Signaling molecules involved in the transition of growth to development of Dictyostelium discoideum

The social amoeba Dictyostelium discoideum, a powerful paradigm provides clear insights into the regulation of growth and development. In addition to possessing complex individual cellular functions like a unicellular eukaryote, D. discoideum cells face the challenge of multicellular development. D. discoideum undergoes a relatively simple differentiation process mainly by cAMP mediated pathway. Despite this relative simplicity, the regulatory signaling pathways are as complex as those seen in metazoan development. However, the introduction of restriction-enzyme-mediated integration (REMI) technique to produce developmental gene knockouts has provided novel insights into the discovery of signaling molecules and their role in D. discoideum development. Cell cycle phase is an important aspect for differentiation of D. discoideum, as cells must reach a specific stage to enter into developmental phase and specific cell cycle regulators are involved in arresting growth phase genes and inducing the developmental genes. In this review, we present an overview of the signaling molecules involved in the regulation of growth to differentiation transition (GDT), molecular mechanism of early developmental events leading to generation of cAMP signal and components of cAMP relay system that operate in this paradigm.     

Specific expression of a gene encoding a novel calcium-binding protein, CAF-1, during transition of Dictyostelium cells from growth to differentiation

The gene expressions involved in the transition from cell proliferation to differentiation were analyzed, using synchronized Dictyostelium discoideum Ax-2 cells and the differential plaque hybridization method. As one of the genes (cDNA) specifically expressed when Ax-2 cells were starved just before the putative shift (PS)-point (putative shift point; a switchover point from growth to differentiation in the cell cycle), calfumirin-1 ( CAF-1 ) was cloned, which encoded a novel calcium-binding protein with E-F hand. Although CAF-1 mRNA was slightly expressed in vegetatively growing cells, the expression was markedly increased in response to starvation of cells just before the PS-point. Northern analysis using non-synchronized Ax-2 cells showed that the CAF-1 mRNA is predominantly expressed within a few hours of starvation. Such a starvation-induced early expression of the CAF-1 mRNA raised a possibility that CAF-1 might be one of Ca²⁺-binding proteins involved in the phase-shift of cells from growth to differentiation.     

Dictyostelium mucoroides cells exhibit cell cycle-dependent sorting behavior as observed in D. discoideum development

Evidence has been obtained indicating that the cell's position in the cell cycle at the onset of starvation is a naturally occurring variable closely involved in the subsequent sorting and pattern formation during the development of Dictyostelium discoideum Ax2. It is of interest to know whether a similar phenomenon is also noticed in species other than D. discoideum and also without any treatment of cells for cell synchronization. For this, the sorting behavior of D. mucoroides-7 ( Dm7 ) cells and its relation to the cell-cycle phase at the onset of starvation were analyzed, using non-synchronized Dm7 cells pulse-labeled with 5'-bromo-2-deoxyuridine (BrdU). The results demonstrate that Dm7 cells starved at the early G2 phase aggregate most rapidly, but are eventually sorted out to the posterior prespore zone of migrating slugs. In contrast, cells starved at the mid late G2 phase exhibited slower aggregation, but were sorted out to the anterior zone (tip), this being basically similar to the sorting behavior of D. discoideum cells. Measurements of cell numbers and nuclearity provided evidence that approximately 80% of cells progressed their cell-cycle after the formation of multicellular structures (mounds), probably coupling with prespore differentiation as in the case of D. discoideum . Thus, cell cycle-dependent sorting during Dictyostelium development is most likely to be a common phenomenon in different species.     

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.     

A Phg2-Adrm1 pathway participates in the nutrient-controlled developmental response in Dictyostelium

Dictyostelium amoebae grow as single cells but upon starvation they initiate multicellular development. Phg2 was characterized previously as a kinase controlling cellular adhesion and the organization of the actin cytoskeleton. Here we report that Phg2 also plays a role during the transition between growth and multicellular development, as evidenced by the fact that phg2 mutant cells can initiate development even in the presence of nutrients. Even at low cell density and in rich medium, phg2 mutant cells express discoidin, one of the earliest predevelopmental markers. Complementation studies indicate that, in addition to the kinase domain, the core region of Phg2 is involved in the initiation of development. In this region, a small domain contiguous with a previously described ras-binding domain was found to interact with the Dictyostelium ortholog of the mammalian adhesion-regulating molecule (ADRM1). In addition, adrm1 knockout cells also exhibit abnormal initiation of development. These results suggest that a Phg2-Adrm1 signaling pathway is involved in the control of the transition from growth to differentiation in Dictyostelium. Phg2 thus plays a dual role in the control of cellular adhesion and initiation of development.     

