrahymena can obtain its energy for nutrient uptake from other sources than the electrochemical Na+ gradient.

Cell Division and DNA Replication in Synchronous Tetrahymena Cultures

Several methods are available for the induction of synchronous cell divisions in pro- and eukaryote cells. The present article deals almost exclusively with the synchronization of the ciliate protozoan Tetrahymena pyriformis, which is one of the cells most intensively studied in synchronous populations. As it will appear from the following pages Tetrahymena has been synchronized with several different procedures. The first of these was the multiple heat shock system which produced well-synchronized cell divisions. Later modification of the original programme synchronized not only cell division but also DNA replication. Since most of the work based on synchronized Tetrahymena cells has been done with heat-synchronized cells, recent results obtained with such cells will take up most of this review.

Autocrine/Paracrine Activator of Cell Proliferation: Purification of a 4–6 kD Compound with Growth-Factor-Like Effects in Tetrahymena thermophila

Extracellular medium from cultures of Tetrahymena thermophila was collected, concentrated and fractionated by ultrafiltration and low-pressure column chromatography. The effect on proliferation of various fractions was tested on cultures of T. thermophila in synthetic medium at low initial cell densities (<250 cells/ml). In unsupplemented cultures, cells failed to thrive and died before proliferation. Addition of the fraction isolated from the extracellular medium in the molecular weight range of 1–10 kD activated the cells to proliferate. The size of the main compound in this fraction, herein referred to as TPAF, was in the range of 4–6 kD as estimated by SDS-PAGE. Evidence of a dialysable factor produced by cells in the stationary phase inhibiting cell proliferation has also been found.