Selection of Nonmotile Tetrahymena with Ficoll Underlayers*,†

The mechanisms regulating the development of cilia in Tetrahymena are poorly understood but might be revealed through the study of ciliogenesis mutants. Failure to regenerate cilia after dibucaine deciliation results in continued absence of motility. Therefore, to isolate ciliogenesis mutants efficiently, methods for separating motile and nonmotile cells are essential. We examined the efficacy of Ficoll underlayers for these separations. Ciliates of T. thermophila strain mpr^-/mpr (6 mp sens IV) (6-methyl purine-sensitive; mating type IV) were mixed with Ficoll and added as underlayers to separatory funnels containing growth medium. At 27 C most of the cells remained motile and were found in the top layer; at 37 C, there was a time-dependent increase in the number of nonmotile cells and the number of cells in the Ficoll layer. After 150 min at 37 C, most of the cells became nonmotile and were found in the Ficoll layer. Other studies indicated that at 37 C, the cells remained alive and capable of regenerating cilia when deciliated. Thus, it is clear that the Ficoll underlayer effectively separates the majority of nonmotile cells from the majority of motile cells. Evidently, however, at 37 C wild-type T. thermophila exhibit temperature-sensitive phenotypic variability with regard to motility which should be minimized when selecting for mutations affecting motility and ciliogenesis.     

THE REGENERATION OF CILIA IN PARTIALLY DECILIATED TETRAHYMENA

Partial deciliation of Tetrahymena resulted in cells losing 75% of their cilia, with the balance being paralyzed. The paralyzed cilia are resorbed in the first 20 min after partial deciliation, and regeneration of cilia begins before resorption is completed. Inhibition of protein synthesis with cycloheximide does not inhibit ciliary resorption or regeneration, whereas vinblastine sulfate inhibits regeneration but not resorption. Inhibition of regeneration occurs in completely deciliated cells when they are treated with cyclohexmimide or vinblastine sulfate. It is concluded that the resorbing cilia contribute materials which allow regeneration to occur in the absence of protein synthesis. The volume of cilia regenerated in the presence of cycloheximide in partially deciliated cells is greater than the ciliary volume which is resorbed. This suggests the Tetrahymena cells have a pool of ciliary precursors. This pool does not contribute materials for regeneration in completely deciliated cells which are treated with cycloheximide. It is concluded that resorbing cilia in partially deciliated cells contribute materials which potentiate assembly of cilia from the pool of precursors.     

CILIA REGENERATION IN TETRAHYMENA AND ITS INHIBITION BY COLCHICINE

The cilia of Tetrahymena were amputated by the use of a procedure in which the cells remained viable and regenerated cilia. Deciliated cells were nonmotile, and cilia regeneration was assessed by scoring the percentage of motile cells at intervals following deciliation. After a 30-min lag, the deciliated cells rapidly recovered motility until more than 90% of the cells were motile at 70 min after amputation. Cycloheximide inhibited both protein synthesis and cilia regeneration. This indicated that cilia formation in Tetrahymena was dependent on protein synthesis after amputation. Conversely, colchicine was found to inhibit cilia regeneration without affecting either RNA or protein synthesis. This observation suggested the action of colchicine to be an interference with the assembly of ciliary subunit proteins. The finding that colchicine binds to microtubule protein subunits isolated from cilia and flagella (13) supports this possibility. The potential of the colchicine-blocked cilia-regenerating system in Tetrahymena for studying the assembly of microtubule protein subunits during cilia formation and for isolating ciliary precursor proteins is discussed.     

