The Dictyostelium cell cycle and its relationship to differentiation

Abstract The Dictyostelium vegetative cell cycle is characterized by a short mitotic period followed immediately by a short S-phase (less than 30 min) and a long and variable G2 phase. The cell cycle continues during differentiation despite a decrease in cell mass: DNA replication and mitosis occur early in development and also at the tipped aggregate stage. Cells that are in mitosis, S-phase or early G2, when starved differentiate into prestalk cells and cells that are in the middle of G2 differentiate into prespore cells. We postulate that there is a restriction point late in the G2 phase, about 1–2 h before mitosis, where the cells can be arrested either by starvation and the initiation of development, by growing into stationary phase, or by prolonged incubation at low temperature. During development, this block persists to the tipped aggregate stage, where it is specifically released in prespore cells, and these cells then go through one more round of cell division. Genes encoding components of the cell cycle machinery have recently been isolated and attemps to specifically block the cell cycle by reverse genetics to study the effects on differentiation have been initiated.     

The relation of cycling of intracellular pH to mitosis in the acellular slime mould Physarum polycephalum.

The relation between intracellular pH and the mitotic cycle of Physarum polycephalum was studied by two-independent techniques. Both techniques revealed a long term cycling of intracellular pH which has the same period as the mitotic cycle, Qualitative detection of the changes in intracellular pH was made by measuring the changes in fluorescence of 4-methylesculetin which had been absorbed by the plasmodium. Quantitative measurements of intracellular pH were made throughout the mitotic cycle with antimony micro pH electrodes. The cycle of intracellular pH is sinusoidal in appearance. The maximum intracellular pH (pH 6.6) occurred at, or very near to, mitosis, and was approximately 0.6 pH units higher than the minimum pH, which occurred near the middle of the mitotic cycle.     

Intracellular pH controls protein synthesis rate in the sea urchine egg and early embryo.

Direct comparisons between intracellular pH and protein synthesis in the sea urchin egg and early embryo show that pH controls protein synthesis rate in a highly sensitive and reversible manner. The entire increase and maintenance of protein synthesis at fertilization or parthenogenetic activation could be accounted for by a permanent increase in intracellular pH. However, unfertilized eggs whose intracellular pH has been raised artificially by ammonia take at least 30 min longer to reach the rate of protein synthesis seen in fertilized eggs. This time lag for ammonia activation and the decrease in protein synthesis rate during mitosis suggest that other unknown factors can also influence protein synthesis rate during fertilization and early embryogenesis.     

Transition of starving Dictyostelium cells to differentiation phase at a particular position of the cell cycle

The relationship between proliferation and differentiation in Dictyostelium discoideum Ax-2 was analyzed with reference to the cell-cycle position at the onset of starvation, using cells synchronized by temperature shift (11.5 degrees C-22.0 degrees C). To examine how far Ax-2 cells at any particular phase of the cell cycle are able to progress through the cycle in response to nutritional deprivation, we measured temporal changes in cell number and nuclearity after starvation. Nuclear DNA synthesis in synchronously developing cells was also monitored by pulse-labeling with [methyl-3H]thymidine. Increase in cell number and subsequent DNA synthesis occurred in cells just before mitosis (referred to as T0.5 cells and T1 cells; 0.5 h and 1 h after the shift-up from 11.5 degrees C to 22.0 degrees C respectively), but not in T3, T5, or T7 cells. When T1 cells were incubated for 6 h in the absence of external nutrients, they (T1 + 6 cells) exhibited developmental features similar to T7 cells, which most rapidly acquired chemotactic sensitivity to 3',5'-cyclic adenosine monophosphate (cAMP) and EDTA-resistant cohesiveness after starvation. Thus, it is quite likely that Ax-2 cells may progress through the cell cycle to a particular point (possibly the cell-cycle position of T7 cells), irrespective of the presence or absence of nutrients, and enter the differentiation phase from this point under conditions of nutritional deprivation. There was no difference in the ratio of prestalk to prespore cells in migratory pseudoplasmodia derived from cells that had been starved at other cell-cycle positions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular and Extracellular pH and Ca Are Bound to Control Mitosis in the Early Sea Urchin Embryo via ERK and MPF Activities

