Re-expression of 117 antigen, a cell surface glycoprotein of aggregating cells, during terminal differentiation of Dictyostelium discoideum prespore cells

117 antigen is a glycoprotein expressed on the surface of D. discoideum cells at aggregation. It then disappears and is later re-expressed on the surface of a subpopulation of cells at culmination, the terminal differentiation stage (Sadeghi et al. 1987). A cDNA clone was used to show that the appearance of cell surface 117 antigen accurately reflects the expression of the 117 gene as measured by mRNA levels. It was also shown that during multicellular development there is a reciprocal relationship between the levels of 117 mRNA and the mRNA which codes for prespore surface glycoprotein, PsA. Dual parameter flow cytometry was used to demonstrate that the 117 antigen is found on the surface of maturing prespore cells after the PsA glycoprotein disappears, but that it is not found on mature spores. Using three monoclonal antibodies which identify respectively 117 antigen, PsA, and MUD3 antigen (a spore coat glycoprotein--probably Sp96), two new stages of final spore maturation were defined. These results indicate that there is a recapitulation of at least one aggregative cell surface glycoprotein in the prespore subpopulation of cells as they rise up the stalk during final spore development. This raises the possibility that culmination, which involves complex three dimensional morphogenetic movements not unlike those observed during animal embryogenesis, involves components of the two-dimensional pattern seen during aggregation.     

Stalk size and altruism investment within and among populations of the social amoeba

Reproductive division of labour is common in many societies, including those of eusocial insects, cooperatively breeding vertebrates, and most forms of multicellularity. However, conflict over what is best for the individual vs. the group can prevent an optimal division of labour from being achieved. In the social amoeba Dictyostelium discoideum, cells aggregate to become multicellular and a fraction behaves altruistically, forming a dead stalk that supports the rest. Theory suggests that intra‐organismal conflict over spore–stalk cell fate can drive rapid evolutionary change in allocation traits, leading to polymorphisms within populations or rapid divergence between them. Here, we assess several proxies for stalk size and spore–stalk allocation as metrics of altruism investment among strains and across geographic regions. We observe geographic divergence in stalk height that can be partly explained by differences in multicellular size, as well as variation among strains in clonal spore–stalk allocation, suggesting within‐population variation in altruism investment. Analyses of chimeras comprised of strains from the same vs. different populations indicated genotype‐by‐genotype epistasis, where the morphology of the chimeras deviated significantly from the average morphology of the strains developed clonally. The significantly negative epistasis observed for allopatric pairings suggests that populations are diverging in their spore–stalk allocation behaviours, generating incompatibilities when they encounter one another. Our results demonstrate divergence in microbial social traits across geographically separated populations and demonstrate how quantification of genotype‐by‐genotype interactions can elucidate the trajectory of social trait evolution in nature.     

Evolutionarily stable stalk to spore ratio in cellular slime molds and the law of equalization in net incomes

The evolutionarily stable stalk ratio (ESSR) in the cellular slime molds is studied when the fruiting body is formed by multiple clones of various size. The survival probability of a spore cell is assumed to depend on the stalk ratio and the fruiting body size. ESSR is obtained as the non-co-operative equilibrium (Nash solution) that maximizes the fitness of each clone. The following two predictions are obtained: (1) the number of spore cells produced by each clone forming a fruiting body tends to be equalized, even if a variation in clone size exists. As a result, the larger clones do not necessarily enjoy higher fitness than the smaller ones. (2) The stalk ratio and the overall fitness of the fruiting body decrease as the genetic diversity in the fruiting body increases. A condition for the stalk to spore ratio to be invariant of overall fruiting body size is also investigated. Finally, "the law of equalization in net incomes" is proposed, extending result (1) into the broader range of resource allocation problems.     

The timing of cell-type-specific differentiation in Dictyostelium discoideum

We have used two-dimensional gel electrophoresis to identify over 30 proteins which are specific to one or other of the two cell types of Dictyostelium discoideum, either at the slug stage or in mature fruiting bodies. Our results support the idea that there is a continuous developmental program that begins in prespore cells at the hemispherical mound stage (10-12 hr) and results in spore differentiation (24 hr). Prestalk differentiation, on the other hand, appeared largely unrelated to stalk differentiation, which was first detectable at the onset of culmination (18 hr). We have also used this approach to study the differentiation of stalk-only mutants and have found that the cells can switch from spore to stalk differentiation as late as 2 hr before the end of the wild-type developmental program.     

