Dictyopyrones, novel alpha-pyronoids isolated from Dictyostelium spp., promote stalk cell differentiation in Dictyostelium discoideum

Dictyopyrones A and B (DpnA and B), whose function(s) is not known, were isolated from fruiting bodies of Dictyostelium discoideum. In the present study, to assess their function(s), we examined the effects of Dpns on in vitro cell differentiation in D. discoideum monolayer cultures with cAMP. Dpns at 1-20 microM promoted stalk cell formation to some extent in the wild-type strain V12M2. Although Dpns by themselves could hardly induce stalk cell formation in a differentiation-inducing factor (DIF)-deficient strain HM44, both of them dose-dependently promoted DIF-1-dependent stalk cell formation in the strain. In the sporogenous strain HM18, Dpns at 1-20 microM suppressed spore formation and promoted stalk cell formation in a dose-dependent manner. Analogs of Dpns were less effective in affecting cell differentiation in both HM44 and HM18 cells, indicating that the activity of Dpns should be chemical structure specific. It was also shown that DpnA at 2-20 microM dose-dependently suppressed spore formation induced with 8-bromo cAMP and promoted stalk cell formation in V12M2 cells. Interestingly, it was shown by the use of RT-PCR that DpnA at 10 microM slightly promoted both prespore- and prestalk-specific gene expressions in an early phase of V12M2 and HM18 in vitro differentiation. The present results suggest that Dpns may have functions (1) to promote both prespore and prestalk cell differentiation in an early stage of development and (2) to suppress spore formation and promote stalk cell formation in a later stage of development in D. discoideum.     

盘基网柄菌中分化诱导因子信号与细胞命运决定

对盘基网柄菌发育过程中分化诱导因子(DIF)的作用及其机制进行了综述,包括DIF对盘基网柄菌前柄细胞、柄细胞分化的作用以及DIF的生物合成、DIF的诱导、降解失活、DIF对细胞命运和细胞比例的调节及其作用机制等。     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

盘基网柄菌细胞分化及细胞比例的调控

本文综述了盘基网柄菌(Dictyosteliumdiscoideum)发育过程中调控细胞分化及细胞比例的一些信号分子,包括分化诱导因子(DIF-1、SDF-2)、糖原合成酶激酶(GSK-3)、环状亮氨酸拉链蛋白(rZIP)等,介绍了这些信号分子的功能及其作用机制。     

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.     

A gene encoding, prespore-cell-inducing factor in Dictyostelium discoideum

Two factors that exist in conditioned medium (CM) of Dictyostelium discoideum induce amoebae to differentiate into prespore cells when they are incubated at a very low cell density in submerged monolayer culture. Previously, we purified one of them, a glycoprotein factor with an apparent molecular mass of 106 kDa, and we named it psi factor (psi, prespore-inducing factor). Based on the partial amino acid sequence of the purified psi factor, we have isolated the corresponding cDNA clone, which is expressed maximally at the loose mound stage. The cDNA encodes a novel protein and the predicted molecular mass of the mature secreted protein is 60 kDa. Knockout mutant strains of the psi factor gene, psiA(-), were created by targeted integration. Although these mutant strains appear to develop normally, CM from these mutants showed reduced prespore-cell-inducing activity. Rescuing the mutant strains by expression of psi factor under control of a constitutive promoter causes overproduction of psi factor protein and CM from such cells showed a 20-fold higher level of prespore-cell-inducing activity than that from wild-type cells. Further, CM from parental cells induced prespore cell division, while that from psiA null strains showed no cell division inducing activity. Our results indicate that psi factor protein is a novel type of growth factor that does not belong to any of the families of growth factor so far identified in animals.     

