The development of the relaying competence in Dictyostelium discoideum.

The fraction, X2, of a population of D. discoideum cells competent to relay an aggregative signal has been measured as a function of time, t, spent in interphase. X2(t) is less than 0.I up to 5 h, increases slowly to 0.4 by 8 h 20 min, and saturates at I by 10 h. The hypothesis that cellular interactions might enhance X2(t) was tested by mixing populations of cells set into interphase at different times. No interactions were found. External stimulation did not affect X2(t).     

The phosphorylation of membranal proteins in Dictyostelium discoideum during development

The pattern of membranal phosphoproteins in Dictyostelium discoideum changes during development (D. S. Coffman, B. H. Leichtling, and H. V. Rickenberg, 1981, J. Supramol. Struct. Cell. Biochem. 15, 369–385). Phosphorylation of six membranal proteins occurred concomitantly with their synthesis. Cyclic AMP stimulated the precocious synthesis of a phosphoprotein, of molecular weight 80,000, which corresponds to contact sites A. Phosphoserine was the only phosphorylated amino acid found in the five phosphoproteins examined. In at least two phosphoproteins, that corresponding to contact sites A and a phosphoprotein of molecular weight 64,000, the phosphate moiety did not turn over.     

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.     

Mutations causing rapid development of Dictyostelium discoideum

A mutation affecting the speed of slime mold development has been genetically analyzed. Strain FR17 carries a recessive mutation on linkage group IV. A selection procedure for isolating more mutants of this type has been developed and new mutations have been tested for complementation. The aberrant morphology of these strains can be partially corrected by development in the presence of glucose.     

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.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

The regulation of membrane fusion during sexual development in Dictyostelium discoideum

During the first 24 h of sexual development in Dictyostelium discoideum, three sequential events of membrane fusion occur: gamete fusion, pronuclear fusion, and phagocytosis. The early events of sexual development are regulated by a diverse group of endogenous molecules: (i) a volatile sexual pheromone, (ii) a protein cell fusion inducing factor (CFIF), (iii) a low molecular weight autoinhibitor, (iv) and cyclic AMP. CFIC enhances cell fusion while the autoinhibitor and cyclic AMP both inhibit the event. Both extracellular and intracellular calcium ions are essential for cell and pronuclear fusion. Pharmacological analyses show that the intracellular functions of the divalent cation in these processes are mediated by calmodulin. The autoinhibitor appears to function by inhibiting calmodulin activity. Glucose, mannose, and N-acetylglucosamine containing glycoproteins have been shown to function in both cell fusion and phagocytosis. The interplay between all of these diverse molecules is examined and a review of all of the recent literature is presented.     

Phagocytic specificity during sexual development in Dictyostelium discoideum

During the sexual cycle of Dictyostelium discoideum, zygote giant cells develop and serve as foci for further development by chemoattracting and cannibalizing hundreds of local amoebae. Previous work has shown that the phagocytic process bears similarities to and differences from asexual endocytosis. In the present study, sexual phagocytosis in D. discoideum was found to be species and developmental stage specific. It was inhibited selectively by glucose and concanavalin A. Although a partial, inhibitory effect of mannose on phagocytosis was not statistically significant, alpha-methylmannosamine, like alpha-methyl-glucose, significantly restored the phagocytic competence of giant cells treated with concanavalin A. Other sugars (N-acetyl-glucosamine, N-acetylgalactosamine, and galactose) and lectins (wheat germ agglutinin, Ulex europus type I, and Ricinis communis agglutinin type I) had no significant effect on sexual phagocytosis. Together these data indicate that a glucose-type receptor is involved in selective uptake of D. discoideum amoebae by giant cells.     

Pheromones do regulate sexual development in Dictyostelium discoideum

Based on negative data, Douglas et al refuted earlier work by the O’Day laboratory on the role of pheromones in the sexual development of Dictyostelium discoideum. This letter addresses some of the issues with their study.     

Changes in phospholipid composition during the development of Dictyostelium discoideum

The phospholipid composition of Dictyostelium discoideum cells was determined at various stages of development by two-dimensional, thin-layer chromatography and reaction thin-layer chromatography. Major phospholipids of D. discoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride and choline phosphoglyceride. Ethanolamine phosphoglyceride and choline phosphoglyceride were found as their plasmalogen forms at 45–58 and 10–24%, respectively. There were no qualitative changes in phospholipid composition during the development, but quantitative changes did occur. The relative content of ethanolamine phosphoglyceride in the total phospholipids gradually decreased from 60% at the vegetative stage to 44% at the 1-day-sorocarp stage. In contrast, choline phosphoglyceride gradually increased from 27% at the vegetative stage to 48% at the preculmination stage, and then gradually decreased to 43% during the culmination. The decrease in ethanolamine phosphoglyceride content during the middle and late development was due mainly to the decreased amount of its plasmalogen form but the increase of choline phosphoglyceride was independent of quantitative changes of its plasmalogen form. Other minor components of phospholipid did not show significant changes in their levels. The causes of these changes in contents of ethanolamine phosphoglyceride and choline phosphoglyceride were examined by label and chase experiments with [³H]ethanolamine and [¹⁴C]choline. It seems that one-third to one-half of the increased amount of choline phosphoglyceride was due to stepwise methylation of ethanolamine phosphoglyceride, and the remaining two-thirds to one-half was caused by de novo synthesis of choline phosphoglyceride from CDP-choline and diglyceride.     

