Rates of accumulation of glycosidase activities during growth and differentiation of Dictyostelium discoideum.

1. The rates of accumulation (enzyme units/h per 10(8) cells) of a number of glycosidase activities were studied in Dictyostelium discoideum cells during the growth and differentiation phases of this organism's life cycle. 2. The rates of accumulation of the enzymes beta-N-acetylglucosaminidase, alpha-glucosidase and beta-galactosidase remain unchanged during the growth and early differentiation phases. 3. The considerable changes in specific activity of the enzymes which occur in the early differentiation phase are due to the massive loss of total cellular protein which occurs at this time. 4. Significant alterations can occur in the rates of accumulation of alpha-mannosidase during both the growth and differentiation phases, and since, on the onset of differentiation, beta-glucosidase activity is excreted and degraded, the rate of accumulation of this enzyme differs in the growth and differentiation phases. 5. The characteristic rates of accumulation of all these glycosidases change markedly with changes in the growth conditions of the myxamoebae, and thus these rates of synthesis must be regulated independently; however, addition of cyclic AMP to the growth medium has no effect on them.     

Half-lives of messenger RNA species during growth and differentiation of Dictyostelium discoideum

The half-lives of functional messenger RNAs were determined by a method employing the drugs actinomycin D and daunomycin for the inhibition of mRNA synthesis; the activity of extracted mRNAs was determined by an in vitro translation assay. Several controls indicated that this method yielded reliable values for mRNA half-lives; in particular, the declining rate of protein synthesis in the presence of the drugs is due predominantly to the decay of translatable mRNA. This method was used to determine the half-lives of two specific mRNAs—encoding actin and a protein of MW 51,000—as well as that of total cytoplasmic mRNA activity during growth and at several times in differentiation. The half-lives of at least these two mRNAs were shown to be distinctly different from that of the total mRNA population—about 4 hr. However, no significant change in any of these half-lives was observed between growing and developing cells. Therefore wholesale alterations in the degradation rates of total and at least specific messages do not appear to play a role in the regulation of gene expression during Dictyostelium development.     

Extracellular cyclic AMP-phosphodiesterase accelerates differentiation in Dictyostelium discoideum.

Extracellular cyclic AMP-phosphodiesterase accelerates the development of aggregation competence in Dictyostelium discoideum when present during the preaggregation stage. The effect on development appears to depend only on hydrolysis of extracellular cyclic AMP and not on other properties of the phosphodiesterase molecule. Extracellular cyclic AMP-phosphodiesterase, as a promoter of differentiation, acts mainly throughout the first half of interphase. Our evidence supports the proposal that cyclic AMP oscillations control the rate and possibly the initiation of development. Since extracellular cyclic AMP-phosphodiesterase acts from the beginning of interphase cyclic AMP oscillations may also occur from early interphase, at least in the presence of this enzyme. This would imply that the cyclic AMP oscillator is a determinant, but not a product, of the developmental programme.     

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.     

Changes in the basic nuclear proteins of the cellular slime mould Dictyostelium discoideum during growth and differentiation.

Nuclei isolated from myxamoebae and differentiating cells (slug stage) of Dictyostelium discoideum contain similar ratios of DNA, RNA and protein (1:8:29) and acid soluble proteins present in amounts equal in weight to the nuclear DNA can be extracted therefrom. On urea polyacrylamide gels these basic proteins were shown to be very similar with the exception of one band, present in the myxamoebae, which was virtually absent from the differentiating cells.     

Rates of degradation and synthesis of glycosidases de novo during growth and differentiation of Dictyostelium discoideum.