Induction of Cell Differentiation of Isolated Cells in Dictyostelium discoideum by Low Extracellular pH

In Dictyostelium discoideum , the formation of multicellular masses is necessary for cell differentiation. However, the present study shows that amoebae of strain V12M2 efficiently differentiate to prespore or stalk cells under submerged incubation in a simple medium containing cAMP and salts without cell contact, only if the pH of the medium is maintained at acidic values; differentiation scarcely occurs in the neutral pH range. The optimum pH values for prespore and stalk cell differentiation are 5.1 and 4.5, respectively. In addition to the extracellular pH, Mg ions and the concentration of cAMP also affect the choice of the differentiation pathway. The time courses of differentiation of both cell types under optimum conditions are also presented.     

Developmental characterization of the wheat germ agglutinin binding proteins, wst31 and wst34, enriched in prestalk and stalk cells of Dictyostelium discoideum

Abstract. The onset of prestalk differentiation of Dictyostelium discoideum has been thought to be triggered by differentiation inducing factor (DIF), which is secreted by differentiating cells. We characterized the cell-type specific proteins, wst31 (prestalk and stalk specific) and wst34 (stalk specific), using the mutant HM44 which is defective in DIF-production, and examined the effects of DIF and cAMP on the formation of the proteins. In the mutant HM44, wst34 was formed only in the presence of exogenous DIF as reported for other prestalk/stalk markers (e.g. pDd63 and acid phosphatase-2), which indicates the DIF-requirement for this protein. By contrast, the accumulation of wst31 in this mutant occurred in the presence of cAMP regardless of the presence of exogenous DIF. Thus, we propose a new and distinct state (or stage) in prestalk differentiation, where the expression of wst31 occurs but not that of pDd63 or acid phosphatase-2.     

Self- and non-self-recognition in bisexual mating of Dictyostelium discoideum

Cell recognition plays a central part in the sexual process. Although cell-surface molecules involved in gamete recognition have been identified in several organisms, our knowledge of the molecular basis of sexual cell recognition is still limited. We have been studying molecular mechanisms of sexual cell fusion using the lower eukaryote Dictyostelium discoideum . There are homothallic, heterothallic, bisexual and asexual strains in D. discoideum , and how they distinguish between each other to find out proper partners is an interesting and important question. However, analytical studies of sexuality in D. discoideum have been carried out mostly on heterothallic strains, and the polymorphism of the mating system has not yet been thoroughly investigated. In the present study, we extended our analysis to the bisexual mating phenomenon paying special attention to the mechanism of self-incompatibility. We showed that a bisexual strain WS2162 was self-incompatible at the step of sexual cell fusion. Results of antibody inhibition of cell fusion and detection of gp138, a cell-fusion-related protein found in heterothallic strains, suggest that a molecular basis for bisexual and heterothallic mating are common. We propose two models to clarify the mechanisms of self- and non-self discrimination in bisexual mating patterns of D. discoideum .     

Chemiluminescence in Dictyostelium discoideum

Abstract Oxygen radicals generated during oxidative metabolism participate in chemical reactions resulting in light emission. Chemiluminescence is used therefore to measure their production. We have shown that starvation and heat shock induce chemiluminescence in Dictyostelium discoideum . The peak light emission was found to occur about 4 h after the onset of starvation. The optimum temperature for chemiluminescence by starving amoebae was about 33°C. The heat shock inducibility of chemiluminescence was maximal at the beginning of development. Our results are consistent with suggestions that the product(s) of perturbed mitochondrial metabolism might be intracellular signal(s) controlling gene expression in stressed cells. They also suggest a role for intracellular stress signal(s) in the initiation of development in Dictyostelium by starvation.     

The role of the cell cycle in differentiation of the cellular slime mould Dictyostelium discoideum

Summary During development and differentiation of the cellular slime mould Dictyostelium discoideum there appears to be a relationship between the cell cycle and cell fate: amoebae halted in G2 phase during early development differentiate into spores whereas stalk cells are formed from amoebae halted in GI phase. It is proposed that this is because a major effect of the cell cycle is to generate heterogeneity in the cell surface properties of the developing amoebae.     

