Reverse genetic analyses of gamete-enriched genes revealed a novel regulator of the cAMP signaling pathway in Dictyostelium discoideum

Sexual development in Dictyostelium discoideum is initiated by the fusion of opposite mating type cells to form zygote giant cells, which subsequently gather and phagocytose surrounding cells for nutrition to form macrocysts. Here we performed the targeting of 24 highly gamete-enriched genes we previously isolated, and successfully generated knockout mutants for 16 genes and RNAi mutants for 20 genes including 6 genes without disruptants. In the knockout mutants of two genes, cell aggregation toward the giant cells was much less extensive and many cells remained around poorly formed macrocysts. We named these genes tmcB and tmcC. Although macrocyst formation of wild type cells was suppressed by the addition of exogenous cAMP, that of knockout mutants of tmcB was much less sensitive. The mRNA level of phosphodiesterase (pde) was higher and that of its inhibitor (pdi) was lower in the latter cells compared to the parental strains during sexual development. Thus, tmcB appeared to be a novel regulator of the cAMP signaling pathway specific to sexual development. Knockout mutants of tmcC were indistinguishable from the wild type cells with respect to the cAMP response, suggesting that this gene is relevant to other processes.     

Regulation of the developmental modes in Dictyostelium mucoroides by cAMP and ethylene

The cellular slime mold Dictyostelium mucoroides-7 (Dm7) and a mutant (MF1) derived from it exhibit clear dimorphism in development depending upon environmental conditions: macrocyst formation occurs during the sexual cycle, and sorocarp formation during the asexual process. As previously reported, exposure of cells to ethylene gas is favorable to macrocyst formation, while exogenously added 3',5'-cyclic adenosine monophosphate (cAMP) induces sorocarp formation. The significance of ethylene and cAMP for the mechanism involved in selection of the developmental pathways was further confirmed by determining the amounts of these substances in macrocyst- or sorocarp-forming cells. Aminooxy-acetic acid (AOA), an inhibitor of ethylene synthesis, was found to switch development of Dm7 and MF1 cells from macrocyst to sorocarp formation by decreasing ethylene production. The cAMP content was shown to be always higher in cells destined for sorocarp formation than in those destined for macrocyst formation, particularly at the aggregation stage. All of the results obtained strongly suggested that the amounts of cAMP and ethylene present, and possibly the ratio between them, may be of great importance for determining which mode of development will be realized.     

Synthesis of specific proteins for sexual development in Dictyostelium discoideum

The development of Dictyostelium discoideum may proceed by two pathways, macrocyst or fruiting-body formation, the former being the sexual and the latter the asexual cycle. The pathway of development depends on the presence or absence of zygote giant cells which are produced through fusion of opposite mating-type cells in a population, in heterothallic strains. During the early stages of macrocyst development the patterns of developmentally regulated proteins were noted to differ considerably from those during fruiting-body development. Furthermore, the haploid cells around zygote giant cells synthesized a large number of specific proteins for macrocyst development through the influence of giant cells.     

Cyclic nucleotides and cyclic nucleotide phosphodiesterase during development of Polysphondylium violaceum

Polysphondylium violaceum is shown to produce and excrete cyclic nucleotides and to produce a cell-associated cyclic nucleotide phosphodiesterase(s). The amount of adenosine 3′,5′-cyclic monophosphate (cAMP) excreted by the amebae reaches a maximum during development when aggregation centers are just forming and then falls off rapidly. Measurements of total cAMP show that the amount synthesized increases more than 15-fold throughout development with the majority of the increase coming during the culmination stages. Guanosine 3′,5′-cyclic monophosphate (cGMP) is either not excreted or is excreted at levels below our limits of detection. An increase in the total cGMP synthesized occurs at mid-aggregation when two or three sharp peaks of synthesis are observed. However, development of P. violaceum is not affected by the addition of high concentrations of either cAMP or cGMP (or their dibutyryl derivatives) to the medium despite the fact that the cells produce these nucleotides. Cell-associated cyclic nucleotide phosphodiesterase activity, which hydrolyses both cAMP and cGMP, is greatest at the onset of starvation with a second increase in activity during aggregation.     