1. Injection of a purified preparation of beta-N-acetylglucosaminidase from the spent growth medium of myxamoebae of Dictyostelium discoideum into rabbits gave rise to an antibody preparation containing both anti-alpha-glucosidase and anti-beta-acetylglucosaminidase activities. 2. These two activities were shown to reside in different immunoglobulin molecules and it was concluded that the beta-N-acetylglucosaminidase preparation contained trace amounts of highly antigenic alpha-glucosidase. 3. A single precipitin band having beta-N-acetylglucosaminidase activity was formed in Ouchterlony plates when this antibody preparation was tested against extracts obtained from differentiated cells or from myxamoebae grown either axenically or on bacteria. 4. The antibody preparation was used to show that both beta-N-acetylglucosaminidase and alpha-glucosidase molecules are synthesized de novo from isotopically labelled amino acids during both the growth and differentiation phases of the life cycle and to show that neither of these proteins is significantly degraded during the growth phase or during the first 9h of differentiation. 5. The rates of accumulation of these assayable enzyme activities are thus equal to their rates of synthesis during growth and early differentiation. 6. The factors regulating cellular enzyme activity during the life cycle of D. discoideum are discussed.     

Acquisition process of differentiation competence in Dictyostelium discoideum

It was previously shown [K. Okamoto, J. Gen. Microbiol. 127, 301 (1981)] that Dictyostelium discoideum cells dissociated from early aggregates, but not aggregation competent cells obtained in a suspension culture, undergo prespore differentiation, when transferred into a medium containing glucose, albumin, and cAMP. Therefore, the former, but not the latter, is considered to have been acquired "differentiation competence." In the present work, the requirements for cells to acquire the differentiation competence are investigated with D. discoideum NC4 strain. On solid substratum, the incubation above a threshold density is absolutely required for this process, while cell aggregation itself is not essential. In suspension cultures, the competence is acquired only under hypertonic conditions. Inhibition of protein synthesis or depletion of cAMP does not affect the acquisition process of the competence. The requirement of hypertonic treatment was also investigated with several other D. discoideum strains.     

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.     

Cell-type-specific responsiveness to cAMP in cell differentiation of Dictyostelium discoideum.

In Dictyostelium discoideum, both prespore and prestalk differentiation require extracellular cAMP. We investigated the difference in inducibility of the two cell types by cAMP. Previous studies indicate that cAMP added in the early stage of development inhibits prespore differentiation, and this was confirmed using three species of prespore specific mRNAs. By contrast, early treatment with cAMP did not inhibit, but induced the expression of prestalk-specific mRNA. These results indicate that differentiation pathways of the two cell types have different processes in the early stage of development.     

Effect of arsenic on cell growth of the cellular slime mould, Dictyostelium discoideum

The growing D. discoideum cells were killed in a dose-dependent manner when exposed to 100 and 140 ppm of arsenic (As2O3) at mid-log phase for 20 min. Reduced plaque sizes and changed cell and colony morphologies were observed in the treated cells. Endocytotic functions (both phagocytosis and pinocytosis) were also inhibited in the treated cells. Arsenic treated cell showed a lower DNA and protein synthetic activities. These findings are discussed in relation to known mechanism of action of the heavy metal on growth-related cellular functions.     

Protocols for growth and development of Dictyostelium discoideum

Dictyostelium discoideum, a unicellular organism capable of developing into a multicellular structure, is a powerful model system to study a variety of biological processes. Because it is inexpensive and relatively easy to grow, Dictyostelium is also frequently used in teaching laboratories. Here we describe conditions for successfully growing and developing Dictyostelium cells and methods for long-term storage of Dictyostelium amoebae and spores.     

The synthesis and degradation of ras-related gene products during growth and differentiation in Dictyostelium discoideum

A Dictyostelium discoideum protein with an Mr of 23,000 (p23dd-ras) is structurally related to the mammalian proto-oncogene ras-gene product, p21ras, and is specifically precipitated from cell-free extracts of D. discoideum by the Y13-259 monoclonal antibody against p21ras. p23dd-ras was degraded at rates that were very similar to those observed for total protein during both growth and differentiation, suggesting that the previously reported decline in p23dd-ras levels during differentiation is due to a change in the rate of synthesis rather than a change in the rate of degradation. p23dd-ras synthesis did not decrease immediately after the initiation of differentiation, but rather its rate of synthesis increased for the first 1-2 h, suggesting that p23dd-ras is not rapidly down-regulated in response to nutrient deprivation. There were differences in the extent of p23dd-ras turnover during the differentiation of the three tested strains, A-3, NC4, and V12-M2. The relative level of p23dd-ras dropped most rapidly in V12-M2, which may reflect the slightly faster differentiation process exhibited by this strain. In all three strains, very little p23dd-ras was present by the end of the differentiation process. A second protein with an Mr of 24,000 (p24dd-ras) was also immunoprecipitated using the Y13-259 antibody. The amount of p24dd-ras was small or undetectable in vegetative cells, but relatively larger amounts of p24dd-ras were synthesized in pseudoplasmodial cells. We found no evidence to suggest that p24dd-ras is a precursor of p23dd-ras.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of growth factors in Dictyostelium discoideum

