Two forms of membrane-bound sphingosine kinase in Tetrahymena and activity changes during growth and the cell cycle

Sphingosine kinase is responsible for the formation of sphingosine-1-phosphate, a sphingolipid mediator with important roles in numerous physiological processes. The sphingosine kinase activity of Tetrahymena pyriformis was recovered predominantly in the particulate fraction and it could be solubilised in 1% beta-octylglucoside. Anion-exchange chromatography resolved the beta-octylglucoside-solubilised sphingosine kinase activity into two peaks corresponding to proteins of Mr 140,000 and 80,000 respectively, as determined by subsequent size exclusion chromatography on Superdex 200. N,N-dimethylsphingosine did not inhibit the sphingosine kinase activity in either fraction, whereas D,L-threo-dihydrosphingosine inhibited sphingosine phosphorylation by the Mr 80,000 kinase but had no effect on the Mr 140,000 kinase activity. The activities also showed different stimulatory responses to Triton X-100 or NaCl. Overall, the results suggest the existence in Tetrahymena of two distinct membrane-associated sphingosine kinases. The kinase activity determined at the different culture stages showed a transient elevation at the mid-logarithmic phase. Further, the sphingosine kinase activity was examined during the synchronous cell division induced by cyclic heat treatments in T. pyriformis. We report for the first time that the sphingosine kinase activity greatly increased at 30 to 45 min after the end of heat treatment prior to the synchronous cell division (75 min), suggesting that the activity changes were associated with the cell cycle and that the up-regulated sphingosine kinase activity would be required for the initiation of the oncoming synchronous cell division in Tetrahymena cells.     

The action of 5-amino uracil on log growth and division-synchronized Tetrahymena

The influence of 5-amino uracil (5-AU) was investigated on the cell cycle of log growth and division-synchronized Tetrahymena pyriformis GL. The division index of log growth phase Tetrahymena was suppressed by 50% after 40 min in 8 mM 5-AU. Cells division-synthronized by one heat shock per generation were also treated with 5-AU. Cells treated either prior to the first synchronous division (80 min EH) or up to 25 min prior to the second synchronous division (after 160 min EH) were not delayed in their progress through the cell cycle. Cells treated during the S phase of the first free running cell cycle, however, were delayed 5-30 min from reaching the second synchronous division. The effect of 5-AU on DNA and RNA synthesis was also examined. Incorporation of [3H]thymidine into acid-precipitable material was reduced in the presence of 5-AU; the rate of DNA synthesis was also reduced. The depression in the rate of DNA synthesis was greater at the beginning of S than at the end of S. The size of the thymidine pool (nucleosides + nucleotides) did not change during 5-AU treatment; however, an accumulation of thymidine tri-phosphate and a decrease in the amount of thymidine nucleoside was observed. A suppression of [14C]uridine incorporation resulting from 5-AU treatment was observed throughout the cell cycle. The rate of RNA synthesis as monitored by [14C]uridine incorporation into acid precipitable material was also reduced during 5-AU treatment. No change in either the size or the composition of the pool of uridine (nucleoside + nucleotide) was detected in 5-AU treated cells as compared to controls.     

Heavy Water and Hydrostatic Pressure Effects on Tetrahymena pyriformis1

SYNOPSIS. Heat-synchronized cultures of Tetrahymena pyriformis strain GL subjected to pulses of high hydrostatic pressure (10,000 psi for 2 min) had increasing division delays during the 1st 40 min after the last heat shock (40 min after heat treatment). Pressure treatment during the subsequent 10-min interval disrupted cell synchrony. Comparable pressures applied to the cells at later stages, before the 1st synchronous division, caused negligible division delay. Continuous exposure to 10% (v/v) heavy water hardly affected division; higher concentrations delayed or blocked division. Ten-min pulses with heavy water (40%, 50%, 70%) resulted in increasing division delays depending on the stage of the cell cycle during which the heavy water was applied. Amelioration of the division-delaying effects of pressure was observed in cells treated simultaneously with pressure (3,000 psi for 30 min), and 30% D₂O. The results are consistent with the hypothesis that some of the pressure and D₂O effects could be attributed to changes in the sol-gel state of the cytoplasm.     

