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.     

The effects of cell density and starvation on early developmental events in Dictyostelium discoideum

We show that removal of yeast extract and trypticase from growth medium is sufficient for induction of several key events which occur during the early stages of Dictyostelium differentiation: run-off of polysomes, the earliest known change in macromolecular metabolism; appearance of the cell surface cAMP receptor; and aggregation itself. Starvation of glucose has little effect on these parameters. These results are consistent with those of other investigators who showed that starvation only of amino acids will induce other activities associated with cAMP-mediated cell signaling and cell-cell adhesion. We show, in contrast, that other factors are involved in the increase in the relative rates of synthesis of three polypeptides very early in differentiation: actin, and two proteins (“45-min” proteins) which are synthesized only during the period of 45–90 min. The induction of synthesis of these three proteins and presumably, of their mRNAs, is not the result of starvation for glucose or amino acids but is the result of plating cells at high density. The increases in the synthesis of these proteins are dependent on the density at which cells are plated and do not occur at a density 75-fold lower than the density used in standard experiments. Cells growing at high density or near stationary phase do not show the induction of increased synthesis of actin or the “45-min” proteins. These experiments suggest that these early developmental changes may be dependent on a threshold level of a diffusible factor excreted early in development.     

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.     

Separation of enzymatic activities in Dictyostelium discoideum by high-pressure gel permeation chromatography.

High-pressure gel permeation chromatography was used to separate the cyclic AMP phosphodiesterase and ATP pyrophosphohydrolase activities of Dictyostelium discoideum. Two types of column packings, with different functional groups on the silica-bonded carbon side chains, were used to separate the two activities in approximately the same amount of time and with the same elution pattern. Recovery of both activities was enhanced when acetate, rather than sulfate, was the mobile phase. This recovery of activity following chromatography at high pressure demonstrates that high-pressure gel permeation chromatography can be used for the purification of enzymes.     

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.     

Comparison of proteins synthesized by anterior and posterior regions of Dictyostelium discoideum pseudoplasmodia.

In migrating pseudoplasmodia of the cellular slime mold Dictyostelium discoideum, cells in approximately the anterior quarter of the structure give rise to stalk cells, while the remainder produce spore cells. Certain biochemical and structural components have been found to differ between cells occupying these two positions, indicating that some differentiation has already occurred by this stage. To evaluate the degree of this differentiation we have compared the proteins being synthesized in different regions of the pseudoplasmodia. Migrating pseudoplasmodia were labeled with [³⁵S]methionine and cut into five segments, and the labeled proteins were resolved by two-dimensional gel electrophoresis and visualized by autofluorography. Of 500 polypeptides detected, 57 showed regional variations in labeling. Nearly all of these differences in labeling occurred between the anterior fifth and posterior four-fifths of the pseudoplasmodia, indicating that by this stage a marked degree of differentiation has occurred between the two cell types.     

Distribution of IP25 in chromatin and its possible involvement in chromatin condensation

Friend leukaemic cells (FLC) were induced to differentiate with dimethyl sulfoxyde (DMSO), hexamethylenbis-acetamide (HMBA) and sodium butyrate (SB) and the phospholipid composition was analyzed. The phospholipid composition of differentiated cells differed from that of non differentiated cells and also varied according to inducer. The ratios of the percentage of phosphatidyl choline (PC) to that of phosphatidyl ethanolamine (PE) or sphingomyelin (SPH) increased by about 2-fold in DMSO or SB induced FLC. These ratios did not vary in HMBa induced FLC. Furthermore the fatty acid composition of PC and PE obtained from differentiated cells varied according to the inducer. Although these changes appeared to be related to the inducers, it can not be excluded that the differentiated state also contributes to these changes.     

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.     

Similarity of the primary sequences of ribosomal RNAs isolated from vegetative and developing cells of Dictyostelium discoideum

Differentiating cell aggregates of Dictyostelium discoideum exhibit a pattern of rRNA metabolism quite different from that observed in the single-celled vegetative amoebas of this organism. We have examined whether the differences are related to a requirement for the production of new types of ribosomal RNA during development. Oligonucleotide maps and supplementary sequence data for 25 S, 17 S, and 5 S rRNAs from vegetative and developing cells have revealed no detectable alterations in primary sequence distinguishing any species of rRNA in developing cells from its vegetative cell counterpart.     

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 sequence of an atriopeptigen: A precursor of the bioactive atrial peptides

The high molecular weight fraction (atriopeptigen-APG) obtained by gel filtration chromatography of rat atrial extracts was fractionated by isoelectric focusing and reverse phase HPLC to obtain a pure APG. Purification of cyanogen bromide digests of the crude high molecular weight fraction resulted in the isolation of a single biologically active cyanogen bromide cleavage peptide. Sequence analyses of these peptides coupled with recent reports of sequence analyses of intermediate molecular weight atrial peptides (Thibault, et al. (1984) FEBS Letters 167, 352–356, and Kangwa, et al., Biochem. Biophys. Res. Commun 119, 933–940) provide the complete primary structure of an 111 residue APG.     