Growth factors and their binding proteins are important proteins regulating mammalian cell proliferation and differentiation so there is considerable interest in producing them as recombinant proteins, especially in hosts that do not already produce a complex mixture of growth factors. Many growth factors require post-translational modifications making them unsuitable for production in Escherichia coli or other prokaryotes. Since several expression vector systems have been recently developed for foreign protein production in the cellular slime mould, Dictyostelium discoideum, we attempted to use two of these systems to express human insulin-like growth factor binding protein 6 (hIGFBP6) and bovine beta-cellulin (bBTC) as secreted proteins. Although both proteins were successfully produced in stably transformed amoebae, no secretion was detected in spite of several attempts to facilitate this occurring.     

盘基网柄菌细胞分化和凋亡的形态特征

本文用透射电镜和DAPI荧光染色法研究了盘基网柄菌(Dictyosteliumdiscoideum)细胞分化和柄细胞的凋亡特征,结果显示:细胞丘中绝大部分细胞的线粒体内出现一小空泡,随着发育进程,空泡逐渐增大,线粒体的嵴随之变少,直至线粒体完全空泡化,最后形成单层膜的空泡。据此我们推测前孢子细胞特有的空泡来源于线粒体,并且这种细胞器水平上的内自噬现象与前孢子细胞分化密切相关。在前柄细胞分化阶段,前柄细胞中出现数个自噬泡,最初吞噬的线粒体嵴结构完整;随着前柄细胞进一步分化,部分线粒体内出现类似于前孢子细胞中的内自噬现象,并且自噬泡只吞噬这种线粒体。在凋亡后期,细胞核内核仁消失,染色体固缩形成高电子密度斑块,自噬泡采用与细胞核膜融合的方式来完成核的清除,最后柄细胞完全空泡化且包被一层纤维素壁。作者认为前柄细胞凋亡过程实质上是一种分化过程,所以有其鲜明特点:细胞出现自噬泡,标志着凋亡开始,用自噬而不是凋亡小体来清除胞内各种细胞器,直到分化最后阶段才清除细胞核和形成纤维素壁。这些特点不仅是前柄细胞凋亡的形态学指标,也和细胞发育和分化相关。     

Adenylyl cyclase expression and regulation during the differentiation of Dictyostelium discoideum

Cyclic AMP metabolism is essential for the survival of the social amoebae Dictyostelium discoideum. Three distinct adenylyl cyclases are expressed and required for the normal development of this simple eukaryote. The adenylyl cyclase expressed during aggregation, ACA, is related to the mammalian and Drosophila G protein-coupled enzymes and is responsible for the synthesis of cAMP that is required for cell-cell signaling in early development. ACB harbors histidine kinase and response-regulator domains and is required for terminal differentiation. Finally, the adenylyl cyclase expressed during germination, ACG, acts as an osmosensor and is involved in controlling spore germination. Together, these enzymes generate the various levels of cAMP that are required for D. discoideum to transition from uni- to multi-cellularity. This review will highlight the properties of these enzymes and describe the signaling cascades that lead to their activation.     

Cell growth and morphology of Dictyostelium discoideum in space environment.

Two strains of cellular slime mold Dictyostelium discoideum, a radiation-sensitive mutant and the parental wild-type strain, were used to investigate the effects of microgravity and/or cosmic radiation on their morphology through the whole life span from spores to fruiting bodies for about 7 days in space shuttle of NASA. We found almost no effect of space environment on amoeba cell growth in both strains. It was also observed that almost the same number and shape of fruiting bodies in space compared to the control experiments on earth. These results suggest that there is little effect of microgravity and space radiation on germination, cell aggregation, cell differentiation and cell morphology in the cellular slime mold.