Differential Cell Cohesiveness Expressed by Prespore and Prestalk Cells of Dictyostelium discoideum

Pseudoplasmodia of Dictyostelium discoideum at the culmination stage were separated into two cell populations by sedimentation in a discontinuous renografin gradient. The two lighter fractions (I and II) had enzymatic activities characteristic of the anterior prestalk cells, while the heaviest fraction (III) showed enzyme activities characteristic of the posterior prespore cells. Cell-cell adhesion among prespore cells is much more resistant to EDTA dissociation than 10-h cells and prestalk cells. Fab fragments prepared from antibodies directed against a specific cell surface glycoprotein gp150 were more effective in dissociating prespore cells than prestalk cells. In addition, prespore cells contained an approximately 2-fold higher concentration of the endogenous carbohydrate binding protein discoidin-I than prestalk cells. These differences may account for the differential cohesiveness of these two cell populations and provide a basis for cell recognition and cell sorting at the slug stage.     

Pattern Formation in Dictyostelium discoideum: Temporal and Spatial Distribution of Prespore Vacuoles

Cell differentiation, cell determination and pattern formation in the pseudoplasmodium of Dictyostelium discoideum , was investigated using the prespore specific vacuole (PV) as a morphological marker. Concomitantly, measurements of cell movement within the pseudoplasmodium were made by tracing radioactively labelled cells. The main results indicate that 1) prespore cells appear first during late aggregation and occur randomly throughout the pseudoplasmodium with the exception of the very tip which stays free of prespore cells throughout development; 2) after late aggregation the number of prespore cells increases over a period of several hours; 3) each prespore cell takes on a progressively more prespore-like character as judged by the increase in number of PVs it contains; 4) establishment of the distribution pattern of prespore and prestalk cells takes place within the first 2 h, mainly by a sorting out mechanism; 5) presumptive spore areas are likely to contain a small proportion of cells lacking PVs (prestalk-cells?) while presumptive stalk cell areas are homogeneous throughout; 6) maintenance of the pattern during migration may be facilitated by a circulation at low level of prestalk cells between prestalk and prespore areas; and 7) during the development of this organism the events of cell determination, cell differentiation and pattern formation overlap substantially in time.     

Ligand-induced phosphorylation of the cAMP receptor from Dictyostelium discoideum

The cell surface cAMP receptor of Dictyostelium discoideum exists as a doublet of low (D) and high (R) electrophoretic mobility forms, both of which are phosphorylated in vivo. The R form is phosphorylated in a ligand-independent manner, while conversion of the R to D forms, induced by the chemoattractant, is accompanied by at least a 4-fold increase in the level of phosphorylation. When cells are stimulated with saturating levels of cAMP, increased phosphorylation is detectable within 5 s and reaches maximum levels by 5 min with a t1/2 of 45 s. Dephosphorylation of receptor, initiated by removal of the stimulus, is detectable within 30 s, has a half-time of 2 min, and reaches a plateau by 20 min. At half-maximal occupancy, phosphorylation occurred more slowly than at saturation, t1/2 = 1.5 min, and remained at intermediate levels until the cAMP concentration was increased. Accompanying electrophoretic mobility shifts occurred in all cases with similar, though not identical, kinetics. Both phosphorylation and mobility shift were half-maximal at 5 nM cAMP and saturated at 100 nM. Estimation of the specific activity of each receptor form indicates that not all sites are phosphorylated during the R to D transition; at least half of the sites are phosphorylated after the transition is completed. The rate of incorporation of phosphates into the receptor, held in the D form by cAMP, was less than one-third the rate of ligand-induced incorporation starting with the R form and was approximately twice the basal rate of incorporation. These results are compatible with ligand-induced receptor phosphorylation being an early event in the adaptation of other cAMP-induced responses.     

Early Recognition of Prespore Differentiation in Dictyostelium discoideum and its Significance for Models on Pattern Formation

An electron microscopic study revealed that during aggregation the cytoplasm of a number of cells increases in electron density. Increased electron density is shown to be the consequence of cell shrinkage, which causes a closer packing of cytoplasmic components. Originally electron-dense cells are spread randomly over the aggregate. The anterior prestalk region of the slug is almost devoid of electron-dense cells. In the posterior prespore region, cells with varying degrees of electron density are intermixed with 15–20% electron-lucent cells. During culmination all cells of the prespore region become very electron dense. Besides introducing a new criterion to recognize prespore cells at an early stage of development, our data give further evidence that induction of prespore cell differentiation is not necessarily position dependent.     

Prespore cell fate bias in G1 phase of the cell cycle in Dictyostelium discoideum

By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.     