A genetic melanotic neoplasm of Drosophila melanogaster

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.     

Evidence that the fertilization envelope blocks sperm entry in eggs of Xenopus laevis: interaction of sperm with isolated envelopes.

Single-celled myxamoebae undergo differentiation into either stalk cells or spore cells during a 24-hr period in Dictyostelium discoideum. This study employed ultramicrochemical techniques and enzymatic cycling to assess the presence of cell-specific events in spore and stalk cells. Freeze-dried sections of one organism were assayed in 0.1 μl of reaction mixture. This method was used to determine the extent of localization of trehalose in spore cells and stalk cells during development.Trehalose was low in the early stages of differentiation to about 20 hr when the level started to increase. In developing spore cells, the trehalose level increased sixfold during the last 5 hr of development. Likewise, the entire stalk contained trehalose when the stalk was first formed. At mature sorocarp, trehalose levels were the same in spores and the apex of the stalk. There was a decreasing gradient of trehalose down the stalk. The bottom one-fourth of the stalk was devoid of this disaccharide. Therefore, trehalose was degraded from an area of the stalk where it was localized earlier in development.The results of this investigation negate the assumption that trehalose is never present in the stalk. Although trehalose was found in spore cells, prestalk cells also contained high trehalose levels. The stalk cell-specific trehalose was not retained during differentiation, however, but was apparently degraded in the mature stalk cell.     

Cell specific activity of trehalose-6-phosphate synthetase during differentiation of Dictyostelium discoideum.

Trehalose-6-P synthetase activity was low at the beginning of the life cycle of Dictyostelium discoideum, reached maximum activity at 20 h, and decreased at late sorocarp. Enzyme activity in developing spore cells increased 10-fold during differentiation from myxamoebae (0 h) to the culmination stage (20 h) and decreased slightly at sorocarp (24 h). Activity was similar in spore cells at the apex of the stalk. The activities in the stalk cells were dependent upon their position in the developing stalk. There was a decreasing gradient of activity from the apex to the base of the stalk.     

Stability in by-product formation as a strain selection tool of Saccharomyces cerevisiae wine yeasts

A strain of Saccharomyces cerevisiae homozygous for different physiological and metabolic characters was inoculated into two grape musts and the stability of the characters was tested by isolating clones at different fermentation stages. A total of 60 cell-clones were collected and asci dissected from each, yielding a total of 1200 single spore cultures, which were then tested for the segregation of several genetically controlled traits. From the parental strain, 10 asci were dissected and the 40 single spore cultures obtained were used as controls. Micro-fermentations were performed with the 200 single spore cultures obtained from clones isolated at the end of Trebbiano and Aglianico must fermentations. The majority of these spore cultures produced amounts of the secondary compounds at the same level as the parental strain. The progeny of three clones from the Trebbiano fermentation exhibited a significant increase in the production of isoamyl alcohol, whereas the progeny of one clone from the Aglianico fermentation differed in the production of acetoin and amyl alcohols. The variability found in the levels of by-products can also affect the organoleptic properties of the final product. The introduction of the 'metabolic characteristics stability' as a selective index for industrial strains is advised.     

A new class of rapidly developing mutants in Dictyostelium discoideum: implications for cyclic AMP metabolism and cell differentiation

