Endocytic transit inDictyostelium discoideum

Summary The cells ofDictyostelium discoideum are soil amoebae with a simple endocytic pathway: Particles or fluid are taken up at the plasma membrane in a process dependent on the actin cytoskeleton. After rapid acidification and subsequent neutralisation of the food vacuoles during which breakdown of the contents occurs, indigestible remnants are exocytosed. This tight coupling between endocytosis and exocytosis is thought to maintain membrane homeostasis. In spite of the apparent overall difference between the endocytic pathways of mammalian cells andD. discoideum, conserved proteins are involved in individual steps of endocytic transport, possibly indicating that in mammalian cells it is only the routing of marker that has evolved from a simple transit to a complex, branched pathway.     

Oxygen and differentiation inDictyostelium discoideum

We showed previously that ifDictyostelium discoideum cells are sucked up into a small glass capillary with air at one end and plugged with mineral oil at the other, a sharp band of fast moving cells with prestalk characteristics formed within a minute at the air end of the cell mass. We now demonstrate that oxygen inside the capillary is responsible for the initiation and positioning of the sharp division line between prestalk-like and prespore-like cells, and that the length of the prestalk zone is regulated by the oxygen concentration. Our results are compared to a quantitative theory, showing good agreement with the experiments. We also discuss the relevance of these observations to the differentiation of prestalk and prespore zones in normal slugs and the origins of polarity in this organism.     

Developmental age-related cell sorting inDictyostelium discoideum

Summary The sorting behavior of mixtures ofD. discoideum cells which had been developed for different lengths of time was examined. Cells developed for 4 and 8 h were mixed together and allowed to form slugs. Within the slugs, 8 h cells sorted to the anterior prestalk region while 4 h cells sorted to the posterior prespore region. These results indicate that the more developed a cell is, the more likely it is to become part of the prestalk zone in the slug. They are also consistent with the differential adhesion and chemotaxis hypotheses as a mechanism for cell sorting since cells become more adhesive and chemotactically responsive as development proceeds.     

Changes in β-Galactosidase activity during differentiation and dedifferentiation inDictyostelium discoideum

β-Galactosidase (EC 3.2.1.23) ofDictyostelium discoideum was investigated for its properties and activity during differentiation and dedifferentiation. β-Galactosidase of this organism had a pH optimum at 3.5. The specific activity of this enzyme was increased gradually from the time of initiation of differentiation and reached a peak at the aggregation stage. Then the activity of the enzyme showed a slight decrease followed by a further increase and reached a maximum at early culmination. During dedifferentiation of cells disaggregated from a slug, the activity of the enzyme was increased, reached a maximum after 3 hr of incubation and then decreased nearly to the original level of activity after completion of dedifferentiation. This increase in the enzyme activity coincided with decomposition of acid mucopolysaccharide contained in the prespore specific vacuoles, and both processes were sensitive to cycloheximide. No increase in the activity of acetylglucosaminidase (EC 3.2.1.30), another lysosomal enzyme, was observed during the process. Possible roles of β-galactosidase in cell type conversion as well as in dedifferentiation of the prespore cell were discussed.     

The determination of spatial pattern inDictyostelium discoideum

Free-living amoebae of the cellular slime mouldDictyostelium discoideum aggregate when starved and give rise to a long and thin multicellular structure, the slug. The slug resembles a metazoan embryo, and as with other embryos it is possible to specify a fate map. In the case ofDictyostelium discoideum the map is especially simple: cells in the anterior fifth of the slug die and form a stalk while the majority of those in the posterior differentiate into spores. The genesis of this anterior-posterior distinction is the subject of our review. In particular, we ask: what are the relative roles of individual pre-aggregative predispositions and post-aggregative position in determining cell fate? We review the literature on the subject and conclude that both factors are important. Variations in nutritional status, or in cell cycle phase at starvation, can bias the probability that an amoeba differentiates into a stalk cell or a spore. On the other hand, isolates, or slug fragments, consisting of only prestalk cells or only prespore cells can regulate so as to result in a normal range of both cell types. We identify three levels of control, each being responsible for guiding patterning in normal development: (i) ‘coin tossing’, whereby a cell autonomously exhibits a preference for developing along either the stalk or the spore pathway with relative probabilities that can be influenced by the environment; (ii) ‘chemical kinetics’, whereby prestalk and prespore cells originate from undifferentiated amoebae on a probabilistic basis but, having originated, interact (e.g. via positive and negative feedbacks), and the interaction influences the possibility of conversion of one cell type into the other; and (iii) ‘positional information’, in which the spatial distribution of morphogens in the slug influences the pathway of differentiation. In the case of possibilities (i) and (ii), sorting out of like cell types leads to the final spatial pattern. In the case of possibility (iii), the pattern arisesin situ     