Factors Affecting the Rate of Heat-induced Spore Germination in Dictyostelium discoideum

Washed spores of Dictyostelium discoideum, strains NC-4H, NC-4D, and V-12, germinated rapidly after being heat shocked at or near 45.0 C for 30 min. Cultures of the slime molds were grown in association with Escherichia coli B/r as the host bacterium; spores taken from plates of synthetic medium had a higher final germination value than spores from complex medium containing peptone and yeast extract. Young spores germinated more rapidly than older spores. Optimal germination occurred between pH 6.0 and 7.0, and, of the buffers tested, potassium phosphate allowed the most rapid germination. After heat shocking, spores were diluted into fresh oxygenated buffer to provide enough oxygen for completion of germination. Germination occurred most rapidly between incubation temperatures of 22 and 25 C.     

Prestalk/prespore Differentiation of Dictyostelium Cells under the Conditions Favoring Stalk or Spore Cell Formation*

The differentiation processes of Dictyostelium discoideum cells under the conditions which favored either stalk or spore cell formation were examined by the use of prestalk- and prespore-specific antibodies. In stalk cell-forming conditions, cells reactive with prestalk-specific monoclonal antibody (C1) increased rapidly early in development and later differentiated into stalk cells. No or only a few cells became reactive with prespore-specific monoclonal (B6) and polyclonal (antispore) antibodies. Despite the fact that most cells terminally became spores under spore cell-forming conditions, cells were first stained with the C1 antibody before becoming reactive with the B6 antibody. Unlike the case of normal development where cells coincidentally become reactive with the B6 and antispore antibodies, the appearance of the cells reactive with the latter was either delayed or suppressed. In conclusion, under either spore or stalk cell-forming conditions, the appearance of the prestalk antigen preceded that of the prespore one, which is consistent with normal development.     

cAMP regulation of cell differentiation in Dictyostelium discoideum and the role of the cAMP receptor

DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.     

Spore Germination in Strains of Dictyostelium discoideum and Other Members of the Dictyosteliaceae

Spores of all strains of Dictyostelium discoideum tested in this study germinated after a heat shock of 45 C for 30 min. Whereas the strains differed in their rates of germination, the rate for each strain was constant. A correlation existed between the rate of germination and the rate of vegetative growth when spores were inoculated into bacterial streaks. Heat shock clearly increased spore germination in D. purpureum, but the response was less dramatic than in D. discoideum. Enhancement also occurred in D. rosarium, but only in media containing peptone. Strains of D. mucoroides gave varied responses, and these could be divided into those which required mutrients for spore germination and those which did not. The spores of Polysphondylium pallidum were resistant to mild heat (45 C), but were not activated; peptone was required for germination. In contrast, the microcysts of this species were heat-labile and required no added nutrients for excystment.     

Spore coat genes SP60 and SP70 of Dictyostelium discoideum.

We cloned and sequenced the genes for two of the major proteins found in spore coats of Dictyostelium discoideum. The predicted translation product of each of these genes starts with a hydrophobic signal sequence that is subsequently cleaved. Expression of these spore coat genes is coordinate in prespore cells.     

Specific photoaffinity labeling of the cAMP surface receptor in Dictyostelium discoideum

The recent observation that ammonium sulfate stabilizes cell-surface [3H]cyclic AMP binding in Dictyostelium discoideum (Van Haastert, P., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) led us to attempt to identify the surface cAMP receptor by photoaffinity labeling with 8-azido-[32P]cAMP using this stabilization technique. 8-azido-[32P]cAMP specifically labeled a polypeptide which migrates as a closely spaced doublet (Mr = 40,000 to 43,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 60% of the labeled polypeptide was found associated with membranes. This protein was distinguished from the cytosolic regulatory subunit of the cAMP-dependent protein kinase (Mr = 41,000) by differences in developmental regulation, specificity, and subcellular localization. No kinase regulatory subunit was detected in membranes by western blot analysis. Our preliminary observations show that labeling of this doublet correlates closely with cAMP-binding activity, suggesting that it is the surface receptor which mediates chemotaxis and cAMP signaling.     