Nuclear fusion in multinucleated giant cells during the sexual development of Dictyostelium discoideum

Macrocyst development in Dictyostelium discoideum, is generally considered a sexual phase. This development is initiated by the formation of a giant cell, the result of the fusion of two different mating type haploid cells, such as NC4 and HM1. The giant cell engulfs unfused surrounding cells to develop into a macrocyst. Therefore, if the macrocyst is a sexual structure, the giant cell must be a diploid zygote. However, under certain conditions, a very large multinucleated giant cell containing several dozens of nuclei is formed, followed by normal development into a macrocyst. In such a multinucleated giant cell, it was found that only two nuclei fuse together to produce a diploid zygote and all others disappear at the early stage of development. The diploid nucleus undergoes meiosis and subsequently subdivides into a number of haploid progeny cells later released from the macrocyst to initiate new life cycles.     

Protein Synthesis Initiated by Cell Fusion in Dictyostelium discoideum

D. discoideum has two alternative developmental pathways. If cells of two complement mating-type strains, NC4 and HM1, fuse sexually, a giant cell is produced which subsequently develops into a macrocyst, the sexual structure of this organism. However, if fusion fails to occur and cells are starved, a fruiting-body is produced instead of a macrocyst. In this paper, a two-dimensional polypeptide gel electrophoresis study showed that giant cells produce specific polypeptides which may possibly be involved in macrocyst development. Out of total 497 polypeptides which appeared in a giant cell during an incubation period of 13 hr, 92 were the specific for giant cells. Four of these polypeptides were appeared within only 1 hr after the cell fusion. The other 405 were non-specific polypeptides which appeared in both giant cells and NC4 or/and HM1 cells. However, the patterns and the rates of production of each polypeptide during the incubation period were different between these cells.     

Aggregation during sexual development in Dictyostelium discoideum.

A microcinematographic analysis of the behaviour and movements of cells and cell masses in mated cultures (NC4 X VI2) of Dictyostelium discoideum indicates that a chemotactic process directs cell aggregation during macrocyst development. Zygote giant cells form before aggregation begins and act as the aggregation centres. Young multicellular macrocyst stages are sources of cyclic AMP, and amoebae from macrocyst cultures orient chemotactically to cyclic AMP. The data, coupled with other characteristics such as pulsatile streaming, suggest that the aggregation process leading to macrycyst development is the same as that occurring during fruit construction. Other aspects of sexual development are also discussed. Based upon these data, we propose a model for the sequence of events leading to macrocyst development in D. discoideum.     

Induction of Macrocyst Formation by Factors Secreted by Giant Cells in Dictyostelium discoideum

The fusion of cells of complementary mating types to produce giant cells has been shown to be the critical event to induce macrocyst formation in Dictyostelium discoideum. We have examined the way in which giant cells use diffusible factors to influence the developmental mode of nearby cells using an experimental design in which NC4 cells are allowed to develop on a dialysis membrane above a suspension of giant cells. We have observed that giant cells are able to inhibit independent aggregation and stream formation in the upper cells and become the dominant aggregation centers. In addition giant cells are able to redirect local amoeba away from the fruiting-body and toward the macrocyst mode of development. We show that these effects are mediated by diffusible factors of under 2,000 MW. and discuss possible mechanisms of action.     

Dissimilar cyclic nucleotide phosphodiesterase activities in subcellular fractions from normal and SV40-transformed WI-38 fibroblasts.