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.     

Environmental factors inducing sexual development inDictyostelium discoideum

In the heterothallic strains NC4 and HM1 ofDictyostelium discoideum, sexual development is initiated by the formation of diploid zygotic giant cells produced through the fusion of these two opposite mating-type haploid cells. For sexual cell fusion, amoeboid cells must first acquire fusion competence, which requires culture under certain environmental conditions, such as darkness, excessive water, and sufficient bacteria as food. However, in the subsequent stages of cell fusion and development of the giant cells into mature macrocysts, cells do not require the above conditions. Cell fusion and development into macrocysts were able to occur even in light with minimum water and in the absence of bacteria. For cell fusion calcium ions were required.     

Rapid identification of non-allelic nystatin resistance mutations inDictyostelium discoideum

Spontaneous nystatin resistance mutations inDictyostelium discoideum fall into three complementation groups;nys A. nys B andnys C. We demonstrate three methods for rapidly distinguishing mutations in the three complementation groups. In the first methodnys B andnys C mutations are identified by their sensitivity to the sterol biosynthesis inhibitors azasterol A25822B and fenarimol respectively. The second method exploits the differential sensitivities of thenys mutations to the polyene antibiotic pimaricin. In the last method we show thatnys C and non-nys C mutants can be distinguished on the basis of the Lieberman-Burchard color reaction for sterols.     

The dependence of glycosyltransferases inDictyostelium discoideum on the structure of polyisoprenols

Summary Mannosyltransferases in plasma membranes ofDictyostelium discoideum synthesize polyisoprenyl-phosphomannosides from exogenous polyisoprenylphosphates and GDP-mannose. The specificity of the enzymes depends on the chain length and the saturation of the polyisoprenols. Maximum activity is reached by a α-saturated C-55 polyisoprenylphosphate.     

Periodic stimuli are more successful than randomly spaced ones for inducing development inDictyostelium discoideum

Aggregation in the cellular slime moldDictyostelium discoideum is due to chemotaxis. The chemoattractant, cyclic AMP, is synthesised and released periodically by the cells. Externally applied periodic pulses of cyclic AMP can also induce differentiation in this organism. The present work examines the role of periodicityper se in cyclic AMP-mediated stimulation of cell differentiation. For this purpose we use Agip53, aDictyostelium mutant which does not develop beyond the vegetative state but can be made to aggregate and differentiate by reiterated applications of cyclic AMP. Importantly, Agip53 cells do not make or release any cyclic AMP themselves even in response to an increase in extracellular cyclic AMP. A comparison of the relative efficiencies of periodic and aperiodic stimulation shows that whereas the two patterns of stimulation are equally effective in inducing the formation of EDTA-stable cell contacts, periodic stimuli are significantly superior for inducing terminal differentiation. This suggests that there must be molecular pathways which can only function when stimulation occurs at regular intervals.     

Accumulation of unsulphated precursors inDictyostelium discoideum during selenate inhibition of growth

Incubation ofDictyostelium discoideum cells with selenate is known to inhibit vegetative growth. In this paper we show that in the presence of selenate macromolecules accumulate which can be converted to sulphated products once the selenate is removed. The presence of cycloheximide, an inhibitor of protein synthesis, during the subsequent incubation does not prevent this conversion but tunicamycin, an inhibitor of glycosylation does. It is concluded that, in the presence of selenate, precursors accumulate as unglycosylated proteins, suggesting that feedback inhibition of glycosylation may be operated.     

