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.     

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.     

Hsp70 protects mitotic cells against heat-induced centrosome damage and division abnormalities

The effect of heat shock on centrosomes has been mainly studied in interphase cells. Centrosomes play a key role in proper segregation of DNA during mitosis. However, the direct effect and consequences of heat shock on mitotic cells and a possible cellular defense system against proteotoxic stress during mitosis have not been described in detail. Here, we show that mild heat shock, applied during mitosis, causes loss of dynamitin/p50 antibody staining from centrosomes and kinetochores. In addition, it induces division errors in most cells and in the remaining cells progression through mitosis is delayed. Expression of heat shock protein (Hsp)70 protects against most heat-induced division abnormalities. On heat shock, Hsp70 is rapidly recruited to mitotic centrosomes and normal progression through mitosis is observed immediately after release of Hsp70 from centrosomes. In addition, Hsp70 expression coincides with restoration of dynamitin/p50 antibody staining at centrosomes but not at kinetochores. Our data show that during mitosis, centrosomes are particularly affected resulting in abnormal mitosis. Hsp70 is sufficient to protect against most division abnormalities, demonstrating the involvement of Hsp70 in a repair mechanism of heat-damaged mitotic centrosomes.     

Delay In Entry Into S Phase After Heat Shock

Synchronized cells of the Harding Passey melanoma grown in culture were given a heat shock treatment of 44° C for 36 min. Thymidine incorporation was measured at frequent intervals after heat shock to determine the time of onset of the next DNA synthetic period. If the heat shock was given at the end of G¹, the following S was delayed by 20 hr. Heating at other times in the cell cycle resulted in an even longer interval before the onset of S. the end of G¹ was also the most resistant to hyperthermic killing and to the effect of heat on the magnitude of thymidine incorporation in the following S. Heating the cells a second time did not repeat the effect of the first treatment unless the second heat shock treatment was at a considerably higher temperature. Thus thermotolerance to heat shock killing also applies to cell-cycle delay.     

The production of cloned fish in the medaka (Oryzias latipes)

The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41 degrees C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85-95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41 degrees C for 2 min at 2-3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85-95 min after insemination arrests the first cleavage, while heat shock at 2-3 min after insemination arrests the second meiotic division. Medaka clones have been produced by the following method: Eggs from orange-red or variegated variety were activated by UV-irradiated, genetically impotent sperm of wild-type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone-containing diet (40 micrograms/gm diet) to produce sex-reversed males, which were mated with brood females, and thus two cloned lines were obtained.     

ELECTROPHORETIC MOBILITY OF SEA-URCHIN EGGS DURING THE DIVISION CYCLE

The electrokinetic potential of fertilized sea-urchin eggs, without the fertilization membrane and hyaline layer, was investigated by measuring the electrophoretic mobility of the eggs from fertilization to the second cleavage. A cyclic change in mobility was found to accompany the division cycle: the peak of the change was observed about 15 min before the appearance of both the first and second cleavage furrows.
A smaller peak was observed at 20–30 min after fertilization, but such a peak was not repeated between the first and the second cleavage.
Fertilized eggs with the fertilization membrane intact did not show a significant change in electrophoretic mobility throughout the division cycle.     

Observations on intracellular pH during cleavage of eggs of Xenopus laevis

Direct measurements of intracellular pH was made with recessed-tip pH microelectrodes in fertilized eggs of the frog, Xenopus laevis, from approximately 1 h after fertilization to mid-blastula. The intracellular pH just before first cleavage was 7.65 +/- 0.04 (SD; n = 9). By stage 5 to the middle of stage 6, average intracellular pH was 7.70 +/- 0.06 (SD; n = 16). A statistically significant alkalization of 0.18 +/- 0.03 pH unit (SD; n = 5) was observed beginning in early blastula. A cycle of less than or equal to 0.05 pH unit was occasionally observed during the pre-blastula period, but its significance is unknown. By exposing the early cleavage embryo to saline buffered with sodium propionate, pH 4.7-5.0, it was possible to lower intracellular pH with some degree of control. Apparently, normal cleavage continued to occur when intracellular pH had been forced as much as 0.3 unit below normal. We conclude that this implies no specific involvement of intracellular pH in mitosis and cytokinesis. If intracellular pH was lowered further, cell division ceased at about pH 7.2, and furrow regression began at about pH 7.0. Once furrow regression occurred, subsequent development was usually arrested or abnormal when the embryo was transferred back to normal saline.     