Rapidly developing (rde) mutants of Dictyostelium discoideum, in which cells precociously differentiated into stalk and spore cells without normal morphogenesis, were investigated genetically and biochemically. Genetic complementation tests demonstrated that the 16 rde mutants isolated could be classified into at least two groups (groups A and C) and that the first described rde mutant FR17 (D. R. Sonneborn, G. J. White, and M. Sussman, 1963, Dev. Biol. 7, 79-93) belongs to group A. Morphological studies revealed several differences in development and final morphology between group A and group C mutants. In group A mutants, the time required for cell differentiation from vegetative cells to aggregation competent cells is reduced, whereas the time required for spore and stalk cell differentiation following the completion of aggregation is shortened in group C mutants. This suggests that group C mutants represent a new class of rde mutants and that there exist at least two mechanisms involved in regulating the timing of development in D. discoideum. Measurements of cell-associated and extracellular phosphodiesterase activities, and intracellular and total cAMP levels revealed that cAMP metabolism in both groups is significantly altered during development. Group A mutants showed precocious and excessive production of phosphodiesterase and cAMP during the entire course of development; intracellular cAMP levels in group C mutants were extremely low, and spore and stalk cell differentiation occurred without an apparent increase in these levels. Thus, while cAMP metabolism is abnormal in all the rde mutants studied, there exist several distinct types of derangement, not necessarily involving the overproduction of cAMP.     

Cell type specific inhibition of cAMP phosphodiesterase activity during terminal differential in Dictyostelium discoideum

Cyclic AMP phosphodiesterase (PDE) activity reaches a peak during the aggregation stage of development where it functions to regulate extracellular levels of cAMP. During the subsequent differentiation of the two cell types at the culmination stage, the activity reappears but only in stalk cells. We found that extracts from the culmination stage contained PDE which could be activated by preincubation with Mg2+ and dithiothreitol (DTT), a treatment which is known to release an endogenous inhibitor from the aggregation stage enzyme. When the culmination stage extracts were subjected to chromatography on Biogel P300, two peaks of activity were eluted, PDE-I (Mr greater than 260,000) and PDE-II (Mr 100,000). Treatment of the fractions with Mg-DTT did not affect the low-molecular-weight enzyme but caused activation of the high-molecular-weight enzyme and the appearance of a third, intermediate form. Kinetic analysis of the two peaks revealed Km values for cAMP of 2 mM and 10 microM for PDE-I and PDE-II, respectively. We tested the possibility that these forms of the enzyme might be distributed differently in the two cell types by measuring the Km for cAMP and the effect of Mg-DTT treatment on isolated sections of stalk and spore cells. The spore sections contained a high Km form of the enzyme (0.3 mM) which was activated by preincubation with Mg . DTT whereas stalk sections contained a low Km form (3 microM) which was not affected by the activation treatment. We conclude that both cell types contain enzyme protein and that the apparent localization of PDE activity in stalk cells is due to the inhibition of activity in spore cells.     

A stalk-specific localization of trehalase activity in Dictyostelium discoideum.

By utilizing ultra-microtechniques, trehalase activity was followed in specific cell types during the differentiation cycle of Dictyostelium discoideum. When whole organisms were assayed, trehalase activity was found to be high in the early stages of differentiation, decreased to its lowest point at 14 h, and then increased at the end of the cycle. By microdissection of freeze-dried individuals, the activity of trehalase could be followed during the migration of pre-stalk and pre-spore cells. No activity was observed at any stage of spore cell development, whereas stalk cells showed a rapid increase in activity upon maturation. An increasing gradient of activity was found from the apex of the stalk toward the base. This localization of trehalase in stalk cells resolves some contradictory results in the literature concerning the role of the enzyme during differentiation.     

The induction of spore cells in vitro by membranes of Dictyostelium discoideum

A mutant of Dictyostelium discoideum, HM18, will differentiate into both stalk and spore cells when plated at high cell density (10⁵ cells/cm²) as a monolayer on non-nutrient agar containing 5 mM cAMP [6]. At low cell density (10³ cells/cm²) neither stalk nor spore cells are produced, but the addition of a cytosol fraction leads to stalk cell formation, and the addition of a membrane fraction leads to spore cell formation. The spore cell-inducing activity of the cell membranes is developmentally regulated; it is first detectable during late aggregation and increases to a maximum level in the pseudoplasmodial stage of development. The activity is sensitive to proteolysis and insensitive to periodate treatment. It is partially inactivated by incubation at 100 °C for 5 min. Variable amounts of the activity can be removed from the membrane by washing, suggesting that at least part of the activity is loosely membrane-bound. Activity is enriched in plasma membrane fractions, suggesting that the inducing factor is located at the cell surface. It is possible that the membranes are replacing a cell-cell contact requirement for spore formation.     