How does environment modify the spatial structure of Dictyostelium discoideum population

The transformation of the spatial structure of a Dictyostelium discoideum population in response to environmental changes induced by this population was investigated. A comparative analysis of the spatial and temporal characteristics of the D. discoideum colony is given for two cases: (a) when the colony is cultivated on a bacterial lawn, i.e. under conditions close to natural, and (b) in the absence of the bacterial lawn when the colony grows on the nutrient substrate enriched with folic acid. It is shown that the environmental changes induced by cell metabolism modify the spatial structure of the D. discoideum population first, the rate of population propagation falls drastically, which correlates with a decrease in the substrate pH; second, the spatial redistribution of the D. discoideum cell density correlates with the redistribution of folic acid in the substrate. The mechanism of the environment impact on the D. discoideum colony transformation is discussed.     

Temporal and spatial expression of ammonium transporter genes during growth and development of Dictyostelium discoideum

Ammonia is an important signaling molecule involved in the regulation of development in Dictyostelium. During aggregation, ammonia gradients are established, and the ammonia concentration in the immediate environment or within a particular cell throughout development may vary. This is due to the rate of cellular ammonia production, its rate of loss by evaporation to the atmosphere or by diffusion into the substratum, and perhaps to cellular transport by ammonium transporters (AMTs). Recent efforts in genome and cDNA sequencing have identified three ammonium transporters in Dictyostelium. In addition to physically altering the levels of ammonia within cells, AMTs also may play a role in ammonia signaling. As an initial step in identifying such a function, the temporal and spatial expression of the three amt genes is examined. RT-PCR demonstrates that each of the three amt mRNAs is present and relatively constant throughout growth and development. The spatial expression of these three amt genes is examined during multiple stages of Dictyostelium development using in situ hybridization. A distinct and dynamic pattern of expression is seen for the three genes. In general, amtA is expressed heavily in pre-stalk cells in a dynamic way, while amtB and amtC are expressed in pre-spore regions consistently throughout development. AmtC also is expressed in the most anterior tip of fingers and slugs, corresponding to cells that mediate ammonia's effect on the choice between slug migration and culmination. Indeed, amtC null cells have a slugger phenotype, suggesting AmtC functions in the signaling pathway underlying the mechanics of this choice.     

The regulation of''early'' enzymes during the development and dedifferentiation of Dictyostelium discoideum

The specific activities of the enzymes alpha-mannosidase and N-acetylglucosaminidase increase immediately after the initiation of the development of bacterially grown cell cultures of Dictyostelium discoideum. The regulation of these two enzymes was found to be dissociable in the developmental timer mutant, FM-1, which aggregates 4.5 h earlier than wild-type cells due to the absence of the first rate-limiting component of the preaggregative period. The increase in alpha-mannosidase activity occurs in the absence of the first rate-limiting component, but the increase in N-acetylglucosaminidase activity does not. These results indicate the following: (1) the increase in the specific activity of alpha-mannosidase is not related to the timing of subsequent developmental stages; (2) the increase in the specific activity of N-acetylglucosaminidase is not necessary for the subsequent developmental program; and (3) either the increase in the specific activity of N-acetylglucosaminidase is dependent upon progress through the first rate-limiting component, or the increase in this enzyme activity and the first rate-limiting component are both dependent upon an early event for which FM-1 is defective. In addition to early development, we monitored the two enzyme activities during dedifferentiation. The results demonstrate that there is no difference between dedifferentiating wild-type cells and dedifferentiation-defective mutant HI-4 cells. Changes in enzyme specific activity accompanying dedifferentiation are dependent upon the composition of the dedifferentiation-inducing media and are consistent with the levels of these enzymes observed in cells growing in the different nutrient media.     

Inhibition of development in Dictyostelium discoideum by sugars.

Sugars such as glucose, maltose, and trehalose, which are metabolized by Dictyostelium discoideum and which enhance vegetative growth, inhibit the development of the slime mold at concentrations which stimulate growth maximally. They block the acquisition of aggregation competence as well as aggregation. The same sugars also inhibit the degradation of preformed glycogen ribonucleic acid, and protein, which is characteristic of development and which occurs when the amoebas are starved by incubation in dilute phosphate buffer.     

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.     

Heat shock protein 90 regulates development in Dictyostelium discoideum

Cytosolic heat shock protein 90 (Hsp90) has been implicated in diverse biological processes such as protein folding, cell cycle control, signal transduction, development, and morphological evolution. Model systems available for studying Hsp90 function either allow ease of manipulation for biochemical studies or facilitate a phenomenological study of its role in influencing phenotype. In this work, we have explored the use of the cellular slime mold Dictyostelium discoideum to examine cellular functions of Hsp90 in relation to its multicellular development. In addition to cloning, purification, biochemical characterization, and examination of its crystal structure, our studies, using a pharmacological inhibitor of Hsp90, demonstrate a role for the cytoplasmic isoform (HspD) in D. discoideum development. Inhibition of HspD function using geldanamycin (GA) resulted in delayed aggregation and arrest of D. discoideum development at the ‘mound’ stage. Crystal structure of the amino-terminal domain of HspD showed a binding pocket similar to that described for yeast Hsp90. Fluorescence spectroscopy, as well as GA-coupled beads affinity pulldown, confirmed a specific interaction between HspD and GA. The results presented here provide an important insight into the function of HspD in D. discoideum development and emphasize the potential of the cellular slime mold to serve as an effective model for studying the many roles of Hsp90 at cellular and organismal levels.