Possible involvements of 101 kDa, 90 kDa, and 32 kDa phosphoproteins in the phase-shift of Dictyostelium cells from growth to differentiation

Abstract. As demonstrated previously, the transition of starving Dictyostelium cells from growth to differentiation phase occurs at a particular position (putative shift point; PS-point) in G₂-phase of the cell cycle of Dictyostelium discoideum Ax-2. In this study we examined what proteins are phosphorylated or dephosphorylated at the onset of starvation, with special emphasis on changes of phosphoproteins near the PS-point. When AX-2 cells at any particular phase of the cell cycle were pulse-labeled with inorganic ³²P (³²P_i) in the presence or absence of nutrients, it was found that 101 kDa and 90 kDa phosphoproteins exhibit specific changes around the PS-point. From the chase-experiments of ³²P-labeled cells, the 101 kDa and 90 kDa proteins were found to fail to be phosphorylated at the PS-point under starvation conditions. The protein phosphatase inhibitors such as okadaic acid and calyculin A inhibited completely entry of starving Ax-2 cells to differentiation, and also blocked perfectly dephosphorylation of 32 kDa protein. Taken together it is likely that dephosphorylation of 32 kDa protein as well as low phosphorylation levels of 101 kDa and 90 kDa proteins may be required for the phase-shift of Ax-2 cells from growth to differentiation. Subcellular fractionation showed the 101 kDa phosphoprotein to be located in cytoplasm, while parts, at least, of the 90 kDa and 32 kDa phosproproteins were in the nucleus. In addition, the results of cellulose thin-layer electrophoresis of digested 101 kDa and 90 kDa phosphoproteins show that in both proteins only serine residues are phosphorylated. The significance of phosphorylation states of 101 kDa, 90 kDa, and 32 kDa proteins is discussed in relation to a breakaway of cells from proliferation to differentiation.     

Bimodal distribution of motility and cell fate in Dictyostelium discoideum

Pre-starvation amoebae of Dictyostelium discoideum exhibit random movements. Starved cells aggregate by directed movements (chemotaxis) towards cyclic AMP and differentiate into live spores or dead stalk cells. Many differences between presumptive spore and stalk cells precede differentiation. We have examined whether cell motility-related factors are also among them. Cell speeds and localisation of motility-related signalling molecules were monitored by live cell imaging and immunostaining (a) in nutrient medium during growth, (b) immediately following transfer to starvation medium and (c) in nutrient medium that was re-introduced after a brief period of starvation. Cells moved randomly under all three conditions but mean speeds increased following transfer from nutrient medium to starvation medium; the transition occurred within 15 min. The distribution of speeds in starvation medium was bimodal: about 20% of the cells moved significantly faster than the remaining 80%. The motility-related molecules F-actin, PTEN and PI3 kinase were distributed differently in slow and fast cells. Among starved cells, the calcium content of slower cells was lower than that of the faster cells. All differences reverted within 15 min after restoration of the nutrient medium. The slow/fast distinction was missing in Polysphondylium pallidum, a cellular slime mould that lacks the presumptive stalk and spore cell classes, and in the trishanku (triA(-)) mutant of D. discoideum, in which the classes exist but are unstable. The transition from growth to starvation triggers a spontaneous and reversible switch in the distribution of D. discoideum cell speeds. Cells whose calcium content is relatively low (known to be presumptive spore cells) move slower than those whose calcium levels are higher (known to be presumptive stalk cells). Slow and fast cells show different distributions of motility-related proteins. The switch is indicative of a bistable mechanism underlying cell motility.     