Effect of delta 9-tetrahydrocannabinol on cyclic nucleotides in synchronously dividing Tetrahymena

Cyclic nucleotide levels were determined in division-synchronized Tetrahymena and the effect of delta 9-tetrahydrocannabinol (THC) on the cyclic nucleotide levels was studied. In non-drug-treated division-synchronized cells, there was no statistically significant variation in the level of cAMP and cGMP during the G2 period, preceding the first division. During the free running cell cycle (the interval of time between the first and second synchronous division) the twofold increase in the level of cAMP was statistically significant; however the variation in the level of cGMP was not statistically significant. THC caused a lowering of cAMP and cGMP levels throughout the 4-experimental treatment. The suppression of cAMP and cGMP levels altered the cyclic nucleotide pattern of the cell cycle. The cAMP pattern was changed particularly in the G2 period preceding the first synchronous division, and immediately after division during the free running cell cycle. THC treatment caused division delays of approximately 8-15 min in the onset of the first and second synchronous division. However, the duration of the free running cell cycle (110-120 min) was unchanged. The suppression of cyclic nucleotide levels resulting from THC treatment is discussed in relation to delays in the division schedule.     

GTP cyclohydrolase I from Tetrahymena pyriformis: cloning of cDNA and expression

A full-length cDNA clone for GTP cyclohydrolase I (EC 3.5.4.16) was isolated from a Tetrahymena pyriformis cDNA library by plaque hybridization. The nucleotide sequence determination revealed that the length of the cDNA insert was 1516 bp. The coding region encoded a protein of 223 amino acid residues with a calculated molecular mass of 25 416 Da. The deduced amino acid sequence of Tetrahrymena GTP cyclohydrolase I showed sequence identity with that of Escherichia coli (55%). The identity of T. pyriformis GTP cyclohydrolase I with sequences of Dictyostelium discoideum, Saccharomyces cerevisiae, Drosophila melanogaster, mouse, rat, and human enzymes was less marked and was 30, 30, 25, 28, 28, and 27%, respectively. RNA blot analysis showed a single mRNA species of 2.1 kb in this protozoan. The mRNA level of GTP cyclohydrolase I increased during synchronous cell division induced by intermittent heat treatment. The results suggest that the mRNA expression is associated with the cell cycle of T. pyriformis.     

Control of tubulin and actin gene expression in Tetrahymena pyriformis during the cell cycle

Poly(A)-containing mRNA was isolated from division synchronized populations of the ciliated protozoan, Tetrahymena pyriformis. The level of tubulin and actin mRNA at specific cell cycle stages was analyzed by hybridization to tubulin and actin cDNA probes and by gel analysis of their in vitro translation products. The pattern of fluctuation of tubulin mRNA levels was similar to that observed for the in vivo tubulin synthesis previously reported [1]. This suggests that as the cells progress through the cell cycle, tubulin synthesis is controlled at the mRNA level. There was little fluctuation of actin synthesis or actin mRNA levels during the cell cycle, which may be indicative of a different regulatory mechanism for actin than for tubulin.     

Requirements for DNA replication preceding cell division in Tetrahymena pyriformis

Populations of Tetrahymena pyriformis were synchronized by 30 min heat shocks at 34 °C separated by 160 min intervals at the normal growth temperature. The cells initiate DNA synthesis immediately after the cellular division, and the S period of the population lasts about 80 min. It was found that DNA replication is a prerequisite for the following synchronous division. Inhibition of the DNA synthesis in early S by starvation of the cells for thymidine prevents the forthcoming division. However, inhibition in the latter half of S does not prevent the subsequent division. Thus the cells have synthesized enough DNA to permit cell division before the end of a normal S period. These results are discussed in relation to the organization of the genome replication in the highly polyploid macronucleus.     