Studies aiming to predict the impact on marine life of ocean acidification and of altered salinity have shown altered development in various species including sea urchins. We have analyzed how external Na, Ca, pH and bicarbonate control the first mitotic divisions of sea urchin embryos. Intracellular free Ca (Ca_i) and pH (pH_i) and the activities of the MAP kinase ERK and of MPF regulate mitosis in various types of cells including oocytes and early embryos. We found that intracellular acidification of fertilized eggs by Na-acetate induces a huge activation of ERK at time of mitosis. This also stops the cell cycle and leads to cell death, which can be bypassed by treatment with the MEK inhibitor U0126. Similar intracellular acidification induced in external medium containing low sodium or 5-(N-Methyl-N-isobutyl) amiloride, an inhibitor of the Na⁺/H⁺ exchanger, also stops the cell cycle and leads to cell death. In that case, an increase in Ca_i and in the phosphorylation of tyr-cdc2 occurs during mitosis, modifications that depend on external Ca. Our results indicate that the levels of pH_i and Ca_i determine accurate levels of Ptyr-Cdc2 and P-ERK capable of ensuring progression through the first mitotic cycles. These intracellular parameters rely on external Ca, Na and bicarbonate, alterations of which during climate changes could act synergistically to perturb the early marine life.     

A link between mitochondrial gene expression and life stage morphologies in Trypanosoma cruzi

The protozoan Trypanosoma cruzi has a complicated dual-host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalian-infectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here, we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation-induced changes in gene expression. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation in insect stage cells, despite the previously observed increases in mitochondrial mRNAs encoding electron transport chain (ETC) components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short-flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet-unknown signaling mechanism.     

CELL PROLIFERATION IN COMPLEX TISSUES: THE CONTROL OF THE MITOTIC CYCLE OF CELL POPULATIONS IN THE CULTURED ROOT MERISTEM OF SUNFLOWER (HELIANTHUS)

Experiments were performed with cultured primary root tips of sunflower (Helianthus annuus var. Russian Mammoth) to determine: (1) if progression in the mitotic cycle of meristematic cells was nutritionally controllable by carbohydrate starvation and replenishment; (2) where in the mitotic cycle control was effected; and (3) whether nutritional deprivation could be used to detect phenotypically different subpopulations in a complex tissue. Meristematic cells were rendered stationary by carbohydrate starvation, as indicated by the absence of cell division; this condition was reversed by carbohydrate provision. After 72 or 96 hr of starvation most cells stopped in G1 (80–90%) and G2 (10–20%), and a very few (“leaky” cells) continued to enter S. “Leaky” cells represent a small population with an S period of approximately 4.1 hr that either lack a principal control point in G1 or have an unusual metabolism whereby the control point requirements are met and have a carbohydrate dependence for mitosis. Though phenotypically different, no specific functions can be attributed to “leaky” cells at this time.     

Diphospho-myo-inositol phosphates during the life cycle of Dictyostelium and Polysphondylium.

The intracellular amounts of diphospho-myo-inositol phosphates and InsP6 were determined in Dictyostelium discoideum AX2 throughout the life cycle, including exponential growth, starvation, differentiation, sporulation and spore germination. Similar experiments were performed with the closely related species Polysphondylium pallidum under conditions resulting in microcyst formation. A distinct accumulation of these compounds is observed during the early starvation phase of the cell population before the onset of the actual differentiation program. When exponentially growing D. discoideum cells were shifted to starvation conditions, a 25-fold accumulation of 5,6-bis-PP-InsP4 within 3 h was observed. In P. pallidum, the 5,6-bis-PP-InsP4 pool rises around 20-fold within 8 h during the formation of microcysts from vegetative cells. Finally, the diphosphoinositol phosphates are deposited in spores or microcysts and are degraded when spores or microcysts germinate at low cell density.     

Aluminium effects on Scenedesmus obtusiusculus with different phosphorus status. I. Mineral uptake