Broken cell preparations of WI-38 and SV40-transformed WI-38 (VA13) fibroblasts were used to compare the cyclic nucleotide phosphodiesterase activities of the two cell strains. The bulk of the cAMP or cGMP phosphodiesterase activity of WI-38 and VA13 homogenates was found in the 100,000 x g fibroblast supernatant fractions. WI-38 and VA13 soluble phosphodiesterase activities showed anomalous kinetic behavior with either cAMP or cGMP as the substrate. At low substrate concentrations, e.g., 0.1 muM, WI-38 supernatant fractions hydrolyzed cGMP much more rapidly than cAMP. At high substrate concentrations, e.g., 100muM, the same enzyme preparations degraded cAMP more than twice as fast as cGMP. In contrast, VA13 soluble phosphodiesterase activity catalyzed the hydrolysis of a wide range of cAMP and cGMP concentrations at similar rates. Phosphodiesterase activity in WI-38 supernatant fractions was generally more sensitive than that of the comparable VA13 enzyme activity to inhibition by MIX and papaverine. The cAMP phosphodiesterase activity of both WI-38 and VA13 supernatant preparations was decreased by cGMP in a concentration-dependent manner. cAMP was an effective inhibitor of cGMP hydrolysis by VA13 soluble phosphodiesterase activity. Yet, the cGMP phosphodiesterase activity of WI-38 supernatant fractions was only slightly reduced in the presence of cAMP. DEAE-cellulose chromatography of WI-38 and VA13 supernatant preparations revealed two major peaks of phosphodiesterase activity for each cell type. WI-38 peak I showed much greater activity with 1muM cGMP than with 1muM cAMP and appeared to be composed of two different phosphodiesterase activities. WI-38 peak Ia included phosphodiesterase activity which could be stimulated by boiled, dialyzed fibroblast homogenates while WI-38 peak Ib coincided with column fractions which contained most of the cyclic GMP hydrolytic activity. VA13 peak I phosphodiesterase activity was eluted from DEAE cellulose columns at the same ionic strength as WI-38 peak Ia and hydrolyzed these two substrates at nearly identical rates. This enzyme activity was also increased in the presence of boiled, dialyzed fibroblast preparations. Peak II phosphodiesterase activities from both WI-38 and VA13 fibroblasts were relatively specific for cAMP as the substrate. Phosphodiesterase activity with the properties of WI-38 peak Ib was not isolated from VA13 supernatant fractions. These results suggested that the dissimilar patterns of cAMP accumulation in WI-38 and VA13 cultures may be at least partially related to different phosphodiesterase activities in the normal and the transformed fibroblasts.     

Concentration and metabolism of cyclic AMP during the early stages of hepatoma 22a growth]

Intracellular levels of cyclic AMP (cAMP), adenylate cyclase, and cAMP-phosphodiesterase activities at lag-period, exponential and stationary growth phases of hepatoma 22a were determined. It was shown that the transition of tumour cells from the lag-period to the exponential phase of growth was accompanied by the two-fold decrease of intracellular cAMP level on account of drastic activation of cAMP phosphodiesterase. Subsequently the cAMP level lowered more slowly until the cells entered the stationary phase of growth. In view of the fact that the adenylate cyclase activity failed to change at different growth phases of hepatoma 22a, it seems very proballe that the rise of cAMP phosphodiesterase activity could be a signal for the exit of tumour cells from the lag-period and their entrance into the mitotic cycle.     

Adenosine 3':5'-Cyclic Monophosphate in Normal and Habituated Helianthus Callus

A compound with properties identical to adenosine 3’:5′-cyclic monophosphate (cAMP) has been isolated from callus of Helianthus annuus. The compound accompanied cAMP during an extensive purification procedure and co-chromatographed with cAMP in three solvent systems. The compound stimulated cAMP-dependent protein kinase and was active in a cAMP protein-binding assay. Its activity was abolished by cAMP phosphodiesterase. It is concluded that the compound is cAMP. The variation in cAMP content in the callus during its development was determined. In 1974 when the stock callus was grown on auxin-containing medium the content was c. 2 pmol cAMP/g when the cells were vigorously dividing and c. 125 pmol cAMP/g two weeks after the transfer. In 1976 the callus had attained hormone autotrophy. Its cAMP content was generally low during the whole developmental cycle. The results might indicate that cAMP played a role in the development of the normal callus of 1974, while it has no regulatory function in the tumour-like tissues of 1976. The results are discussed on the basis of recent literature.     

Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis

Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA- cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA- cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.     