Seven Dictyostelium discoideum phosphodiesterases degrade three pools of cAMP and cGMP

The Dictyostelium discoideum genome uncovers seven cyclic nucleotide PDEs (phosphodiesterases), of which six have been characterized previously and the seventh is characterized in the present paper. Three enzymes belong to the ubiquitous class I PDEs, common in all eukaryotes, whereas four enzymes belong to the rare class II PDEs that are present in bacteria and lower eukaryotes. Since all D. discoideum PDEs are now characterized we have calculated the contribution of each enzyme in the degradation of the three important pools of cyclic nucleotides: (i) extracellular cAMP that induces chemotaxis during aggregation and differentiation in slugs; (ii) intracellular cAMP that mediates development; and (iii) intracellular cGMP that mediates chemotaxis. It appears that each cyclic nucleotide pool is degraded by a combination of enzymes that have different affinities, allowing a broad range of substrate concentrations to be degraded with first-order kinetics. Extracellular cAMP is degraded predominantly by the class II high-affinity enzyme DdPDE1 and its close homologue DdPDE7, and in the multicellular stage also by the low-affinity transmembrane class I enzyme DdPDE4. Intracellular cAMP is degraded by the DdPDE2, a class I enzyme regulated by histidine kinase/phospho-relay, and by the cAMP-/cGMP-stimulated class II DdPDE6. Finally, basal intracellular cGMP is degraded predominantly by the high-affinity class I DdPDE3, while the elevated cGMP levels that arise after receptor stimulation are degraded predominantly by a cGMP-stimulated cGMP-specific class II DdPDE5. The analysis shows that the combination of enzymes is tuned to keep the concentration and lifetime of the substrate within a functional range.     

Adenosine and its derivatives inhibit the cAMP signaling response in Dictyostelium discoideum

In developmentally competent Dictyostelium discoideum amoebae, binding of cAMP to high-affinity surface receptors produces a rapid activation of adenylate cyclase which adapts within minutes. The result is a transient increase in intracellular cAMP which is rapidly secreted. Adenosine inhibited this cAMP signaling response with an apparent Ki of 300 microM. The apparent Ki's for 2'-O-methyladenosine and 2-chloroadenosine were approximately 30 and 100 microM, respectively. Inhibition by adenosine was rapid, reversible, and depended on the cAMP stimulus concentration. In addition, the adaptation of the cAMP signaling response was blocked by adenosine. As has been previously reported, adenosine inhibits cAMP binding to intact cells. Under the same developmental conditions as in the perfusion studies, we find the binding inhibition depends on both the cAMP and adenosine concentrations, with an apparent Ki of 100 microM. The apparent Ki's for 2'-O-methyl- and 2-chloroadenosine were approximately 8 and 35 microM, respectively. However, with cells developed for short times and with an axenic strain, inhibition was independent of cAMP concentration or cells showed mixed-type binding inhibition. The effect of adenosine on the cAMP signaling response is consistent with the reported effects of adenosine on other cAMP-mediated processes such as chemotaxis and the increase in intracellular cGMP, and may provide an explanation for the reported inhibition of center formation.     

Localization of functional domains of the cAMP chemotactic receptor of Dictyostelium discoideum

The topography and functional domains of the cAMP chemotactic receptor of Dictyostelium discoideum were investigated by protease sensitivity to chymotrypsin. Proteolytic digestion of intact cells produced a 23-kDa fragment of the receptor that retained the photoaffinity label used to identify the receptor. Additionally, this fragment contained the sites phosphorylated by CAR-kinase, the enzyme that phosphorylates the ligand-occupied form of the receptor. The fragment was also found to be phosphorylated in response to cAMP stimulation of cells. Proteolytic digestion of either intact cells or membrane preparations did not appreciably alter the binding properties of the receptor, indicating that the domains which determine the cAMP binding pocket are likely to be transmembrane regions of the protein. Additionally, the sensitivity of down-regulated receptors to chymotrypsin digestion suggests that the initial loss of cAMP binding activity upon incubation of cells with high concentrations of ligand does not require receptor internalization.     

Ultraviolet Irradiation of the Vegetative Cells of Dictyostelium discoideum

Experiments on the effect of ultraviolet (UV) light on the survival of vegetative Dictyostelium discoideum cells indicate that this is a relatively UV-resistant organism. Several factors suggest the presence of some type of repair process. Experiments to test for liquid-holding recovery and simple photoreactivation yielded negative results. Acriflavine and caffeine were utilized to possibly interfere with dark repair. Acriflavine produced no UV sensitization, but caffeine did cause a concentration-dependent decrease in survival of irradiated cells. When UV-irradiated cells were illuminated with photoreactivating light while suspended in caffeine, the survival increased above that for cells treated with caffeine alone, suggesting an overlap between lesions repaired by photorepair and dark repair. Growth experiments showed that UV light induced a dose-dependent division delay, followed by a period of retarded growth characterized by the presence of a constant fraction of nonviable cells in the irradiated population. The delayed exposure of cells to caffeine after irradiation showed that the magnitude of the caffeine sensitization diminished throughout the division-delay period. An action spectrum indicated probable nucleoprotein involvement in the induction of division delay. UV light retarded ribonucleic acid and protein synthesis and temporarily blocked deoxyribonucleic acid synthesis. However, synthesis of all three accelerated prior to the end of the division-delay period and then closely paralleled the increase in cell number.