A Simple Method for Determining Antitubulinic Activities of Ansamitocins and Related Compounds Using a Cilia Regeneration System with Deciliated Tetrahymena

Cilia regeneration with deciliated Tetrahymena pyriformis W was tested to determine the antitubulinic activities of ansamitocins and related compounds in a microbial system. Various factors interfered with the regeneration process, i.e. excess shearing force in deciliation procedure, high temperature (32°C or above), high or low pH (pH 9 or 5), and exogenous divalent cations, such as Zn²⁺, Mn²⁺, Cu²⁺ and Co²⁺. Under suitable conditions, cilia regeneration was completed in about 60 min of incubation, and a number of newly formed cilia were observed around the cell surface. When ansamitocins were added to the recovery solution, cilia regeneration was completely suppressed without alterations in cell shape or the surface structure of deciliated Tetrahymena. In this assay system, the inhibitory activity of ansamitocins was slightly stronger than that of maytansine.     

Low concentrations of dimethyl sulphoxide accelerate conjugation and incorporation of glycine and glucosamine intetrahymena

Summary Dimethyl sulphoxide at relatively low comentrations, 0.01 to 1 mM, enhanced the conjugation and cell-to-cell adhesion of complementary strains of matingTetrahymena thermophila. The time required to form stable conjugates was reduced by dimethyl sulphoxide. This chemical stimulated the uptake of glycine and glucosamine from the suspending media. Incorporation of 2-¹⁴C-glycine and 6-³H-D-glucosamine into protein and glycoprotein was enhanced in whole cells, surface membrane and cilia. Incorporation of glucosamine into the microsomal fraction was increased in the dimethyl sulphoxidetreated cells while there was little change in glycine incorporation. There were no detectable changes in glycine and glucosamine incorporation into the nuclear fractions isolated from conjugatingTetrahymena exposed to dimethyl sulphoxide.     

Isolation of a Δ7-cholesterol desaturase from Tetrahymena thermophila

Cell-free preparations of Tetrahymena thermophila catalyze the direct desaturation of cholesterol to Δ⁷-dehydrocholesterol (provitamin D₃). The activity was isolated in the microsomal fraction from Tetrahymena homogenates. Δ⁷-Desaturase activity was stimulated fivefold by the addition of 6 mM ATP. Other cofactors assayed, including NAD, NADP, NADH or NADPH, had no significant effect. The activity was found in microsomes prepared from stationary-phase cultures of the ciliate, grown either with or without added cholesterol, thus indicating that it is constitutively expressed in T. thermophila cells. Received: 17 May 1999 / Accepted: 24 September 1999     

Induction of tubulin synthesis during cilia regeneration in growing Tetrahymena

Log growth Tetrahymena pyriformis GL were deciliated by means of a calcium pulse and allowed to regenerate their cilia in a non-nutrient recovery medium. Polyribosome profiles show only small amounts of polysomes up to 30 min after suspension in recovery medium. After this time the number of polysomes increases continuously as protein synthetic activity and motility are recovered. Labeling of whole cells with l-[³⁵S]methionine and comparison of the resulting electrophoretic patterns reveals a marked induction of tubulin synthesis as cilia regeneration proceeds. At its peak, tubulin accounts for 7–10% of the incorporated label but this peak occurs 35 min after the cells become greater than 90% motile and about 25 min after the cilia reach full length. These results are discussed with respect to the regulatory mechanism of tubulin induction and induction of tubulin synthesis in starved Tetrahymena.     

Lanthanum(III) Impacts on Metallothionein MTT1 and MTT2 from <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Metallothionein MTT1 and MTT2 from Tetrahymena thermophila are sulfydryl-rich proteins that can bind to and are inducible by heavy metals such as mercury, cadmium, zinc, and copper. However, little is known about the induction and binding of T. thermophila metallothionein by trivalent metals. In this study, we found that 10–80 μM La³⁺ can promote Tetrahymena cells proliferation, and fluorescence spectrum analysis showed that La³⁺ can enter T. thermophila cells. Real-time quantitative polymerase chain reaction showed La³⁺ induced the expression of MTT1 and MTT2. Furthermore, Fluorescence analysis indicated La³⁺ bind to MTT1 and MTT2. These results implied that La³⁺ could interact with MTT1 and MTT2 via aspartic or glutamic acid oxygen atoms.     

Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I.