Tetrahymena actin: localization and possible biological roles of actin in Tetrahymena cells

Though actin is ubiquitous in eukaryotes, its existence has not been clearly proven in Tetrahymena. Recently, we have succeeded in cloning and sequencing the Tetrahymena actin gene using a Dictyostelium actin probe (Hirono, M. et al. (1987) J. Mol. Biol. 194, 181-192). The primary structure of the Tetrahymena actin deduced from the nucleotide sequence of its gene is greatly divergent from those of other known actins, making it necessary to ascertain whether the predicted Tetrahymena actin is indeed an actin. In this paper, we investigated the localization of the predicted Tetrahymena actin by an immunofluorescence technique using antibody against its synthetic N-terminal peptide, in order to elucidate its possible biological roles. The results showed that immunofluorescence was localized in the division furrow of the dividing cell, and in the intranuclear filament bundles formed in cells exposed to heat shock or DMSO. In addition, the oral apparatus and the proximity of the cytoproct, which are organelles involved in endocytosis and exocytosis, respectively, also fluoresced. Thus, we conclude that the Tetrahymena actin we identified is indeed an actin and plays the same biological roles as ubiquitous actins do, although it is considerably divergent in its amino acid sequence.     

Accumulation of a Protein Required for Division During the Cell Cycle of Escherichia coli

A heat-labile protein required for division accumulates during the duplication cycle of Escherichia coli. Its formation appears to commence shortly after the cell divides, and it reaches a maximal amount shortly before the next division. A plausible mechanism for timing cell division depends on building up the critical amount of this protein. Completion of deoxyribonucleic acid (DNA) replication is also necessary for division to occur, but it does not uniquely initiate division. The evidence for these conclusions comes from heat-shock experiments; heating to 45 C for 15 min delays division increasingly with the age of a cell. A heat shock given near the end of a cycle delays division for about 30 min, whereas at the beginning of the cycle it hardly affects division. The net result is synchronization of cell division. The effect of heat is increased in bacteria which have incorporated p-fluoro-phenylalanine into their proteins. When the incorporation is early and the heat shock is late in the cycle, division is delayed by about 30 min, indicating that the division protein is synthesized early even though its sensitivity is not observed until later. At any time in the cell cycle, heat shock simply delays total protein and DNA synthesis ((3)H-thymidine uptake) for approximately 14 min. DNA replication and cell division are thus discoordinated, since DNA replication is not synchronized by the treatment.     

TpMRK regulates cell division of Tetrahymena in response to oxidative stress

TpMRK was identified as a stress‐responsive mitogen activated protein kinase (MAPK)‐related kinase and has been shown to play a critical role in the stress signalling in Tetrahymena cells. Here, we found that the mRNA expression of TpMRK was correlated with cell division of Tetrahymena with decreased expression occurring in cells prior to entering synchronous cell division induced by heat treatment. Notably, cell division was delayed with a lower division index of 40% after exposure to hydrogen peroxide while 85% of cells underwent cell division synchronously at 75 min after heat treatment without the oxidative exposure. Furthermore, inactivation of TpMRK signalling by p38 MAPK inhibitor SB203580 or MEK inhibitor PD 98059 partially derepressed cell division induced by hydrogen peroxide. Our data suggest that oxidative stimuli might cause aberration of synchronous cell division of Tetrahymena through activating the TpMRK cascade. Copyright © 2009 John Wiley & Sons, Ltd.     

Changes in cAMP levels in bacterial cells during the cell cycle

The changes in the cAMP level during the cell cycle in the synchronous cultures of E. coli were demonstrated. Two maxima in the cAMP level were revealed during each generation period. In the cell cycle of 40-45 min duration the first increase was observed approximately in the middle of the cycle, i. e., it was coincident with the initiation of DNA synthesis. Under these conditions the cAMP level increased 8-10 times (from 0.5 to 5.0 pmole per ml of cell suspension). The second, less pronounced increase in the cAMP level was observed immediately before or during the cell division and was probably related to the regulation of the cell wall formation.     