The cyclic nucleotide phosphodiesterase of Dictyostelium discoideum: the structure of the gene and its regulation and role in development

The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum plays an essential role in development by hydrolyzing the cAMP used as a chemoattractant by aggregating cells. We have studied the biochemistry of the phosphodiesterase and a functionally related protein, the phosphodiesterase inhibitor protein, and have cloned the cognate genes. A 1.8-kb and a 2.2-kb mRNA are transcribed from the single-phosphodiesterase gene. The 2.2-kb mRNA comprises the majority of the phosphodiesterase mRNA found in differentiating cells and is transcribed only during development from a promoter at least 2.5 kb upstream of the translational start site. The 1.8-kb phosphodiesterase mRNA is detected at all stages of growth and development, is present at lower levels than the developmentally induced mRNA, and is transcribed from a site proximal to the protein-coding region. The phosphodiesterase gene contains a minimum of three exons, and a 2.3-kb intron, the longest yet reported for this organism. We have shown that the pdsA gene and four fgd genes affect the accumulation of the phosphodiesterase mRNAs, and we believe that these loci represent a significant portion of the genes regulating expression of the phosphodiesterase. The phosphodiesterase gene was introduced into cells by transformation and used as a tool to explore the effects of cAMP on the terminal stages of development. In cells expressing high levels of phosphodiesterase activity, final morphogenesis cannot be completed, and differentiated spore and stalk cells do not form. We interpret these results to support the hypothesis that cAMP plays an essential role in organizing cell movements in late development as well as in controlling the aggregation of cells in the initial phase of the developmental program.     

Fusion of cell ghosts with sexually opposite type cells in Dictyostelium discoideum

In the sexual cycle of Dictyostelium discoideum, haploid cells of two opposite mating types, strains HM1 and NC4, acquire fusion-competence under certain conditions, such as suspension culture in the dark, and fuse specifically to form giant zygote cells. Each giant cell engulfs the surrounding cells, gradually increases in size, and finally develops into a macrocyst that is a sexual structure in D. discoideum. Fusion-competent HM1 cells suspended in a solution were frozen and thawed to make cell ghosts. When cell ghosts were introduced into fusion-competent and -incompetent intact NC4 cells, the cell ghosts killed them in a short time, but the fusion-competent cells were killed in preference to the fusion-incompetent cells. This killing occurred through the fusion of the cell ghosts directly to intact cell membranes. Since the fusion was specific, the fusion between ghosts and cells appears to be essentially the same as that between intact cells during the sexual cycle in molecular mechanisms.     

Cyclic AMP and theophylline enhance DNA synthesis in L cells stimulated with anti-actin IgG and [(IgG)2protein A]2 complex by recruiting cells into S-phase

Surface binding of anti-actin IgG alone or in a M_r = 716 000 [(IgG)₂Protein A]₂ complex results in a stimulation of DNA synthesis and cell growth in L cells. Cyclic-AMP (0.01–1.0 mM) added to such cell cultures augmented DNA synthesis as measured by incorporation of [³H]thymidine into DNA. Theophylline (0.1–1.0 mM), a phosphodiesterase inhibitor which prevents enzymatic breakdown of cAMP, had similar effects, but cGMP (0.01–1.0 μM) reversed the effects of cAMP and theophylline upon DNA synthesis. Analysis of the cell cycle by flow cytometry revealed that antibody produced a shift (7%) of cells from the G₁-phase to the S-phase (DNA-synthetic) of the cell cycle at 72 hr of incubation. Addition of cAMP (0.5 mM) to cell cultures, however, produced significant shifts of antibody stimulated cells from G₁-phase to S-phase at all time points measured, i.e., 24 (12%),48 (22%),72 hr (23%). Thus, antibody recruited cells into S-phase of the cell cycle and cAMP greatly augmented the effect. These observations suggest that the mechanism of activation of L cell growth by antibody to surface antigens involves a recruitment of cells into the DNA-synthetic phase and that the effect may be mediated by cAMP.     

Cell cycle traverse and macromolecular synthesis in BHK fibroblasts as affected by insulin

Insulin produced a 10-fold activation of cAMP phosphodiesterase activity and a 3-fold reduction in cAMP content in serum-deprived, growth-arrested BHK fibroblasts. Insulin did not promote growth of these cells, as judged by measurement of cell number, but it did promote reentry of cells into the cell cycle and progression into S phase, as determined by rates of macromolecular synthesis, and autoradiographic and microfluorometric analyses. Insulin stimulation of macromolecular synthesis can be blocked by the addition of a phosphodiesterase inhibitor, suggesting that these effects of insulin on the cell cycle may be mediated through its activation of cAMP phosphodiesterase.     