We first identified GTP cyclohydrolase I activity (EC 3.5.4.16) in the ciliated protozoa, Tetrahymena pyriformis. The V_max value of the enzyme in the cellular extract of T. pyriformis was 255 pmol mg⁻¹ protein h⁻¹. Michaelis–Menten kinetics indicated a positive cooperative binding of GTP to the enzyme. The GTP concentration producing half-maximal velocity was 0.8 mM. By high-performance liquid chromatography (HPLC) with fluorescence detection, a major peak corresponding to -monapterin (2-amino-4-hydroxy-6-[(1′R,2′R)-1′,2′,3′-trihydroxypropyl]pteridine, -threo-neopterin) and minor peaks of -erythro-neopterin and -erythro-biopterin were found to be present in the cellular extract of Tetrahymena. Thus, it is strongly suggested that Tetrahymena converts GTP into unconjugated pteridine derivatives. In this study, dopamine was detected as the major catecholamine, while neither epinephrine nor norepinephrine was identified. Indeed, this protozoa was shown to possess the activity of a dopamine synthesizing enzyme, aromatic -amino acid decarboxylase. On the other hand, activities of tyrosine hydroxylase or tyrosinase which converts tyrosine into dopa, the substrate of aromatic -amino acid decarboxylase, could not be detected in this protozoa. Furthermore, neither dopamine β-hydroxylase activity nor phenylethanolamine N-methyltransferase activity could be identified by the HPLC methods.     

Tetrahymena: An Alternative Model Host for Evaluating Virulence of Aeromonas Strains

An easier assessment model would be helpful for high-throughput screening of Aeromonas virulence. The previous study indicated the potential of Tetrahymena as a permissive model to examine virulence of Aeromonas hydrophila. Here our aim was to assess virulence of Aeromonas spp. using two model hosts, a zebrafish assay and Tetrahymena-Aeromonas co-culture, and to examine whether data from the Tetrahymena thermophila model reflects infections in the well-established animal model. First, virulence of 39 Aeromonas strains was assessed by determining the 50% lethal dose (LD₅₀) in zebrafish. LD₅₀ values ranging from 1.3×10² to 3.0×10⁷ indicated that these strains represent a high to moderate degree of virulence and could be useful to assess virulence in the Tetrahymena model. In Tetrahymena-Aeromonas co-culture, we evaluated the virulence of Aeromonas by detecting relative survival of Aeromonas and Tetrahymena. An Aeromonas isolate was considered virulent when its relative survival was greater than 60%, while the Aeromonas isolate was considered avirulent if its relative survival was below 40%. When relative survival of T. thermophila was lower than 40% after co-culture with an Aeromonas isolate, the bacterial strain was regarded as virulent. In contrast, the strain was classified as avirulent if relative survival of T. thermophila was greater than 50%. Encouragingly, data from the 39 Aeromonas strains showed good correlation in zebrafish and Tetrahymena-Aeromonas co-culture models. The results provide sufficient data to demonstrate that Tetrahymena can be a comparable alternative to zebrafish for determining the virulence of Aeromonas isolates.     

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.     

The use of <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis> mutant cell line for removal of cholesterol from milk

The nonpathogenic ciliate Tetrahymena thermophila converts cholesterol from foodstuffs into provitamin D compounds in high yields. However, prolonged incubation with wild-type strain CU-399 at high densities results in a final deterioration of milk properties, possibly as a result of secreted hydrolases. Here we attempted to solve this problem using MS-1 Tetrahymena strain, a stable mutant with a low rate of hydrolase secretion. Densities of to 2 × 10 ⁶ cells/ml can be incubated for up to 5 h in milk, without any clotting or change in appearance. Moreover, centrifugation of this suspension eliminates most of the cells, and results in an about 75% ± 10 (n = 10) decrease of the initial cholesterol. Sterols are recovered in the cell pellets, which show that Tetrahymena is able to avidly capture them from the medium. Therefore, this mutant strain is optimal for milk cholesterol depletion, avoiding unfavorable sensory alterations.     