Relationship of the DNA content in the nuclei of Amoeba proteus to the food regimen]

The relative DNA content of isolated Amoeba proteus nuclei has been measured by cytofluorometry. With the amoeba strain studied, the generation time is roughly equal to 48 hours at 25 degrees C, and with the presence of food in the medium. After the synchronous divisions, amoebae were maintained in the medium either with or without food organisms (Tetrahymena pyriformis). DNA contents in the nuclei of both the amoebae groups were measured within 4 and 48 hours after division. Before 16 hours, the nuclear DNA contents did not differ in either group. Starting from 20 hours, the DNA amount in fed amoebae exceeded that in starved animals. On the whole, the differences in DNA quantity increased by a 48th hour after division, when the nuclei of the former contained 145% DNA of the latter. The results obtained suggest that the DNA synthesis in amoeba nuclei may proceed during the whole interphase, and that during the second half of interphase the content of DNA may depend on the feeding intensity in amoebae. After refeeding the starved animals, DNA contents in their nuclei increased to reach the same level as in the constantly fed amoebae seen in the end of interphase.     

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Abundance of Tubulin Mrna On Poly Somes Following Deciliation and During the Synchronous Cell Cycle of Tetrahymena

The protozoan, Tetrahymena pyriformis GL, was used as a model system for studying polysomal mRNA during the cell cycle and during cilia regeneration. Our previous work has shown a substantial induction of tubulin synthesis following deciliation and during G₂ of the synchronous cell cycle. In the present study, the abundance of tubulin mRNA on polysomes was examined in order to determine whether more tubulin mRNA was being translated during the periods of peak tubulin synthesis. Polysomes isolated at sequential times following deciliation and during the synchronous cell cycle were translated in a cell-free translation system derived from wheat germ. the abundance of tubulin mRNA on polysomes was inferred from the amount of tubulin translated in vitro. Following deciliation and prior to the peak period of tubulin synthesis, the abundance of tubulin mRNA (at 140 min post-deciliation) increases to 25 times the initial value observed (at 20 min post-deciliation). Since the increase in tubulin mRNA abundance precedes the peak in tubulin synthesis, induction of tubulin synthesis appears to be mRNA-dependent. A similar analysis of tubulin mRNA abundance on polysomes during the synchronous cell cycle revealed a peak of tubulin mRNA prior to each peak of tubulin synthesis. These studies suggest that the periodic fluctuations in the synthesis of tubulin are dependent upon fluctuating levels of tubulin mRNA on polysomes.     

Expression of GFP-actin leads to failure of nuclear elongation and cytokinesis in Tetrahymena thermophila

Green fluorescent protein (GFP)-tagged actin was used to investigate the distribution and function of actin in Tetrahymena. A strain that expresses both GFP-actin and endogenous actin was developed by transformation of Tetrahymena thermophila with a ribosomal DNA-based replicative vector. Confocal microscopy of living cells and immunogold electron microscopy confirmed localization of GFP-actin to basal bodies and the contractile ring. Incorporation of the fusion protein into these and other actin-related structures correlated with severe impairment of macronuclear elongation and cytokinesis. At 30 degrees C macronuclear elongation failed to occur in 25% of the transformants despite completion of micronuclear division. At 20 degrees C macronuclear elongation failed to occur in 2% of the population. Arrest of cytokinesis coincided with failure of macronuclear elongation. Arrested cells developed into homopolar doublets with two sets of oral structures. This study indicates a requirement for actin in nuclear elongation and cytokinesis. Although GFP-actin can interfere with the functioning of actin-containing structures, the GFP-actin transformant strain can be used to monitor actin distribution and dynamics and is therefore an important new tool for further studies of Tetrahymena actin.     

Cell Division of Escherichia coli: Control by Membrane Organization

Cells of certain strains of Escherichia coli, after transfer from 37 to 45 C and incubation for 16 min, were observed to swell and subsequently divide synchronously. This swelling and the resulting stretching of the membrane are proposed to be the basis for the synchronous division. Four lines of evidence support this hypothesis. First, osmotic protection by the addition of either sodium chloride or sucrose at the time of heat shock prevents both swelling and synchrony. Second, a mutant neither swelled nor divided synchronously after heat shock. Third, cells grown for several generations with 10% sucrose in the medium swelled and divided synchronously upon transfer to medium without sucrose. Fourth, the mutant not synchronized by heat shock also swelled and underwent synchronous division after the osmotic shift. A tentative model is suggested for the normal control of division, based on membrane configuration at the septation site.