Lectin binding and inhibition studies reveal the importance of D-glucose, D-mannose and N-acetylglucosamine during early sexual development of Dictyostelium discoideum

Fluorescein-conjugated and non-conjugated lectins were used to determine which surface sugars are involved in the early events of sexual (macrocyst) development in Dictyostelium discoideum. Only zygote giant cells showed unique binding of FITC-WGA and FITC-PNA while all cell types (amoebae, gametes, binucleates, giant cells) showed identical patterns of FITC-Con A, -Gorse and -RCA II binding. In spite of its non-selective labelling of all cell types, Con A inhibited macrocyst formation. The temporal addition of Con A with and without specific hapten sugars indicates the importance of both D-mannose and D-glucose in phagocytosis and, possibly, cell fusion. WGA also inhibited macrocyst formation. Varying the time of addition of the lectin plus/minus its primary hapten sugar implicates N-acetylglucosamine as being important in cell fusion. Neither Gorse, RCA II nor PNA had any detectable inhibitory effects on macrocyst development leaving the appearance of increased PNA receptors at the giant cell surface as an enigma.     

Hormonal regulation of a plasma membrane phosphodiesterase in differentiating granulosa cells. Reciprocal actions of follicle-stimulating hormone and a gonadotropin-releasing hormone agonist on cAMP degradation

The activity of a plasma membrane cAMP-phosphodiesterase in cultured ovarian granulosa cells was regulated by follicle-stimulating hormone (FSH) and the gonadotropin-releasing hormone (GnRH) agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). Degradation of cAMP was similar in cultures treated with FSH alone or FSH plus GnRHa when the labeled cyclic nucleotide was added from 24 to 42 h of culture. However, at 48 h and subsequent times of incubation, cAMP phosphodiesterase activity was significantly higher in cells incubated with FSH plus GnRHa. Phosphodiesterase activity was progressively increased by GnRHa concentrations between 10(-13) and 10(-10) M, and was maximally stimulated by 10(-9) M GnRHa. In comparison with control cells, FSH lowered the Vmax of cAMP catabolism by the high (1 microM cAMP substrate) and the low (50 microM) affinity phosphodiesterase, while GnRHa raised enzyme activity toward control levels. These actions of FSH and GnRHa were specific for a plasma membrane phosphodiesterase that was accessible to extracellular cAMP, since extracellular substrate was hydrolyzed, no intracellular uptake of [3H]cAMP was observed, and only a small fraction (10%) of cAMP was catabolized in the incubation medium in the absence of cells. Further, the actions of FSH and GnRHa on the membrane enzyme were the opposite of those observed when total phosphodiesterase activity was measured in cellular sonicates. Hormonal changes in phosphodiesterase activity were not due to leakage of the enzyme from damaged cells since a constant percentage of cAMP hydrolysis in the medium was observed during culture. Analysis of cAMP catabolites in granulosa cells indicated that the phosphodiesterase reaction product, 5'-AMP, was rapidly converted to adenosine by a plasma membrane 5'-nucleotidase, independent of the cellular hormonal status. These results indicate that the opposing actions of FSH and GnRHa upon granulosa cell differentiation include modulation of cAMP degradation at the plasma membrane level.     

ENZYMATIC DISSOCIATION OF NASCENT MACROCYSTS AND PARTITION OF THE LIBERATED CYTOPHAGIC GIANT CELLS IN DICTYOSTELIUM MUCOROIDES*

Nascent macrocysts of the cellular slime mold Dictyostelium mucoroides were dissociated enzymatically and the liberated cytophagic giant cells were partitioned by dextrin density gradient centrifugation. Enzymatic and cytochemical studies revealed that the primary wall is composed mainly of cellulose (β-1,4-glucan) associated with polysaccharides including hemicellulose, pectic substances and á-1,4-glucan. The buoyant density of the liberated cytophagic giant cells and peripheral cells was determined by density gradient centrifugation, and partitioning of the cells was possible due to the difference in this property. The process of macrocyst reconstitution was investigated using dissociated cells. The isolated cytophagic giant cell has a specific affinity for other cytophagic giant cells and predominantly ingests them by phagocytosis, while it retains the ability to ingest peripheral cells. The present study provides a clue for investigating the differentiation and development of sexual cells, since only the cytophagic giant cell gives rise to a zygote in macrocyst formation.