Guanine nucleotides modulate the function of chemotactic cyclic AMP receptors inDictyostelium discoideum

Summary Guanosine di- and triphosphates specifically decrease the affinity of chemotactic cAMP receptors in isolatedDictyostelium discoideum membranes. The K_0.5 was increased from 50 nM to 150 nM. Receptors were shown to be heterogeneous in dissociation kinetics. In the absence of guanine nucleotides three dissociation processes could be resolved, having first order rate constants of 8.7 x 10⁻⁴, 1.3 X 10⁻², and higher than 0.1 s⁻¹. Guanine nucleotides decreased the affinity for cAMP by transforming the slowest dissociating receptor form (K_D is 8 nM) to forms dissociating more rapidly. Our data indicate that a guanine nucleotide binding protein (G-protein) is involved in the transduction of the cAMP signal inD. discoideum.     

The purification,specificity, and role of dipeptidyl peptidase III inDictyostelium discoideum

Dipeptidyl peptidase III was purified from culminating cells ofDictyostelium discoideum to apparent homogeneity by salt fractionation, isoelectric focusing, hydrophobic interaction, gel filtration, and ion-exchange chromatography. The enzyme was also found in and purified from cells growing on bacteria or in axenic media. The enzyme from the growth phase was indistinguishable from the culmination enzyme during all purification procedures. Thus dipeptidyl peptidase III is not a developmentally associated enzyme and probably is involved in peptide breakdown throughout the life cycle. The enzyme cleaved arginyl-arginyl-2-naphthylamide most effectively and showed no action on leucyl-glycyl-naphthylamide, thus resembling the mammalian enzyme and differing from the yeast enzyme. Aspartyl-arginyl-naphthylamide was cleaved at 6% of the rate observed with arginyl-arginyl-naphthylamide and the enzymein vivo might therefore be capable of removing the N-terminal peptide (aspartyl-arginine) from peptides such as angiotensins I and II. It showed no aminopeptidase or endopeptidase activity. Angiotensin III was an effective inhibitor of the enzyme; peptides with the N-terminal sequences Gly-Gly, Gly-Arg, Arg-Val, and Gly-His inhibited the enzyme weakly.     

Nondegradative pisatin-resistance inDictyostelium discoideum,Neurospora crassa andNectria haematococca: Similarities and differences

Paradoxically, on pisatin-medium (150 μg/ml) the cellular slime mouldDictyostelium discoideum grows only when plated as spores but not when plated as amoebae. The recent discovery of inducible nondegradativc pisatin resistance in amoebae has allowed us to formulate a model that resolves this paradox. In this model, the germinating amoeba is postulated to acquire a pisatin-resistance phenotype while ensconced within the spore wall. This article reviews the findings on which this model is based and extends it to also account for the differences in pisatin sensitivity phenotype that result from sterol alteration in cellular slime mould^s and fungi.     

A flow fluorimetric analysis of the cell cycle during growth and differentiation inDictyostelium discoideum

Summary We report a flow fluorimetric analysis of the DNA content of cells and nuclei from vegetative populations and various developmental stages of the cellular slime mouldDictyostelium discoideum using the dyes Hoechst 33258 and mithramycin. Nuclei from all of these populations showed an identical single DNA-content peak, indicating that most vegetative cells and most cells in all developmental stages are in one phase of the cell cycle. Our own data and findings in the literature indicate that this phase is G₂. On the other hand, we also found that various stages, subpopulations of cells at early stages and the different differentiated cell types in the slug stage differ in DNA content per cell. Any particular population typically has one major peak of DNA content, with a modal value that is characteristic for the cell type and for the developmental stage. These differences presumably reflect differences in mitochondrial DNA content per cell.