当归不同生长时期根际丛枝真菌分布及土壤养分和酶活性的动态变化

【目的】探讨当归不同生长时期丛枝菌根真菌(Arbuscular mycorrhizal fungi,AMF)的分布及土壤养分和土壤酶活的变化,以期了解当归不同生长时期AMF与土壤养分和土壤酶活的关系,为AMF在当归种植的应用提供理论依据。【方法】在当归不同生长时期分别采集根际土壤样品,测定其土壤养分、土壤酶活、AMF孢子密度和球囊霉素等因子,分析当归不同生长时期根际土壤AMF孢子密度、土壤养分和土壤酶活等指标的动态变化和相关性。【结果】随着当归生育期的完成,根际土壤AMF孢子密度先降低后持续升高;易提取球囊霉素(Easily extractable glomalin,EEG)和总球囊霉素(Total glomalin,TG)平稳增加,而且EET与TG和脲酶活性呈显著正相关(P0.01),EET和TG与根际土壤有机质和全氮含量以及酸性和中性磷酸酶活性均显著正相关(P0.05);根际土壤有机质和全氮表现为增加的总趋势;有效磷含量呈现为生长前期保持不变、中期显著降低、后期逐渐升高的趋势,而有效钾含量先逐渐增加,生长中后期显著降低的趋势;根际土壤酸性和中性磷酸酶活性均呈现逐渐增加趋势,而脲酶活性表现为生长前期逐渐增加,中后期显著降低;p H值在当归不同生长时期有所波动。相关性分析结果表明,AMF孢子密度与土壤酸性磷酸酶酶活性呈显著正相关,而酸性磷酸酶酶活性与根际土壤全氮、有机质、易提取球囊霉素和总球囊霉素呈显著正相关,与有效磷和有效钾含量呈显著负相关,表明AMF对根际土壤养分和酶活性具有一定的调节作用;主成分分析结果表明,不同生长时期是影响当归根际土壤理化指标的主要因素。【结论】AMF孢子密度在当归根际的动态变化一定程度上反映出了AMF分泌球囊霉素的能力,以及球囊霉素对增加根际土壤碳氮储存的贡献,同时表明球囊霉素影响了当归根际土壤酶活性和其它养分的代谢循环,对改良土壤和促进当归生长发挥重要的作用。     

ATP, trehalose, glucose and ammonium ion localization in the two cell types of Dictyostelium discoideum

Ultra-microfluorometric techniques were adapted to follow several compounds related to energy metabolism through the developmental cycle of Dictyostelium discoideum. Each compound (ATP, trehalose, glucose, and ammonium ion) was found to be present in stalk and/or spore cells. The accumulation of NH4+ was interpreted as an indication of protein degradation, a source of energy in this organism. During the early stages of differentiation NH4+ was localized only in prestalk cells. However, it accumulated in spore cells during culmination such that levels were comparable in the two cell types by the end of development. Trehalose, an energy source for germinating spores, was found in both cell types but was preferentially degraded in stalk cells late in development. Glucose, the degradation product of trehalose, was localized in prestalk cells and varied inversely with trehalose levels. ATP was not localized in a specific cell type during development. However, ATP declined in stalk cells at an earlier stage of development.     

Sporocarp Ulrastructure and Development in the Protostelid Cavostelium apophysatum1

Ultrastructural observations on the sporocarp of the protostelid Cavostelium apophysatum are presented, including information about the mechanism of sporocarp development. The onset of sporogenesis is marked by cessation of trophic activity and the secretion of a protective sheath. The protoplast gradually molds itself into two distinct zones approximating the shape of the final sporocarp–a hyaloplasmic pre-stalk zone and a globose incipient spore zone. The protoplast in the stalk zone deposits a fibrillar and amorphous stalk acropetally as the stalk protoplast moves into the incipient spore region. The last portion of the stalk to be deposited is the apophysis, a hollow cup-like structure at the stalk apex. Spore wall deposition begins as the stalk nears completion. The wall consists of a single electron-dense layer ornamented with hollow cones and solid projections. The mechanism of sporocarp development in C. apophysatum is compared to the developmental patterns of the protostelids Planoprotostelium aurantium and Schizoplasmodiopsis amoeboidea.     

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.     

