Correlates of developmental cell death in Dictyostelium discoideum

We have studied the correlates of cell death during stalk cell differentiation in Dictyostelium discoideum. Our main findings are four. (i) There is a gradual increase in the number of cells with exposed phosphatidyl serine residues, an indicator of membrane asymmetry loss and increased permeability. Only presumptive stalk cells show this change in membrane asymmetry. Cells also show an increase in cell membrane permeability under conditions of calcium-induced stalk cell differentiation in cell monolayers. (ii) There is a gradual fall in mitochondrial membrane potential during development, again restricted to the presumptive stalk cells. (iii) The fraction of cells showing caspase-3 activity increases as development proceeds and then declines in the terminally differentiated fruiting body. (iv) There is no internucleosomal cleavage of DNA, or DNA fragmentation, in D. discoideum nor is there any calcium- and magnesium-dependent endonucleolytic activity in nuclear extracts from various developmental stages. However, nuclear condensation and peripheralization does occur in stalk cells. Thus, cell death in D. discoideum shows some, but not all, features of apoptotic cell death as recognized in other multicellular systems. These findings argue against the emergence of a single mechanism of 'programmed cell death (PCD)' before multicellularity arose during evolution.     

Role of inositol polyphosphates in programmed cell death

The role of inositol polyphosphates (InsPs) in the mediation of cellular apoptosis was investigated in mouse MC3T3 osteoblastic cell line. Extracellular administration of InsP₄, InsP₅, and InsP₆ increased apoptosis in a dose-dependent manner. InsP₆ was more potent than InsP₅ and InsP₄ in promoting apoptosis. Inositol hexasulfate (InsS₆), a structural analog of InsP₆, was used to determine specificity of InsP₆-induced apoptosis as measured by acridine orange/ethidium bromide, flow cytometry, and DNA degradation. In order to study the effects of endogenous InsPs on apoptosis, we used NaF and antimycin A as treatment agents to manipulate intracellular levels of InsPs. NaF is known to increase levels of higher InsPs by inhibiting InsPs phosphatases, a process that is reversed by antimycin A because InsPs kinases are inhibited as a result of depletion of cellular ATP pools. Apoptosis was induced in MC3T3 cells in a NaF dose- and time-dependent manner. Approximately 50% apoptosis was observed at 1 mM NaF in 8 h. Prior treatment with 10 μM antimycin A for 30 min significantly reduced the NaF-induced apoptosis as compared with its control. Additionally, we measured changes in AKT phosphorylation, cleavage of caspase-3 and caspase-9, and release of cytochrome C from mitochondria into cytosol. These changes coincided with total cellular InsPs under similar conditions. The data indicated that NaF-induced changes in apoptotic markers could be due to an increased endogenous InsPs that were partially reversed by antimycin A treatment.     

The cell cycle and its relationship to development in Dictyostelium discoideum.

When deprived of exogenous nutrients some amoebas of Dictyostelium discoideum do continue to progress through the cell cycle. There are two distinct periods when mitotic cell division occurs. Labeling studies show that during the first period, which begins at the onset of development and ceases at the first visible signs of aggregation (rippling), only those cells which are beyond a certain point in G2 at the initiation of development divide. The second period of mitotic activity begins at tip formation, reaches maximum activity at the grex stage, and ceases during early culmination. Significantly, examination of the development of amoebas harvested when in the stationary phase of growth (and thus arrested in G2) shows that these cells still undergo mitotic cell division during the second period but do not show any such division during the preaggregation phase. The extent to which increases in cell number can be taken to be indicative of mitotic cell division varies from one culture to another due to the presence of variable numbers of multinucleate cells which become mononucleate during the first 10 hr of development. However, when due allowance has been made for the existence of these cells in axenically growing amoebal populations, our data show that by completion of fruiting body construction there has been a doubling in cell number as a direct result of mitotic cell division. Nuclear DNA synthesis also occurs at two distinct periods during development, these coinciding with the periods of mitotic activity. However, since no more than 35% of the cells have undergone nuclear DNA synthesis by the end of the developmental phase, our results are inconsistent with the conclusion that all cells accumulate at a position in G2 at the time of aggregation. Our results do suggest, however, that mitotic cell division of a fraction of the cells may be an integral part of the developmental phase.     

A developmental shift in cell volume regulation in Dictyostelium discoideum

Abstract. We describe developmentally modulated volume regulation phenomena in Dictyostelium discoideum amebae. Transfer of cells to a medium of different osmolarity leads initially to volume shifts in the expected direction (cells shrink in concentrated media, swell in dilute media). Subsequently, the cells may slowly return to the original volume over 30–90 min. The capacity for volume regulation appears and disappears during development. A mutant isolated for lack of regulation shows abnormalities in developmental timing. Mutant enrichment according to volume regulatory properties may thus be useful in some lines of work.     

