Jytte R. Nilsson

Nucleolar organization and ribosome formation in Tetrahymena pyriformis GL

Abstract The multiple peripherally located small nucleoli in the macronuclei of exponentially growing Tetrahymena cells aggregate to a few large fusion bodies within 1 h of starvation. At the same time changes occur in the nucleolar substructure and many small thread-like or ring-shaped elements appear to be associated with the fusion bodies. Refeeding of the cells with fresh growth medium induces dissociation of the fusion bodies into small nucleoli. The typical log phase nucleolar organization is attained within 1–2 h of refeeding; at the same time net-RNA synthesis begins. The nucleolar organization in Tetrahymena , described above, is different from that in metazoan cells where one or few nucleoli enlarge when ribosome formation is accelerated. Since the nucleoli are sites for formation of ribosomes, the particular nature of the nucleolar organization in Tetrahymena may be related, at least in part, to the larger ribosome-forming capacity of this cell compared with mammalian cells. The large number of nucleoli and the high ribosome assembling efficiency of each nucleolus may both account for the high ribosome forming capacity of Tetrahymena . The high efficiency of each nucleolus may be related to its location near the nuclear envelope which is likely to offer easy access for precursor material and favorable conditions for export of nascent ribosomal particles to the cytoplasm. Morphological observations of isolated macronuclei and of centrifuged cells indicate that the nucleoli are associated with a larger structural entity in the periphery of the nucleus. This structure may or may not be identical with the nuclear envelope.

Effects of copper on phagocytosis inTetrahymena

SummaryAddition of copper, corresponding to 100 ppm, to the normal 2% proteose peptone medium is tolerated byTetrahymena. This concentration of copper stimulates phagocytosis to a maximum value which is reached gradually during the first 1 hour exposure, and which is maintained during continuous exposures. Cell proliferation is resumed after a lag period, although at a decreased rate. Cells exposed to copper contain small refractile granules, previously proposed to represent an ion-regulating system; the number of granules remains constant in proliferating cells. Higher concentrations of copper also resulted in an elevated rate of phagocytosis but at the same time cell mortality was observed; this lack of transition between inhibited phagocytosis and cell mortality may be ascribed to the physiological role of copper. The high amount of organic matter in the growth medium protects against the toxic effects of copper, thus in the absence of organic matterTetrahymena tolerated only a 100-fold lower concentration of copper than that tolerated in the growth medium. However, cells which had initiated granule formation (for example for regulation of calcium) prior to starvation and exposure to copper, were more resistant to copper than cells which had not yet activated this mechanism, perhaps because of the low capacity of starved cells for protein synthesis.

On cell organelles in Tetrahymena. With special reference to mitochondria and peroxisomes

In the ciliate, Tetrahymena pyriformis, the population of cell organelles shows variation during the culture cycle and when the cells are subjected to various treatments. New cell constitutents may appear and the fine structure of mitochondria and peroxisomes may change. Such dynamic structural changes are described and reviewed in relation to physiological and biochemical studies on Tetrahymena. The cell organelles discussed are about 1 μm in diameter or smaller; reference is also made to the nucleolar organization which is the most sensitive indicator of the physiological state of the cells. The presumed distribution of the various cell organelles in subcellular fractions of Tetrahymena is mentioned, since dependent on the pretreatment of the cells, especially the mitochondrial and peroxisomal fractions may be contaminated to varying degrees with other cell organelles.The purpose of the review is to demonstrate that a correlation exists between the structure and function of cell organelles, especially mitochondria and peroxisomes, and that the overall fine structure of a cell reflects its physiological state.

On starvation-induced autophagy in Teraahymena

Starvation-induced autophagy in Tetrahymena was studied over a 48-hour period during which the cells decreased in size to a quarter of their original volume. The autophagosomes were divided into 4 types: I) Clearly identifiable cytoplasmic content delimited by 2 membranes. II) Clearly identifiable content delimited by one membrane. III) Content of remnants permitting identification of origin. And, IV) Completely disintegrated content of unknown origin. All types were present in 30-minute starved cells and Type I organelles were seen throughout starvation, thus indicating that autophagy occurred continuously. The number of autophagosomes per unit cytoplasmic area remained constant but with a decrease of the first 3 types and an accumu-lation of Type IV. Acid phosphatase was found in some Type II organelles and in most Types III and IV; these organelles are autolysosomes but Type I is not. Disintegration of the inner autophagosomal membrane (passage from Type I to Type II) thus occurs in the absence of lysosomal enzymes but could be ascribed to an assumed acidification of the organelles. In addition to autophagosomes, profiles were observed of double membrane systems, isolation membranes, enveloping cell constituents as an indication of autophagy in progress. These isolation membranes seemed to derive from Golgi complexes as tubula, or sac-like structures; moreover, such structures were seen in association with microtubules which indicates a possible transport mechanism to the site of autophagy. An analysis of the origin of the autophagosomal contents, revealed that cytoplasm was enclosed throughout starvation, whereas enclosure of mitochondria, peroxisomes, and membrane whorls occurred in a time-dependent manner. Hence in this ciliate, autophagy occurs in a well-defined and controlled manner.