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)     

Regulation of development in Dictyostelium discoideum: I. Initiation of the growth to development transition by amino acid starvation

Evidence is presented which indicates that amino acid starvation is the specific stimulus initiating the developmental phase of the life cycle of Dictyostelium discoideum: (i) Amoebae were washed free of complex growth medium and placed in buffer supplemented with specific nutrients; amino acids were the only nutrients that specifically inhibited the initiation of development. (ii) A partially defined growth medium allowing selective starvation for amino acids or glucose during growth is described. Amoebae initiated development only when starved for amino acids. Any effect of glucose on the primary control of the initiation of development is an indirect result of its utilization as a source of precursors for endogenously synthesized amino acids.     

Involvement of the glucose-regulated protein 94 (Dd-GRP94) in starvation response of Dictyostelium discoideum cells

Upon deprivation of nutrients, Dictyostelium discoideum Ax-2 cells arrest proliferation and initiate a metamorphosed developmental program including induction of altered gene expressions which are necessary for differentiation. In Ax-2 cells, we found out a member of Hsp90 family usually contained in the endoplasmic reticulum (ER), Dd-GRP94 (Dictyostelium discoideum glucose-regulated protein 94). In general, GRP94 are induced either by glucose-depletion or by depletion of Ca(2+) in intracellular Ca(2+) stores. Unexpectedly, however, the expression of Dd-grp94 was greatly reduced within 60 min of starvation. Dd-grp94-overexpressing cells (GRP94(OE) cells) collected without forming distinct aggregation streams, and never formed normal fruiting bodies. Also, prespore differentiation as well as maturation into spores and stalk cells were particularly impaired in the GRP94(OE) cells. Thus Dd-GRP94 seems to be crucial in late differentiation as well as in starvation response.     

Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis

The multicellular development of the single celled eukaryote Dictyostelium discoideum is induced by starvation and consists of initial aggregation of the isolated amoebae, followed by their differentiation into viable spores and dead stalk cells. These stalk cells retain their structural integrity inside a stalk tube that support the spores in the fruiting body. Terminal differentiation into stalk cells has been shown to share several features with programmed cell death (Cornillon et al. (1994), J. Cell Sci. 107, 2691-2704). Here we report that, in the absence of aggregation and differentiation, D. discoideum can undergo another form of programmed cell death that closely resembles apoptosis of most mammalian cells, involves loss of mitochondrial transmembrane potential, phosphatidylserine surface exposure, and engulfment of dying cells by neighboring D. discoideum cells. This death has been studied by various techniques (light microscopy and scanning or transmission electron microscopy, flow cytometry, DNA electrophoresis), in two different conditions inhibiting D. discoideum multicellular development. The first one, corresponding to an induced unicellular cell death, was obtained by starving the cells in a "conditioned" cell-free buffer, prepared by previous starvation of another D. discoideum cell population in potassium phosphate buffer (pH 6.8). The second one, corresponding to death of D. discoideum after axenic growth in suspension, was obtained by keeping stationary cells in their culture medium. In both cases of these unicellular-specific cell deaths, microscopy revealed morphological features known as hallmarks of apoptosis for higher eukaryotic cells and apoptosis was further corroborated by flow cytometry. The occurrence in D. discoideum of programmed cell death with two different phenotypes, depending on its multicellular or unicellular status, is further discussed.     

Plasticity in the development and dedifferentiation of Dictyostelium discoideum

Dictyostelium discoideum has served as a model for development and differentiation for over 70 years. Also regulated in Dictyostelium is the process of dedifferentiation, which consists of multiple cellular events that are separately regulated, providing an excellent model system for studying the return of partially differentiated cells to a more pluripotent state. An interesting aspect of Dictyostelium development is the plasticity between growth and development. Reversibility of the processes of differentiation and dedifferentiation exist, allowing Dictyostelium to adjust to changing conditions by reverting to the growth phase during differentiation or reinitiating development during dedifferentiation. This ability of cells to respond to environmental cues is mediated by the checkpoint-like events "commitment" and "erasure," which occur during differentiation and dedifferentiation, respectively. Our review will discuss the current state of knowledge regarding dedifferentiation and the plasticity of the developmental process in both the forward and reverse directions.     