Cytoplasmic pH and glycolysis in the Dictyostelium discoideum cell cycle

Lactate production measurements during the cell cycle of synchronized populations of Dictyostelium discoideum cells reveal cyclic variations in glycolysis which correspond with pH_i oscillations which were discovered by us previously [(1985) Cell, in press]. Aerobic lactate production varies about 6-fold during the cell cycle and the lactate maxima correlate with (~ 0.25 pH unit) cyclic increases in pH. However, artificially altering pH_i using weak acids or bases does not influence the rate of lactate production in asynchronous cell populations. This result suggests that the cyclic variations in pH_i and those in glycolytic rate are not causally related events.     

The relationship of phosphodiesterase to the developmental cycle of Dictyostelium discoideum.

Determination of the rate of total phosphodiesterase production by Dictyostelium discoideum shows that a dramatic rise in enzyme production occurs after 3 hours of cell starvation. Use of imposed cAMP pulses indicate that this increase is related to the developmental program of the amoebae and is probably due to a stimulation of adenyl cyclase.     

Shortening of the cell cycle and developmental interruption in a Dictyostelium discoideum cell line due to RNAi-silenced expression of allantoicase

The signaling molecules NH(3) (unprotonated volatile ammonia), as well as cyclic adenosine monophosphate and differentiation-inducing factor, play important roles in the multicellular development of the slime mould Dictyostelium discoideum. One of the downstream metabolic products catalyzed by allantoicase (allC) is ammonia. We observed the role of allC by RNAi-mediated manipulation of its expression. The allC gene of D. discoideum was silenced by RNAi. We found significant downregulation of allC mRNA and protein expression levels. Recombinant allC RNAi mutant cell lines had a shortened cell cycle, a reduction in cell size relative to wild-type cells and interrupted development. We conclude that the normal functions of allC include retarding cell division until a specific cell size is reached and coordinating the progression of development.     

An analysis of developmental timing in Dictyostelium discoideum

A new method has been developed to assess the minimum complexity and relationships of those pathways (developmental timers) which time the consecutive stages of a developing system (Soll, 1983). This method has been applied to the morphogenetic program of Dictyostelium discoideum and has resulted in (1) a minimum estimate of the number of components comprising the timers for the first seven stages of morphogenesis, (2) a characterization of the temperature sensitivities of these components including demonstration of a reversible timer component, (3) detailed temporal definition of a number of transition points between rate-limiting components including a major branch point for the onset of several independent timer components coincident with the onset of aggregation, and (4) a temporal model for the relationships between the timers of the seven consecutive morphogenetic stages, including several examples of parallel timers.     

The cell cycle and its relationship to aggregation in the cellular slime mold, Dictyostelium discoideum

Vegetative amoebae of the cellular slime mold Dictyostelium discoideum were synchronized by the use of a temperature-sensitive mutant. The synchronized population was then used to analyze the cell cycle in Dictyostelium discoideum. This in turn enabled us to study the relationship between specific stages of the cell cycle and the initiation of aggregation. It was shown that all cells are at the same position (midway in G₂) at the time of aggregation. Synchronous cells starved at all points in the cell cycle, however, took the same length of time to aggregate. This suggests that the limiting step in the aggregation process is starvation, which is independent of the position of the cells in the cell cycle.     

Calcium levels during cell cycle correlate with cell fate of Dictyostelium discoideum

Levels of intracellular calcium, (Ca(2+))(i), from different stages of cell cycle of Dictyostelium discoideum were monitored using the fluorescent Ca(2+)-sensitive dye, Indo 1. Combinations of Ca(2+)-ionophore (A23187) and Ca(2+)-chelator (EGTA) resulted in the inhibition of progression of cell cycle. This delay was caused due to block in G(2)/M-->S phase transition of the cell cycle. Rescue of the cell cycle progression was made with 0.5 m m of exogenous Ca(2+). High (Ca(2+))(i)levels overlapped with the S-phase, of the cell cycle.Results indicate that a high (Ca(2+))(i)level during S-phase is not required for cell cycle progression but for cell-type choice mechanism at the onset of starvation, and these cells tend to follow the prestalk pathway.     

Evolutionary crossroads in developmental biology: Dictyostelium discoideum

Dictyostelium discoideum belongs to a group of multicellular life forms that can also exist for long periods as single cells. This ability to shift between uni- and multicellularity makes the group ideal for studying the genetic changes that occurred at the crossroads between uni- and multicellular life. In this Primer, I discuss the mechanisms that control multicellular development in Dictyostelium discoideum and reconstruct how some of these mechanisms evolved from a stress response in the unicellular ancestor.     