Phosphonoacetic Acid: Effect on and Disposition by Tetrahymena1

The antiviral agent phosphonoacetic acid inhibits growth of Tetrahymena thermophila at concentrations comparable to those inhibiting growth of other eukaryotic cells, with 50% inhibition at 0.5 mM phosphonoacetic acid. The compound is cytotoxk to Tetrahymena at concentrations greater than 2.0 mM. When a culture of Tetrahymena the growth of which was totally inhibited by 2.0 mM phosphonoacetic acid was diluted with fresh medium, growth resumed in an exponential, rather than synchronous, fashion. [2–¹⁴C]phosphonoacetic acid is not metabolized by Tetrahymena.     

Lysosomal enzymes produced by immobilized Tetrahymena thermophila

Summary The ciliated protozoon Tetrahymena thermophila was immobilized for production of secreted lysosomal enzymes in two ways. Cells entrapped in solid Ca-alginate spheres survived but were unable to grow and multiply. However, when encapsulated in hollow Ca-alginate spheres Tetrahymena multiplied well, reaching 0.9 × 10⁷ cells/ml. These immobilized cells secreted large amounts of lysosomal enzymes when the medium was changed daily. This system was transferred to a reactor scale using a conical bubble column reactor for semicontinuous cultivation of the encapsulated cells. Under these conditions -glucosidase, -glucosidase, -hexosaminidase and acid phosphatase were produced for at least 4 weeks. The hollow spheres were stable for 3 months and contained living and secreting Tetrahymena cells during this time. Immobilized T. thermophila cells can thus serve as a good source for production of commercially interesting enzymes. Offprint requests to: A. Tiedtke     

Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymena pyriformis

Tetrahymena pyriformis reportedly possesses binding structures for the vertebrate hormone insulin that are amplified in cells having prior exposure to the hormone. Conventional and confocal microscopic studies were conducted to verify the validity of the reports and to localize the binding sites. Logarithmic cultures were exposed to insulin concentrations of 0, 3, 6, and 12 μg/ ml for 1 h (receptor induced, RI). After an additional culture period the cells were fixed, exposed to porcine insulin (antigen), immunocytochemically processed, and examined for staining intensity by video image analysis. Observations indicate that T. pyriformis does bind insulin whether or not the cells have prior exposure to insulin. Staining intensity increased at the two highest RI concentrations over 0 μg/ml (P < 0.01) but the staining intensity at 0 μg/ml was not different from that at 3 μg/ml. The results confirm that T. pyriformis does bind insulin and that prior exposure to insulin increases the binding capacity for insulin in what may be a concentration-dependent manner. Confocal microscopy of RI cells that had been labeled with either fluorescein isothiocyanate-insulin or the immunocytochemical technique outlined above revealed labeling of the cytoplasm that appeared to be vesicular. Both techniques produced very similar labeling patterns when optical sections through the cells were viewed. Conventional fluorescence revealed ciliary labeling that could be decreased by incubation with excess unlabeled insulin. Further studies with the exo⁻ mutant of T. thermophila, SB 255, showed that mucocyst discharge and capsule formation are not involved in insulin binding.     

SHORT COMMUNICATIONSTEROID HORMONE (HYDROCORTISONE, OESTRADIOL AND TESTOSTERONE) UPTAKE, STORAGE OR INDUCED SYNTHESIS INTETRAHYMENA

After cyclodextrin-coated 10⁻⁶ steroid hormone treatment for 3 days (hormonal imprinting), Tetrahymena cells and their media were analysed by radioimmunoassay for the same hormone and for the presence of the other two. In the absence of hormone treatment, the cells contained no detectable levels of the three steroids. By 2 days in fresh medium following exposure of cells to a 72 h pretreatment of each specific hormone, correspondingly high quantities of hydrocortisone and oestradiol, but lesser quantities of testosterone, were found in both the media and the cells. One week after treatment only traces of hydrocortisone were found, exclusively within the cells themselves. Oestradiol was present in measurable quantities in both cells and media, whereas testosterone was only present in the medium. The presence of the other two hormones to the one used in the pretreatment were not usually present, except that when testosterone had been given, some oestradiol was also detected at 48 h, suggesting Tetrahymena has a functional cytochrome P₄₅₀aromatase.     

Effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells was investigated. As zinc compounds, we used zinc sulfate, AHZ, di(N-acetyl-β-alanyl-l-histidinato)zinc (AAHZ), and di(histidino)zinc (HZ). Cells were cultured for 72 h in the presence of zinc compounds (10⁻⁸–10⁻⁵M). The effect of AHZ (10⁻⁷ and 10⁻⁶M) to increase protein and deoxyribonucleic acid (DNA) contents in the cells was the greatest in comparison with those of other zinc compounds. Zinc sulfate and HZ at 10⁻⁷M did not have an effect on the cellular protein content. AHZ (10⁻⁶M) had a potent effect on cell proliferation, although zinc sulfate (10⁻⁶M) had no effect. β-Alanyl-l-histidine (10⁻⁶ and 10⁻⁵M) did not have an appreciable effect on the cells. Those effects of AHZ (10⁻⁶M) on osteoblastic cells were completely abolished by the presence of cycloheximide (10⁻⁶M). AHZ (10⁻⁸–10⁻⁵M) directly activated [³H]leucyl-tRNA synthetase in the cell homogenate, whereas the effect of zinc sulfate was seen at 10⁻⁶ and 10⁻⁵M. The present study suggests that the chemical form of zinc-chelating β-alanyl-l-histidine (AHZ) can reveal a potent anabolic effect on osteoblastic cells, and that AHZ directly stimulates protein synthesis.     

Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by improper cryopreservation

Ty1 is a retrotransposon of the yeast Saccharomyces cerevisiae whose transposition at new locations in the host genome is activated by stress conditions, such as exposure to UV light, X-rays, nitrogen starvation. In this communication, we supply evidence that cooling for 2 h at +4 °C followed by freezing for 1 h at −10 °C and 16 h at −20 °C also increased Ty1 transposition. The mobility of Ty1 was induced by cooling at slow rates (3 °C/min) and the accumulation of trehalose inside cells or the cooling at high rates (100 °C/min) inhibited significantly the induction of the transposition. The freeze-induced Ty1 transposition did not occur in mitochondrial mutants (rho⁻) and in cells with disrupted SCO1 gene (Δsco1 cells) evidencing that the Ty1 transposition induced by cooling depends on the mitochondrial oxidative phosphorylation. We also found that the freeze induced Ty1 transposition is associated with increased synthesis and accumulation of superoxide anions (O⁻₂) into the cells. Accumulation of O⁻₂ and activation of Ty1 transposition were not observed after cooling of cells with compromised mitochondrial functions (rho⁻, Δsco1), or in cells pretreated with O⁻₂ scavengers. It is concluded that (i) elevated levels of reactive oxygen species (ROS) have a key role in activation the transposition of Ty1 retrotransposon in yeast cells undergoing freezing and (ii) given the deleterious effect of increased ROS levels on cells, special precautions should be taken to avoid ROS production and accumulation during cryopreservation procedures.     

Phospholipase D in Tetrahymena: activity and significance

We detected phospholipase D in three species of ciliates: Tetrahymena: T. thermophila, T. pyriformis and T. setosa in nutrient medium supplemented with ethanol in in vivo systems, by the appearance of phosphatidylethanol. The calcium ionophore A23187 increased the synthesis of phosphatidylethanol, as compared with untreated controls. We suggest that Tetrahymena possess a calcium sensitive phospholipase D. Propranolol caused the cells in dense cultures to increase their average generation times or die, dependent on the drug concentration. This inhibition could be overcome by the addition of phospholipids or ethanol. Pure phosphatidylethanol had no effect on growth rates or generation times in cultures at high cell density, but postponed cell death in cultures at low cell density by a factor of 10. We suggest that an important role of phospholipase D in Tetrahymena is to supply the cell with diacylglycerol without which it can not enter the mode of proliferation from the lag phase of the culture.