盘基网柄菌细胞发育过程中尿囊酸酶的亚细胞定位

利用胶体金免疫电镜技术,观察了盘基网柄菌细胞分化与凋亡过程中胞内尿囊酸酶的位置变化。结果表明,在细胞聚集期细胞产生的尿囊酸酶主要分布于线粒体及周围细胞质内。到了细胞丘时期,尿囊酸酶只特异地存在于发生内自噬的线粒体内,且仅局限于线粒体因内自噬产生的空泡区域,这些发生线粒体内自噬的细胞将分化成前孢子细胞。随着前孢子细胞分化的进行,尿囊酸酶颗粒在细胞内分布逐渐减少,在靠近质膜处的空泡内还能观察到一些酶颗粒;而另一些细胞内,几乎所有的胞器内都能观察到酶颗粒,一直延续至柄细胞形成。从中可以看到尿囊酸酶在将发育成孢子细胞和柄细胞两种类型细胞内的分布位置明显不同,结果提示了尿囊酸酶蛋白与盘基网柄菌细胞分化和凋亡调控途径有密切关系。     

Rapid patterning of Dictyostelium discoideum cells under confined geometry and its relation to differentiation

The following was recently reported by Bonner et al. (1995): (1) Rapid differentiation occurred into two zones in Dictyostelium discoideum cells confined in a fine glass capillary. The cells in the anterior zone exposed to the air appear similar to prestalk cells, while the posterior zone isolated from the air mimics prespore cells. (2) The volumes of the two zones are proportional to each other for different sized cell masses, and the proportion is the same as that in normal migrating slugs. We investigated the nature of this newly discovered rapid differentiation in a slightly modified geometry. Exponentially growing cells were harvested, washed to remove external nutrients, and pelleted by centrifugation. Subsequently, a small drop of the pelleted (starved) cells was placed on a slide glass and then confined in a two-dimensional space between the slide glass and a coverslip, with help of spacers whose thickness varied from 25 to 100 μm. As a result, a dark zone, which looked optically different, emerged within several minutes in the periphery of the disc of the confined cells, corresponding to the zonation in a capillary as previously reported. When the width of the peripheral zone was measured for more than 30 samples of different diameters for each thickness of the spacers, the width was found to be always about 100 μm, irrespective of the size difference of the cell mass placed. This seems to be contradictory to the previous observation made by Bonner et al. (1995). We also examined oxygen concentration dependence on the zone width. The zone width was found to be independent of the oxygen concentration at low concentrations, but increased rapidly at high concentrations. A reaction-diffusion mechanism for formation of the zone and possible involvement of atmospheric oxygen (O₂) in the initial steps of cell differentiation and pattern formation is discussed.     

盘基网柄菌细胞分化和凋亡中酸性磷酸酶的定位

利用电镜酶细胞化学方法,观察盘基网柄菌细胞分化和凋亡过程中酸性磷酸酶的变化。在细胞丘阶段,酶反应颗粒出现在线粒体内自噬空泡内,随着内自噬空泡的逐渐增大,线粒体内的酶反应颗粒逐渐增多,线粒体内嵴结构不断破坏,直至遍布整个空泡化的线粒体内;当细胞发育至前孢子细胞时,由于嵴结构被完全破坏,酶反应颗粒主要集中在前孢子细胞空泡的单层膜上,空泡化的线粒体内酶反应颗粒逐渐消失。在凋亡的柄细胞中,自噬泡内酶反应强烈,凋亡中期的前柄细胞的细胞核中出现酶反应颗粒,均匀分布在细胞核中,直至细胞核与自噬泡融合。在孢子细胞外被与质膜间也观察到非溶酶体酸性磷酸酶。所得结果证实:线粒体内自噬小泡具有消化功能;自噬泡内酶活性与细胞器消亡有关;细胞核中的酸性磷酸酶可能作为一种非溶酶体酸性磷酸酶参与细胞核中核蛋白的脱磷酸化过程,与发育相关基因表达有关     