Heat-shock gene expression and cell cycle changes during mammalian embryonic development

Synchronized regulation of cell division during gastrulation is essential for the regional proliferation of cells and pattern formation of the early CNS. The neural plate and neuroectoderm cells are a rapidly dividing and differentiating population of cells with a unique and rapid heat-shock response. Heat shock and the heat-shock genes were studied during neural plate development in a whole rat embryo culture system at 9.5-11.5 days. A lethal heat shock can cause cell death and severe developmental defects to the forebrain and eye during organogenesis. Heat shock can also result in acquired thermotolerance whereby cell progression is delayed at the G1/S and S/G2 boundaries of the cell cycle. This delay in cell cycle progression caused an overall lengthening of the cell cycle time of at least 2 hr. The heat shock genes may therefore function as cell cycle regulators in neuroectoderm induction and differentiation. The kinetics and expression of the hsp genes were examined in neuroectodermal cells by flow cytometry and Northern analysis. The levels of hsp mRNA 27, 71, 73, and 88 were identified following exposure at 42°C (nonlethal), 43deg;C (lethal) and 42deg;/43deg;C (thermotolerant) heat shock. Examination of hsp gene expression in the neural plate showed tight regulation in the cell cycle phases. Hsp 88 expression was enhanced at Go and hsp71 induction at G2 + M of the cell cycle. Cells exposed to a thermotolerant heat shock of 42deg;C induced hsp71 mRNA expression in all phases of the cell cycle with the mRNA levels of hsp27, 73, and 88 increased but relatively constant. Following a lethal heat shock, dramatic changes in hsp expression were seen especially enhanced hsp71 induction in late S phase. The regulated expression of hsps during the cell cycle at various phases could play a unique and important role in the fate and recovery of neuroectoderm cells during early mammalian embryo development. © 1993Wiley-Liss, Inc.     

Cyclic activation of histone H1 kinase during sea urchin egg mitotic divisions

Fertilized sea urchin eggs undergo a series of rapid and synchronized mitotic divisions. Extracts were made at various times throughout the first three mitotic divisions and assayed for phosphorylating activity toward histone H1. Histone H1 kinase (HH1K) undergoes a transient activation (8- to 10-fold increase) 20 min before each cleavage. The amplitude of the HH1K peak strongly depends on the synchrony of the egg population. Concomitant cytological observations show that the time-course of HH1K correlates with the time-course of nuclear envelope breakdown and of metaphase. This correlation is observed at each cell division cycle. HH1K from each of the three first mitoses show identical time- and concentration-dependence curves as well as identical dose-inhibition curves with 6-dimethylaminopurine and quercetin, suggesting that the same (group of) kinase(s) is (are) activated before each cleavage. Ionophore A23187 does not trigger, but inhibits, HH1K activation; however, partial activation of the eggs with ammonia at pH 9.0 (but not at pH 8.0) triggers the transient HH1K activation. Appearance of the HH1K cycle requires protein synthesis since it is completely abolished in emetine-treated eggs. Although cytochalasin B blocks egg cleavage, it does not inhibit HH1K activation nor nuclear divisions. A prolonged HH1K activation cycle is observed in eggs arrested in metaphase with colchicine or nocodazole. Despite the existence of a cycle in cAMP concentration during mitosis, forskolin, an activator of adenylate cyclase, does not modify the time-course of HH1K activation and of cell division. The cycling HH1K is independent of calcium-calmodulin, calcium-phospholipids, or cyclic AMP. It clearly resembles the mammalian "growth-associated histone kinase." The relationship between the transient activation of HH1K and the intracellular mitotic factors driving the cell cycle is discussed.     