The effects of cell density and starvation on early developmental events in Dictyostelium discoideum

We show that removal of yeast extract and trypticase from growth medium is sufficient for induction of several key events which occur during the early stages of Dictyostelium differentiation: run-off of polysomes, the earliest known change in macromolecular metabolism; appearance of the cell surface cAMP receptor; and aggregation itself. Starvation of glucose has little effect on these parameters. These results are consistent with those of other investigators who showed that starvation only of amino acids will induce other activities associated with cAMP-mediated cell signaling and cell-cell adhesion. We show, in contrast, that other factors are involved in the increase in the relative rates of synthesis of three polypeptides very early in differentiation: actin, and two proteins (“45-min” proteins) which are synthesized only during the period of 45–90 min. The induction of synthesis of these three proteins and presumably, of their mRNAs, is not the result of starvation for glucose or amino acids but is the result of plating cells at high density. The increases in the synthesis of these proteins are dependent on the density at which cells are plated and do not occur at a density 75-fold lower than the density used in standard experiments. Cells growing at high density or near stationary phase do not show the induction of increased synthesis of actin or the “45-min” proteins. These experiments suggest that these early developmental changes may be dependent on a threshold level of a diffusible factor excreted early in development.     

The developmental regulation of L-ornithine decarboxylase in Dictyostelium discoideum and its induction by cAMP

By the use of an in vivo assay, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) is shown to be developmentally regulated in Dictyostelium discoideum. High levels of cAMP can induce ornithine decarboxylase activity in preaggregative cells kept in shaking suspension, under similar conditions as where other markers for development can also be induced. This induction by cAMP is solely dependent on the total amount of cAMP to which the cells have been exposed, and not on the manner of cAMP addition. Induction of ornithine decarboxylase activity, when measured in vitro, is caused by both an increase in total enzyme activity and by a proportional increase in activity of the high-affinity form for the cofactor pyridoxal phosphate. When measured in vivo, an additional regulatory mechanism seems to be involved. Kinetic studies with the competitive inhibitor putrescine suggest that in cAMP-stimulated cells the low affinity form of the enzyme may also be active in vivo.     

The developmental regulation of single-cell motility in Dictyostelium discoideum

The velocity of single amebae in the absence of a chemotactic signal has been analyzed during growth, development, rapid recapitulation, and dedifferentiation in the cellular slime mold Dictyostelium discoideum. It is demonstrated that (1) the velocity of axenically grown cells in half that of bacterially grown cells, (2) the velocity of bacterially grown cells decreased to roughly the same low level as axenically grown cells approximately 5 hr after the removal of exogeneous bacteria, (3) the velocity remains low for a 7-hr period preceding the onset of aggregation in both axenically and bacterially grown cells, (4) the velocity increases transiently at the onset of aggregation for both axenically and bacterially grown cells, (5) the velocity decreases to a very low level after the formation of loose aggregates and remains at that level at least through the early culminate I stage, (6) the velocity is not stimulated in 13-hr developing cells (finger stage) by inducing rapid recapitulation, (7) the velocity decreases after the erasure event in cultures of 7-hr developing cells (ripple stage) stimulated to undergo dedifferentiation, but the inhibition of the erasure event by the addition of 10(-4) M cAMP does not block this decrease. These results demonstrate that the basal level of single-cell motility in growing cultures is significantly influenced by the nutrient composition of the supporting medium, and that the transient increase in single-cell motility at the onset of aggregation is under the rigid control of the initial developmental program. Both rapid recapitulation and the program of dedifferentiation appear to have no influence on the basal level of single-cell motility.     

The developmental regulation of phosphatidylinositol kinase in Dictyostelium discoideum

Phosphatidylinositol kinase was examined in Dictyostelium discoideum since this organism offers molecular and genetic advantages to study the role of phosphatidylinositol metabolism during cell growth and development. D. discoideum homogenates phosphorylated phosphatidylinositol to form phosphatidylinositol 4-phosphate in a reaction which was found to be linear with time and cell concentration. Optimal activity was obtained in the presence of 1 mM MgCl₂ and pH 7.6 and has an apparent K_m for ATP of about 250 μM. Changes in phosphatidylinositol kinase were examined during D. discoideum development. Activity increased about 2-fold, 4 h after removal of the food source, to decline to almost no activity at late aggregation. During slug formation the activity increased about 15-fold and remained constant during further development. These results suggest a role for D. discoideum phosphatidylinositol kinase during development.