Isolation and characterization of cAMP unresponsive (frigid) aggregation-deficient mutants of Dictyostelium discoideum

To find mutants of Dictyostelium discoideum that are unable to respond to exogenous cAMP signals (frigid mutants), amoebae of 218 independent aggregation-deficient mutants were treated in suspension with artificial pulses of cAMP and screened for the capacity to form EDTA-resistant cohesion sites. Eleven frigid mutants were identified and further characterized. Using parasexual genetic techniques, these strains were assigned to five complementation groups (fgdA-E) and the fgd loci were mapped in three linkage groups: fgdA and D in group II, fgdC in group III, and fgdB and E in group VII. Biochemical and physiological experiments with these strains indicated that fgd mutants are of two general types. When starved, strains in groups fgdB, D, and E failed to produce detectable levels of membrane-associated cAMP phosphodiesterase, surface cAMP receptors, or extracellular phosphodiesterase inhibitor, and the cells continued to respond chemotactically to folate. Thus, these strains are probably arrested in the vegetative phase or very early in development. In contrast, strains in groups fgdA and C produced low levels of cAMP receptors and secreted phosphodiesterase inhibitor. Moreover, after starvation, some of these mutants elicited a weak chemotactic response to cAMP. Therefore, unlike the former group of mutants, these strains appear to initiate development when starved, but the process is blocked at an early stage.     

Inhibitors of intracellular cyclic AMP accumulation affect differentiation of sporogenous mutants of Dictyostelium discoideum

Abstract Sporogenous mutants of Dictyostelium discoideum strain V12M2 were used to determine whether the intracellular levels of cyclic AMP or other second messengers regulate differentiation. Increasing external concentrations of cyclic AMP promoted spore formation. Caffeine and progesterone, which lower intracellular cyclic AMP levels by different mechanisms, blocked spore formation and favored stalk cell formation. In contrast, differentiation of both spore and stalk cells occurred normally in the presence of agents that disrupt calcium/calmodulin or protein kinase C-based second messenger systems. The data are in accord with the view that (1) intracellular cyclic AMP is essential for terminal differentiation of both cell types, and (2) higher levels are required for formation of spores than for stalk cells.     

Isolation and characterization of glycogen synthase in Dictyostelium discoideum

We have partially purified the protein and isolated the glcS gene for glycogen synthase in Dictyostelium. glcS mRNA is present throughout development and is the product of a single gene coding for 775 amino acids, with a predicted molecular mass of 87 kD. The sequence is highly similar to glycogen synthase from human muscle, yeast, and rat liver, diverging significantly only at the amino and carboxy termini. Phosphorylation and UDPG binding sites are conserved, with K_m values for UDPG being comparable to those determined for other organisms, but in vitro phosphorylation failing to convert between the G6P-dependent (D) and -independent (I) forms. Enzyme activity is relatively constant throughout the life cycle: the I form of the enzyme isolates with the soluble fraction in amoebae, switches to the D form, becomes pellet-associated during early development, and finally reverts during late development to the I form, which again localizes to the soluble fraction. Deletion analysis of the promoter reveals a GC-rich element which, when deleted, abolishes expression of glcS. © 1996 Wiley-Liss, Inc.     

Staurosporine induces tyrosine phosphorylation in Dictyostelium discoideum proteins

The treatment of cells with staurosporine results in inhibition and less frequently activation of protein kinases, in a cell-type specific manner. In the social amoeba Dictyostelium discoideum, staurosporine induces marked changes in cell morphology affecting growth and development. Here we describe that incubation of D. discoideum growing or starved cells with staurosporine results in a rapid and unexpected tyrosine phosphorylation on two polypeptides of approximately 64 and approximately 62 kDa. These proteins emerge as novel substrates for tyrosine phosphorylation opening up new perspectives for the study of cell signalling in D. discoideum.