Synchronously growing cultures of the unicellular green alga Scenedesmus obtusiusculus were cultivated for 24 and 72 h in the presence or absence of phosphorus. Aluminium chloride (37, 74, 111, 148, 185, or 222 μmol) was added daily to 1 l cell suspension at the end of the cell division phase. As AlCl₃ decreases the pH of the growth medium, controls were run in media with low pH in the absence of AlCl₃. Samples for analysis of the internal (net uptake) and external (bound to cell surface) levels of Al, Mg, P, Ca, and Fe were taken every second hour during a 24 h period or once after 72 h. The investigation shows that the intracellular aluminium in Scenedesmus affects the nutrient status of the cells. A high intracellular level of Al is in consort with an enhancement of the intracellular fractions of Mg, P, Ca and Fe. The increase in net uptake of the minerals measured in the presence of Al is not due to an Al-induced lowering of the pH, caused by Al. The concentrations of Al, Mg, P, Ca and Fe in the cells are generally lower during the dark period of the cell cycle, when the cells are dividing, than during the light period. A peak in mineral concentration of the cells could be monitored in the middle of the 24 h life cycle of the cells. The intracellular Al level is higher when the growth medium is low in P than in phosphorus-rich medium, due to precipitation of aluminium-phosphate both in medium and at cell surfaces. The extracellular Al and P fractions are thus higher in the presence than in the absence of P. The highest Al content monitored in the cells is about 100 nmol Al (10⁶ cells)^?1. A large fraction of Al initially taken up after addition to the medium is subsequently released from the cells during the 24 h cell cycle. The results are interpreted as Al effects on the plasma membrane, thus indirectly affecting various mechanisms for ion transport across the membrane. There are also indications that a surface covered with aluminium-phosphate, formed at high P level in the medium, may prevent ion uptake.     

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.     

CELL ARREST IN Gl AND G2 OF THE MITOTIC CYCLE OF VICIA FABA ROOT MERISTEMS

Experiments were performed with cultured excised primary root tips of Vicia faba ‘Longpod’ to determine: (1) the proportion of meristematic cells arrested in Gl and in G2 during carbohydrate starvation, and to determine if the proportion is fixed or can be varied experimentally; (2) the effect of increased starvation on the ability of arrested cells in Gl and G2 to initiate DNA synthesis and mitosis, respectively, when exogenous sucrose was supplied; and (3) whether puromycin, cycloheximide, or actinomycin D prevented the initiation of DNA synthesis and the onset of mitosis. Microspectrophotometry of nuclear DNA and autoradiographic measurements of incorporated ³H-thymidine showed that 72 hr of starvation immediately after excision produced tissue with more than 70 % of the cells arrested in G2 and less than 30 % in Gl. If cultured for three days and then starved for 72 hr, the tissue had nearly equal numbers of cells arrested in Gl and G2. As the duration of starvation increased, the time required to initiate DNA synthesis and to divide when carbohydrate was replenished also increased. Inhibition of protein synthesis by puromycin and cycloheximide prevented the initiation of DNA synthesis and mitosis, but actinomycin D, an inhibitor of RNA synthesis, did not prevent division of cells from G2 nor DNA synthesis by cells from Gl. The experiments demonstrated that the mitotic cycle of Vicia has two major controls, one in Gl and another in G2, and that other factors determine how many cells are affected by either of these cycle controls.     

Seasonal trends in body mass, food intake and resting metabolic rate, and induction of metabolic depression in arctic foxes (Alopex lagopus) at Svalbard

 Post-absorptive resting metabolic rates (RMRs), body mass and ad libitum food intake were recorded on an annual cycle in captive arctic foxes (Alopex lagopus) at Svalbard. During the light season in May and in the dark period in November, RMR during starvation and subsequent re-feeding were also measured. In contrast to earlier findings, the present study indicated a seasonal trend in post-absorptive RMR (in W · kg⁻¹ and W · kg^−0.75). The values in the light summer were 15% and 11% higher than the values in the dark winter, suggesting a physiological adaptation aiding energy conservation during winter in arctic foxes. Body mass and ad libitum food intake varied inversely through the year. A significant reduction in RMR (in W and W · kg^−0.75) with starvation (metabolic depression) was recorded both in May and November, indicating an adaptation to starvation in arctic foxes. The lack of metabolic depression during a period of starvation that was concomitant with extremely cold ambient temperatures in November 1994 indicates that metabolic responses to starvation may be masked by thermoregulatory needs. At very low ambient temperatures, arctic foxes may require increased heat production which cannot be achieved via below-average rates of metabolism. Accepted: 7 June 1999     

Intracellular cAMP in Dictyostelium discoideum: Characterization of an aggregation-deficient mutant with elevated cAMP pools

Forty aggregation-deficient mutants of Dictyostelium discoideum were screened for changes in intracellular cAMP during the first 10 hr of starvation. The pools in 39 of the mutants remained low and relatively static during this period. However, amoebae of one mutant, strain HC151, exhibited significantly elevated levels of intracellular cAMP during vegetative growth and for several hours after starvation. A more detailed analysis of this mutant indicated that the elevated cAMP pools in these cells are a consequence of the premature appearance and partial activation of an adenylate cyclase. The mutation(s) altering adenylate cyclase regulation in this strain appears to map in linkage group IV. Complementation tests between strain HC151 and another mutant, HH201, which has recently been shown to produce an adenylate cyclase activity precociously [1], indicated that the mutations affecting adenylate cyclase activity in these strains map at different loci. Although both of these mutations behave recessively in heterozygous diploids with respect to gross development, an examination of early cAMP metabolism and terminal spore differentiation in these diploids suggest that these mutations are at least partially expressed during some stage(s) of the developmental cycle.     