Dictyostelium discoideum gene family contains a long internal amino acid repeat

Two different cDNA clones denoted pTO270-6 and pTO270-11 represent two mRNAs that are developmentally regulated during spore germination in Dictyostelium discoideum. The respective mRNAs are found only during early germination and are not present in other stages of growth or multicellular development. Four different genomic clones that hybridize to sequences that are common to both of the 270 cDNA clones were isolated from Dictyostelium libraries and sequenced. Two are the genes for the two cDNAs, and the other two represent genes that do not seem to be transcribed. All four genomic sequences possess a very unusual internal feature in the deduced protein sequences composed of a monotonous repeat of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The deduced protein corresponding to the 270-6 gene is very similar to avocado (Persea americana) cellulase. Since cellulose in the spore wall has to be digested during spore germination this suggests that this protein may function as an endo-(1,4)-beta-D-glucanase during germination.     

Cheating does not explain selective differences at high and low relatedness in a social amoeba

Background  

Altruism can be favored by high relatedness among interactants. We tested the effect of relatedness in experimental populations of the social amoeba Dictyostelium discoideum, where altruism occurs in a starvation-induced social stage when some amoebae die to form a stalk that lifts the fertile spores above the soil facilitating dispersal. The single cells that aggregate during the social stage can be genetically diverse, which can lead to conflict over spore and stalk allocation. We mixed eight genetically distinct wild isolates and maintained twelve replicated populations at a high and a low relatedness treatment. After one and ten social generations we assessed the strain composition of the populations. We expected that some strains would be out-competed in both treatments. In addition, we expected that low relatedness might allow the persistence of social cheaters as it provides opportunity to exploit other strains.     

Separation of Dictyostelium discoideum cells into density classes throughout their development and their relationship to the later cell types

This paper describes a fast, non-destructive method for the separation of large quantities of Dictyostelium discoideum cells into density classes at all stages of development. The cells were separated by low-speed centrifugation on preformed, linear Percoll density gradients. On these gradients, cells at all developmental stages showed a unimodal variation in density and this variation in density rapidly increased during the first hours of development. The density was affected by the amount of salt present in the gradient medium, which suggests that it is regulated by a permeability property of the cells. Slug cells showed a unimodal variation in density and did not form bands corresponding to the cell types. However, were able to isolate density fractions which showed a good enrichment of prespore and prestalk cells: 95% and 90%, respectively. Preaggregation cells separated on density gradients yielded fractions which contained different amounts of three developmentally regulated enzymes. Hence, cells at this stage are already heterogeneous in their enzymatic content. Sorting experiments showed a strong correlation between density and developmental fate; the least dense (light) cells preferentially became prestalk cells, and the dense (heavy) cells became prespore cells. This was found for cells at all developmental stages; even vegetative-stage cells showed considerable heterogeneity with regard to density, which was related to their developmental fate. The light cells become prestalk cells, and the heavy cells become prespore cells. Vegetative cells from the various density fractions differed in their DNA content and temporal onset of mitotic activity when resuspended in medium. Therefore, we suggest that the separation of vegetative cells on density gradients results in a separation of cells into cell-cycle phases. Hence, there appear to be cell-cycle-linked differences among vegetative cells, which bias their differentiation towards either the spore or stalk pathway.     

Trishanku, a novel regulator of cell-type stability and morphogenesis in Dictyostelium discoideum

We have identified a novel gene, trishanku (triA), by random insertional mutagenesis of Dictyostelium discoideum. TriA is a Broad complex Tramtrack bric-a-brac domain-containing protein that is expressed strongly during the late G2 phase of cell cycle and in presumptive spore (prespore (psp)) cells. Disrupting triA destabilizes cell fate and reduces aggregate size; the fruiting body has a thick stalk, a lowered spore: stalk ratio, a sub-terminal spore mass and small, rounded spores. These changes revert when the wild-type triA gene is re-expressed under a constitutive or a psp-specific promoter. By using short- and long-lived reporter proteins, we show that in triA(-) slugs the prestalk (pst)/psp proportion is normal, but that there is inappropriate transdifferentiation between the two cell types. During culmination, regardless of their current fate, all cells with a history of pst gene expression contribute to the stalk, which could account for the altered cell-type proportion in the mutant.