THE RELATIONSHIP BETWEEN DEOXYRIBONUCLEIC ACID REPLICATION AND CELL DIVISION IN HEAT-SYNCHRONIZED TETRAHYMENA

The effect of supraoptimal temperature on macronuclear DNA synthesis in Tetrahymena was studied by radioautography during prolonged heat and heat-shock synchronization treatments. Prolonged heat treatments (34°C) delayed the initiation of S, but did not appreciably delay DNA synthesis in progress. Return to optimal temperature (28°C) 50 or 100 min later resulted in initiation of S, in delayed cells, at a rate greater than in controls. During the synchronization treatment, most cells were unable to enter S during a heat shock, but initiated S with a slight delay during the following intershock period. These cells were not appreciably delayed in completion of S by subsequent heat shocks. Supraoptimal temperature appears to affect the DNA synthetic cycle near the G₁ to S transition. Cells subjected to the heat-shock treatment in early G₁ all participated in one S period, and many underwent a succession of two S periods. DNA synthesis occurred in about 50% of the cells between EST and the first synchronous division, with the likelihood of DNA synthesis becoming greater the longer the interval between these two events. In some cells no detectable DNA synthesis occurred between EST and the second synchronous division. It was concluded that a precise temporal alternation of DNA replication and cell division is not obligatory in Tetrahymena.     

Cyclic regulation of cytokinesis in amphibian eggs

The effects of amphibian egg cytoplasm extracted at different times after activation and during the first four cleavages on cytokinesis were examined. Extracts of artificially activated or fertilized Xenopus or Pleurodeles eggs taken at the time of activation (T = 0) provoked precocious cleavage furrows in Pleurodeles eggs. Between T = 0.25 and T = 0.75 of the first cell cycle, the period corresponding to interphase, an inhibitory effect was found, and the division of injected eggs was delayed up to 30%. After T = 0.75, that is during mitosis, the cleavage induction effect was observed again. These enhancing and inhibitory effects were also found in the two fractions obtained following gel filtration of the cytoplasmic extracts. These experiments support the hypothesis that two antagonistic factors control cytokinesis. The inhibitory factor is active only during interphase, while the positive factor is present during mitosis and appears to regulate cytokinesis.     

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).     

Role of spindle microtubules in the control of cell cycle timing

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.     

Tetraploid Induction by Inhibiting Mitosis I with Heat Shock, Cold Shock, and Nocodazole in the Hard Clam Mercenaria mercenaria (Linnaeus, 1758)

Tetraploid induction by inhibiting mitosis I with heat shock (32, 35, and 38°C), cold shock (1, 4, and 7°C), and nocodazole (0.02 to 1.6 mg/L) was investigated in the hard clam Mercenaria mercenaria. All treatments were applied to fertilized eggs about 5 min before the first cell division at 22 to 23°C, and lasted for 10, 15, and 20 min. Three replicates were produced for each treatment with different parents. The ploidy of resultant larvae and juveniles was determined with flow cytometry. Heat shock of 35 and 38°C was effective in inhibiting mitosis I, producing 54% to 89% tetraploid larvae. Heat shock of 32°C accelerated embryonic development without inhibiting mitosis or producing tetraploids. In all heat-shock groups, the survival to D-stage larvae was lower than in controls, suggesting that heat-shock treatments and tetraploidy were detrimental to larval development. At the juvenile stage, survivors from heat-shock groups contained no tetraploids. Cold shocks suspended the first cell division during the treatment, but produced no tetraploids in the 4°C and 7°C treatment groups. Cold shock of 1°C produced 31% tetraploid larvae in one replicate, with none surviving to juvenile stage. Nocodazole inhibited mitosis I at concentrations of 0.04 mg/L or higher, but did not produce tetraploids. This study indicates that heat shock is most effective in inducing tetraploids through mitosis I inhibition, although none of the induced tetraploids survived to juvenile stage.     

The effect of heat shock on the cell cycle regulation of tubulin expression in Physarum polycephalum

In the myxomycete Physarum polycephalum, tubulin synthesis is subject to mitotic cycle control. Virtually all tubulin synthesis is limited to a 2-h period immediately preceding mitosis, and the peak of tubulin protein synthesis is accompanied by a parallel increase in the level of tubulin mRNA. The mechanism by which the accumulation of tubulin mRNA is turned on and off is not clear. To probe the relationship between tubulin regulation and cell cycle controls, we have used heat shocks to delay mitosis and have followed the pattern of tubulin synthesis during these delays. Two peaks of tubulin synthesis are observed after a heat shock. One occurs at a time when synthesis would have occurred without a heat shock, and a second peak immediately precedes the eventual delayed mitosis. These results are clearly due to altered cell cycle regulation. No mitotic activity is detected in delayed plasmodia at the time of the control mitosis, and tubulin behavior is shown to be clearly distinct from that of heat shock proteins. We believe that the tubulin family of proteins is subject to regulation by a thermolabile mitotic control mechanism but that once the cell has been committed to a round of tubulin synthesis the "tubulin clock" runs independently of the heat sensitive system. In delayed plasmodia, the second peak of synthesis may be turned on by a repeat of the commitment event.     