Assembling the components of the quorum sensing pathway in African trypanosomes

African trypanosomes, parasites that cause human sleeping sickness, undergo a density‐dependent differentiation in the bloodstream of their mammalian hosts. This process is driven by a released parasite‐derived factor that causes parasites to accumulate in G1 and become quiescent. This is accompanied by morphological transformation to ‘stumpy’ forms that are adapted to survival and further development when taken up in the blood meal of tsetse flies, the vector for trypanosomiasis. Although the soluble signal driving differentiation to stumpy forms is unidentified, a recent genome‐wide RNAi screen identified many of the intracellular signalling and effector molecules required for the response to this signal. These resemble components of nutritional starvation and quiescence pathways in other eukaryotes, suggesting that parasite development shares similarities with the adaptive quiescence of organisms such as yeasts and Dictyostelium in response to nutritional starvation and stress. Here, the trypanosome signalling pathway is discussed in the context of these conserved pathways and the possible contributions of opposing ‘slender retainer’ and ‘stumpy inducer’ arms described. As evolutionarily highly divergent eukaryotes, the organisation and conservation of this developmental pathway can provide insight into the developmental cycle of other protozoan parasites, as well as the adaptive and programmed developmental responses of all eukaryotic cells.     

Intracellular pH shift leads to microtubule assembly and microtubule-mediated motility during sea urchin fertilization: correlations between elevated intracellular pH and microtubule activity and depressed intracellular pH and microtubule disassembly

The regulation of the microtubule-mediated motions within eggs during fertilization was investigated in relation to the shift in intracellular pH (pHi) that occurs during the ionic sequence of egg activation in the sea urchins Lytechinus variegatus and Arbacia punctulata. Microtubule assembly during formation of the sperm aster and mitotic apparatus was detected by anti-tubulin immunofluorescence microscopy, and the microtubule-mediated migrations of the sperm and egg nuclei were studied with time-lapse video differential interference contrast microscopy. Manipulations of intracellular pH were verified by fluorimetric analyses of cytoplasmic fluorescein incorporated as fluorescein diacetate. The ionic sequence of egg activation was manipulated i) to block the pHi shift at fertilization or reduce the pHi of fertilized eggs to unfertilized values, ii) to elevate artificially the pHi of unfertilized eggs to fertilized values, and iii) to elevate artificially or permit the normal pHi shift in fertilized eggs in which the pHi shift at fertilization was previously prevented. Fertilized eggs in which the pHi shift was suppressed did not assemble microtubules or undergo the normal microtubule-mediated motions. In fertilized eggs in which the pHi was reduced to unfertilized levels after the assembly of the sperm aster, no motions were detected. If the intracellular pH was later permitted to rise, normal motile events leading to division and development occurred, delayed by the time during which the pH elevation was blocked. Microtubule-mediated events occurred in eggs in which the intracellular pH was elevated, even in unfertilized eggs in which the pH was artificially increased. These results indicate that the formation and normal functioning of the egg microtubules is initiated, either directly or indirectly, by the shift in intracellular pH that occurs during fertilization.     

Evidence for a homolog of the yeast cell cycle regulatory gene product of cdc2+ in Physarum polycephalum

Evidence for the presence of a Cdc2-like protein in Physarum polycephalum has been obtained using a peptide antibody directed against a highly conserved amino acid sequence near the N-terminal end of Cdc2, Cdc28 and Cdc2HS. The antibody detected a 34 kDa cytoplasmic protein, similar in apparent size to Cdc2 in yeast and Cdc2Hs in HeLa cells. A 60 kDa nuclear band was also detected in Physarum but not in yeast or HeLa. Evidence is presented that this is not related to the 34 kDa protein nor is it found in HeLa nuclei or yeast cells. The Cdc2-like protein level did not fluctuate over more than 10 h of the naturally synchronous cell cycle of Physarum. Several heat-shock experiments using regimens that either: delayed mitosis and S-phase; prevented mitosis or uncoupled S-phase from mitosis were performed. None had any effect on the level of the Cdc2-like protein. The induction of spherulation by starvation was shown to have no effect on the levels of the 34 kDa Cdc2 analog. The invariant level of the 34 kDa protein during the cell cycle and starvation is consistent with previous results obtained with yeast. Three heat-shock regimens which either delay mitosis, eliminate S-phase or uncouple mitosis from S-phase in Physarum also had no effect on the level of the 34 kDa protein. This result emphasizes the stable nature of this protein.     

Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum

In Dictyostelium discoideum, the initial differentiation of cells is regulated by the phase of the cell cycle at starvation. Cells in S and early G2 (or with a low DNA content) have relatively high levels of cellular Ca2+ and display a prestalk tendency after starvation, whereas cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. We found that there is a correlation between cytosolic Ca2+ and cell cycle phase, with high Ca2+ levels being restricted to cells in the S and early G2 phases. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, it has been reported that in the rtoA mutant, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation. In contrast to the wild type, a disproportionately large fraction of rtoA amoebae fall into the high Ca2+ class, possibly due to an altered ability of this mutant to transport Ca2+.     

Zonal expression of the glucokinase gene in rat liver. Dynamics during the daily feeding rhythm and starvation-refeeding cycle demonstrated by in situ hybridization

The abundance and zonal distribution of glucokinase (GK) mRNA were studied in rat liver during a normal 12 h day/12 h night rhythm (dark from 1900 to 0700 hours) and during refeeding after 60 h of starvation. Zonation of GK gene expression was examined by in situ hybridization with a radiolabelled cRNA probe and GK mRNA abundance was determined by Northern blot analysis with a digoxigenin-labelled cRNA probe. GK mRNA appeared to be almost homogeneously distributed throughout the whole daily feeding cycle; yet it was predominantly localized in the perivenous and intermediate zone during refeeding after 60 h of starvation. During the daily feeding rhythm, the total amount of GK mRNA increased quickly with the beginning of the feeding period at 1900 hours reaching a maximum at midnight and then decreased continuously to a basal level at noon. Virtually no GK mRNA was detected after 60 h of starvation. Refeeding caused a rapid increase in GK mRNA to a maximum at 2400 hours followed by a decrease to approximately two-thirds of the maximum value at 0700 hours. If the homogeneous distribution of GK mRNA during the daily feeding rhythm was real rather than apparent because of too low a sensitivity of the cRNA probe, the present results suggest that during the normal circadian cycle the mainly perivenous distribution of GK enzyme activity and protein is regulated preferentially at a translational level. The findings clearly show that during refeeding after 60 h of starvation the GK distribution is controlled predominantly at a pretranslational level.     

ON THE RESTING PERIODS IN THE CELL LIFE CYCLE*

The review is concerned with the transition of cells into a specific physiological state—the resting period—in which they may stay for an indefinite time interval without undergoing division or differentiation but retaining both of these potentials. When stimulated such cells may enter into mitotic cycle, divide and differentiate. No direct correlation between the onset of the differentiated state and the transition of cells through the mitotic cycle has been established. It cannot be excluded that sometimes cells may differentiate directly from the resting period. However, there is a large body of evidence that the entry of cells into mitotic cycle is a necessary prerequisite for subsequent differentiation. The susceptibility of cells to differentiative stimuli is retained during the mitotic cycle. The completion of mitosis itself does not imply that a cell will undergo differentiation; in the absence of adequate stimulus it may pass again into a resting period. According to what is known at present it is suggested that cells may pass into a true resting stage not only after completing mitosis but also after doubling their DNA content. It is also conceivable that a cell may pass into a resting period at different stages of its life cycle. The essential feature of the cell life cycle is the alternation of resting periods and periods of active proliferation. This general principle of organization provides conditions necessary for population-size control, cell differentiation, interaction of a given population with other systems, and the reactions of cells to a changing environment.     

Copine A plays a role in the differentiation of stalk cells and the initiation of culmination in <Emphasis Type="Italic">Dictyostelium</Emphasis> development

Background  

Copines are calcium-dependent phospholipid-binding proteins found in diverse eukaryotic organisms. We are studying the function of copines in Dictyostelium discoideum, a single-celled amoeba that undergoes cell differentiation and morphogenesis to form multicellular fruiting bodies when placed in starvation conditions. Previously, we showed that Dictyostelium cells lacking the copine A (cpnA) gene are not able to complete the developmental cycle, arresting at the slug stage. The aim of this study is to further characterize the developmental defect of the cpnA- cells.