Retention of lead within the digestive vacuole inTetrahymena

SummaryThe 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 μm 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.

Effects of chloramphenicol on the physiology and fine structure ofTetrahymena pyriformis GL: Correlation between diminishing inner mitochondrial membrane and cell doubling

SummaryA time-dependent loss of tubular infolding of the inner mitochondrial membrane was reported recently as an effect of the cytostatic drug, methotrexate (MTX), onTetrahymena (Nilsson 1983); this finding was interpreted as an inhibition of mitochondrial protein synthesis. In the present study, the cells were exposed to chloramphenicol (CAP), an inhibitor of mitochondrial translation, at the same concentrations (1–25 mM) as MTX; the question asked was whether the two drugs acted similarly. CAP affected cell proliferation by causing a dose-dependent prolongation of the generation time, but at 10–25 mM permitted only a limited number of cell doublings, whereas 1 mM MTX inhibits growth after 5 cell doublings. With CAP the inner mitochondrial membrane diminished gradually in accordance with the number of cell doublings at 10–25 mM, but in 2 mM CAP, for example, some tubular infoldings were still present after 17 cell doublings. The gradual loss of the inner mitochondrial membrane correlated with a gradual decrease in the cellular ATP content, irrespective of the concentration of the drug but dependent on the progress of the cells through their first cycle when exposed to the drug; in cells which continued to proliferate, the ATP content remained at a value corresponding to 80% of the control value. With respect to cell proliferation, the two drugs act differently. CAP is less toxic than MTX, reflected in a 10 times shorter recovery time for cell proliferation after removal of CAP. Hence, although the structural manifestation of the action of the two drugs on mitochondria is identical, their target site may differ.

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

SummaryAt 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.

Effects of dimethyl sulphoxide on ATP content and protein synthesis inTetrahymena

SummaryThe content of ATP in cells exposed for 1 hour to 2.5% and 7.5% dimethyl sulphoxide (DMSO) was 90% and about 80%, respectively, of that in control cells. This difference of about 10% in the ATP content cannot explain the previously reported cessation of food vacuole formation in 7.5% DMSO and the uninhibited function in 2.5% DMSO (Nilsson 1974). However, DMSO had a dose-dependent effect on the rate of turnover of ATP in cell extracts, thus the amount of ATP expended per unit time in 7.5% DMSO is only 60% of that expended by extracts of control cells. The rate of protein synthesis was studied by electron microscope autoradiography which revealed considerably less labelled material in DMSO-treated cells than in control cells. Semi-quantitative estimates showed that cells in 2.5%, 5%, and 7.5% DMSO had a rate of incorporation of the labelled amino acid corresponding to 38%, 31%, and 51%, respectively, of that of control cells; the seemingly high rate of incorporation in 7.5% DMSO may reflect a low internal pool of amino acids in these cells. An additional fine structural detail is the induction of intranuclear fibrous bundles in all concentrations of DMSO. The findings are in accord with a random interference of DMSO, presumably by inducing conformational changes in some macromolecules which affect their cellular function.

Synthesis of ribosomal DNA in conjugating Tetrahymena.

Abstract In the ciliate protozoan Tetrahymena thermophila, a single integrated gene coding for ribosomal RNA in the micronucleus is amplified during the sexual cycle to yield many copies of extrachromosomal palindromic rDNA in the macronucleus. Hybridization of newly synthesized DNA with rRNA has shown that extensive rDNA synthesis takes place early in the sexual cycle of Tetrahymena. The number of genes synthesized during this period is sufficient to account for gene amplification. A later period of rDNA synthesis occurs when new macronuclear anlagen are beginning to develop. This synthesis may represent preferential polyploidization of already amplified rDNA.

Vanadate affects nuclear division and induces aberrantly-shaped cells during subsequent cytokinesis in Tetrahymena

ABSTRACT Sodium orthovanadate at 0.1–5.0 mM affected cell proliferation of Tetrahymena in a dose‐dependent manner. At 1 h the cell increment was 76–12% of the control (100%), but after lag periods in 1–5 mM the growth rate remained at 76% of control in 0.1 mM vanadate and at 64–61% of control in 0.2–5.0 mM vanadate. Endocytosis was affected in both a time‐ and dose‐dependent manner; an increasing number of cells did not form vacuoles. Cell motility increased initially in 0.1 mM vanadate but decreased later as it did in 0.5–2.0 mM vanadate where the proportion of immobile cells increased with time. Cell divisions occurred at all concentrations but macronuclear elongation was disturbed and subsequent cytokinesis resulted in daughter cells containing the entire G2 macronucleus, a large or small portion of it, or no nucleus at all. Moreover, odd cell shapes appeared with time. The size of the cell and nucleus increased but there was great variation with disturbed cytoplasm/nucleus ratios. Treated cells had dilated rough endoplasmic reticulum that included dense material, presumed to be vanadate. which was not seen in control cells. Scant amounts of dense material were found in dense granules, small vacuoles, and abundantly in contractile vacuoles. It is argued that interference with proper microtubular function is the main effect of vanadate.