Periodic appearance and disappearance of microvilli associated with cleavage cycles in the egg of the ascidian, Halocynthia roretzi

The surface of eggs of the ascidian Halocynthia roretzi, observed with SEM, is essentially smooth until immediately before cell division when numerous microvilli appear and remain during cytokinesis. As the dividing blastomeres become closely adherent, however, the microvilli disappear and the eggs recover their smooth surface. This periodic appearance-disappearance of microvilli is repeated at each cleavage cycle up to at least the 32-cell stage. During blastomere adhesion, microvilli that have appeared near the plane of the first cleavage or of the bilateral symmetry seem to fuse together across the plane to form a zipper-like complex of cytoplasmic processes, which might be responsible for attachment of the two halves of these bilaterally symmetrical embryos via the blastomeres bordering the plane of symmetry.     

LIGHT-DEPENDENT INHIBITION OF CELL DIVISION AND RHIZOID ELONGATION IN GEMMAE OF THE FERN,VITTARIA GRAMINIFOLIA

Gametophytes of the shoe-string fern Vittaria graminifolia produce linear, six-celled propagules called gemmae. The terminal cells of each gemma elongate into primary rhizoids in culture, and the inner body cells divide asymmetrically to produce prothallial or rhizoid initials. The initiation of both asymmetric cell division and rhizoid elongation is delayed by light intensities greater than 2 w/m². The maximal rates of cell division and rhizoid elongation are unaltered. A 24-hr pulse of high light intensity delays cell division and rhizoid elongation to the same extent, whenever applied during the first 3 d of culture. The model we propose for cell division hypothesizes the existence of a preparatory phase of finite duration prior to mitosis that is sensitive to light intensity. If a cell is irradiated by light intensities greater than 2 w/m² while in the preparatory phase, its entrance into mitosis is delayed. A similar model is proposed for the initiation of rhizoid elongation. Despite the fact that both cell division and rhizoid elongation are dependent on photosynthesis, direct measurements of CO₂-uptake rates show that the inhibitory effects of high light intensities are not due to an inhibition of photosynthesis.     

Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos.

Polyspermy occurs frequently in the fertilization of mammalian eggs, but little is known about whether polyspermic eggs have developmental ability in vitro or in vivo. We previously reported that poly-pronuclear (PPN; 3 or more pronuclei) pig eggs developed normally to the blastocyst stage despite having fewer inner cell mass cell numbers as compared to blastocysts derived from two-pronuclear (2PN) eggs. Here it is shown that most PPN pig eggs have abnormal cleavage patterns (having 3 or more cells) in the first cell division and retarded development of pronuclei prior to syngamy as compared to 2PN eggs. Most blastocysts (14 of 18) that developed from PPN eggs showed abnormal ploidy (were haploid, triploid, and tetraploid) whereas 20 of 22 blastocysts derived from 2PN embryos were diploid. The size and morphology of most Day 40 fetuses that developed from PPN eggs appeared to be normal. Of 8 Day 40 fetuses analyzed, 1 was triploid (XXY) and another was a mosaic with both diploid (XX) and tetraploid cells (frequency of less than 10%, XXXX), and the others were diploid. Anomalies of chromosomal composition were not detected in these fetuses. Five live piglets and one dead piglet were born from two recipients of PPN eggs. It is proposed that not all pronuclei of PPN pig eggs participate in syngamy, resulting in diploid cells in the conceptus. Our data suggest that there are two types of pronuclei location in polyspermic pig eggs and that the resulting ploidy is determined at the zygote stage before the first cell division according to pronuclear location.     

Reversibility of heat shock in Chlamydia trachomatis

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.