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells

The K⁺ content and the K⁺ flux were measured in the cell lines ME₂ and MF₂ isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the same K⁺ content and the same K⁺ steady state flux per unit of surface area.In ME₂ cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K⁺ net movement.The (Na⁺ + K⁺)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K⁺ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells.The K⁺ net flux is considerably increased when the intracellular K⁺ level is reduced after preincubation of the cells in a K⁺-free medium. Thus, internal K⁺ seems to regulate the K⁺ influx.     

Effects of tunicamycin upon glycoprotein synthesis and development of early mouse embryos

Tunicamycin, an antimetabolite which inhibits the N-glycosylation of proteins, does not block the initial cleavages of mouse embryos, even at relatively high concentrations. However, it can interfere with compaction and blastocyst formation. Although tunicamycin treatment from the two-cell or eight-cell stage can cause developmental arrest prior to hatching from the zona pellucida, much higher (sublethal) concentrations of the antimetabolite added at the morula or blastocyst stage do not specifically affect hatching of blastocysts, their attachment to the substratum, or outgrowth of trophoblast cells. The consequence of continuous exposure of embryos to moderate amounts (0.05 to 0.1 μg/ml) of tunicamycin through peri-implantation stages is death of trophoblast cells with little effect upon the cells of the inner cell mass (ICM). The latter give rise to apparently normal early endoderm cells in the presence of the antimetabolite. The incorporation of leucine, mannose, and fucose into acid-insoluble material by ICM cells is only minimally inhibited by tunicamycin. On the other hand, the antimetabolite causes a severe inhibition of incorporation of not only mannose, but also leucine, into acid-insoluble material in trophoblast cells. Thus, trophoblast cells resemble transformed cells by their extreme sensitivity to tunicamycin.     

Activation of cyclic AMP-dependent protein kinases I and II by cyclic 3',5'-phosphates of 9-beta-d-ribofuranosylpurine and 2-beta-D-ribofuranosylbenzimidazole

Analogs of cyclic AMP lacking the 6-amino group—9-β-D-ribofuranosylpurine cyclic 3′,5′-phosphate (I)—or the 1- and 3-nitrogens as well as the 6-amino group—1-β-D-ribofuranosylbenzimidazole cyclic 3′,5′-phosphate (II)—were effective activators of both type I (cAKI) and type II (cAKII) isozymes of cAMP-dependent protein kinase. An analog with a pyrimidine ring fused to the benzimidazole ring system of II—3-β-D-ribofuranosyl-8-aminoimidazo[4,5-g]-quinazoline cyclic 3′,5′-phosphate (III)—was equipotent to I or II as an activator of cAKII but only $\text{1}\text{10}$ as potent as I or II as an activator of cAKII. The results show that neither cAKI nor cAKII requires the 6-amino group and that they may have different sensitivities to the effects of alterations in the electron distribution in the pyrimidine ring.     

Differential effects of detergents upon the soluble and particulate guanylate cyclases from rat lung.

The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O₂. The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition.     

Reducible cross-links in human glomerular basement membrane

Reducible cross-links in purified human glomerular basement membrane (GBM) were examined with an ion exchange chromatographic system that provided complete separation of cross-link standards and glucosylamines. After hydration in phosphate buffer, lyophilized GBM was reduced with tritiated borohydride. Chromatographic separation revealed two major radioactive peaks, identified as di-hydroxylysinonorleucine (di-OHLNL) and hydroxyaldolhistidine (HAH) by coelution with authentic di-OHLNL and HAH standards. Radioactive glucitol-lysine and glucitol-hydroxylysine were also identified on the basis of their co-elution with synthetic standards. The findings document the existence and establish the nature of the major reducible cross-links in adult human GBM.     

Catabolite repression in Bacillus subtilis

The d-gluconate transport system of Bacillus subtilis is optimally induced by exposure of cells for 2 h to 5 mM d-gluconate in the growth medium. d-gluconate transport is subject to catabolite repression, as distinct from inducer exclusion or catabolite inhibition, in a manner parallel to the repression of inducible histidase synthesis, suggesting that the repression is not specific to this transport system. Maximum repression with the repressing carbon source (10 mM) added to cells grown in either casein hydrolysate or amino acid medium is achieved within two doubling times. Urea, the only non-carbon source tested for a repressing effect, was found to act solely by inducer exclusion. The ability of a sugar carbon source to evoke catabolite repression appears to be unrelated to its suitability as a substrate for the sugar: phosphoenolpyruvate phosphotransferase system but nonetheless the conversion to a phosphorylated derivative of the sugar seems essential. Repressed cells fail to synthesize, or do so to a more limited extent, an as yet unidentified phosphorylated compound (probably a highly phosphorylated nucleotide) which is accumulated in the medium of non-repressed cells. Mutant studies imply that inosinic acid synthesis is necessary for catabolite repression whereas the adenosine highly phosphorylated nucleotides required for spurulation are not.