The developmental fate of Dictyostelium discoideum cells depends greatly on the cell-cycle position at the onset of starvation

The relationship between the development of Dictyostelium discoideum Ax-2 and the cell cycle at the onset of starvation was analysed with special reference to sorting behaviors during the formation of polarized cell masses (slugs), using a method for inducing good synchrony. Cells starved at different cell-cycle positions showed different developmental features during further culture. For example, cells just before mitosis and dividing cells were sorted out into the anterior prestalk zone of migrating slugs, while cells starved during most of the G2-phase, into the posterior prespore zone. Time courses of cell aggregation and tip formation were also found to vary greatly in a cell-cycle-related manner, and cells starved during the late G2-phase showed the most rapid development. Differential chemotaxis and cohesiveness are generally considered to be important for cell sorting in Dictyostelium development. In fact, remarkable differences in the chemotactic ability to a chemoattractant, cAMP, were detected among cells starved at any particular phase of the cell cycle. EDTA-resistant cohesiveness was also acquired differently depending on the cell cycle, and it was stronger in the cells showing more rapid aggregation. These findings indicate a close relation of the cell cycle to the cell sorting and pattern formation. The possible significance of the cell-cycle-related events presented here is discussed, with special emphasis on the process of cell aggregation.     

Suppression of the growth/differentiation transition in Dictyostelium development by transient expression of a novel gene, dia1

In Dictyostelium discoideum Ax-2 cells, a specific checkpoint (PS point) from which cells enter the differentiation phase in response to starvation has been specified in the cell cycle. Using the differential display method, we isolated a novel gene, dia1 (differentiation-associated gene 1), that is specifically expressed in cells differentiating from the PS point. The dia1 mRNA has an open reading frame of 1,368 bp and is deduced to code for a 48.6 kDa protein (DIA1). The DIA1 protein is highly serine-rich and the serine residues are predominantly located in the C-terminal region. After the PSORT II search, the protein is predicted to be GPI-anchored at the plasma membrane. Unexpectedly, dia1 overexpression rather impaired the progression of differentiation, possibly coupled with the reduced expression of early genes such as cAMP receptor1 (car1). The inhibitory effect of dia1 expression on early differentiation was almost completely nullified by externally applied cAMP pulses. In contrast to dia1 overexpression, antisense RNA-mediated dia1 inactivation was found to enhance the initial step of cell differentiation, as exemplified by precocious expression of car1 and other early genes. We discuss the unique structure and function of DIA1 in relation to the cooperative development of cells during the establishment of multicellular organization.     

Dictyostelium cell death: early emergence and demise of highly polarized paddle cells

Cell death in the stalk of Dictyostelium discoideum, a prototypic vacuolar cell death, can be studied in vitro using cells differentiating as a monolayer. To identify early events, we examined potentially dying cells at a time when the classical signs of Dictyostelium cell death, such as heavy vacuolization and membrane lesions, were not yet apparent. We observed that most cells proceeded through a stereotyped series of differentiation stages, including the emergence of "paddle" cells showing high motility and strikingly marked subcellular compartmentalization with actin segregation. Paddle cell emergence and subsequent demise with paddle-to-round cell transition may be critical to the cell death process, as they were contemporary with irreversibility assessed through time-lapse videos and clonogenicity tests. Paddle cell demise was not related to formation of the cellulose shell because cells where the cellulose-synthase gene had been inactivated underwent death indistinguishable from that of parental cells. A major subcellular alteration at the paddle-to-round cell transition was the disappearance of F-actin. The Dictyostelium vacuolar cell death pathway thus does not require cellulose synthesis and includes early actin rearrangements (F-actin segregation, then depolymerization), contemporary with irreversibility, corresponding to the emergence and demise of highly polarized paddle cells.     

Effects of chlorpromazine on growth and development of Dictyostelium discoideum.

Chlorpromazine (5 x 10(-3) M) administered to Dictyostelium discoideum cells inhibited its growth and morphogenesis. Cells treated with chlorpromazine were found to have distorted morphology. At lower doses of chlorpromazine the development was delayed. Early developmental events such as cell streaming, cell aggregations, development of EDTA stable cell contacts, cAMP-chemotaxis etc, were inhibited. Chlorpromazine was also found to inhibit spore formation. Culturing D. discoideum cells on chlorpromazine agar, in supernatant taken from the chlorpromazine treated cells, or co-culturing of chlorpromazine-treated and control cells, inhibited the development of normal Dictyostelium cells. Chlorpromazine-treated cells showed a higher cAMP-dependent extracellular phosphodiesterase activity.     

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.