Dictyostelium Discoideum Development Research Articles

Properties of a 5′-AMP specific nucleotidase which accumulates in one cell type during development of Dictyostelium discoideum

5′-AMP nucleotidase activity accumulates during the culmination stage of development in a thin layer of cells at the prestalk-prespore interface of Dictyostelium discoideum. In this report we characterize a highly purified preparation of this enzyme in an attempt to determine the physiological significance of the accumulation and localization of the activity during cellular differentiation. A pH optimum of 9.5 was determined using nine different buffer systems tested over a range of pH from 3 to 13.5. The Michaelis constants for p-nitrophenylphosphate (NPP) and 5′-AMP were 1.8 and 1.2 m m, respectively. Substrate concentrations of 5′-AMP in excess of 2.5 m m were found to inhibit the activity. Little or no effect on the activity of the enzyme was observed in the presence of EDTA, Mg 2+, Mn 2+, Ca 2+, Fe 2+, or Zn 2+ ions. However, the enzyme appears to be a zinc metalloprotein as evidenced by its inhibition with 1,10-phenanthroline and recovery of activity in the presence of zinc. Other inhibitors of enzymatic activity include dithiothreitol and imidazole. The enzyme was bound by calcium phosphate, but could not be immobilized on matricies containing other substrate or product analogs, including 5′-AMP, cyclic AMP, ATP, phenylalanine, blue dextran, and Procion Red HE3B. The hydrophobicity of 5′-AMP nucleotidase was demonstrated by its strong affinity for immobilized alkyl and ω-amino alkyl ligands, as well as phenyl Sepharose. Isoelectric focusing of the enzyme in granulated gel required both the presence of detergent to prevent aggregate formation and precipitation of the enzyme, and the addition of zinc after focusing to reverse Ampholine inhibition. Apparently, Ampholine chelates zinc away from the enzyme much like 1,10-phenanthroline. Using this method, the isoelectric point of 5′-AMP nucleotidase was found to be 4.5–4.9, with a 30% recovery of the applied activity.

Differential expression of the members of the discoidin I multigene family during growth and development of Dictyostelium discoideum

Discoidin I and II are lectins synthesized during the aggregation of Dictyostelium discoideum amoebae which may play a role in cellular cohesion. Discoidin I was thought to consist of two major polypeptides, but we show that there are three. The N-terminal amino acid sequence of the polypeptides has been predicted by determining part of the nucleotide sequence of their respective mRNAs. We obtained the nucleotide sequences by reverse transcription of the mRNAs, using as primers, fragments derived from the coding region of two cloned discoidin I sequences, and utilizing cross hybridization to the various mRNA species and differences in the length of their 5′ noncoding regions to isolate fragments for DNA sequencing. We used primer extension to measure the relative concentration of the three major discoidin I mRNA sequences. We show that during development changes in the abundance of all three mRNA sequences occur coordinately. In cells growing in nutrient broth, however, only two of the three major discoidin I mRNA sequences accumulate, and if such cells are grown to a very high density, both sequences disappear. These results indicate that the coordination of discoidin I gene expression is not obligatory and that the members of this multigene family may differ in the mode of their induction during normal development.

Cell fusion, nuclear fusion, and zygote differentiation during sexual development of Dictyostelium discoideum

The fluorescent nuclear stain Hoechst 33258 was used to study the nuclear events during mating of Dictyostelium discoideum in liquid culture. These studies revealed that cell fusion begins about 11 hr after the sexually compatible cultures are mixed and continues until 26 hr. Approximately 37% of the cells fuse during this 15-hr period. At first the fused cells are relatively small, but by 20 hr the fusion products become evident as morphologically distinct giant cells. Starting at 22 hr these giant cells are transformed into true zygotes as nuclear fusion begins. Both the fusion of amebae and the differentiation of zygote giant cells are Ca 2+-dependent events as revealed by studies using EGTA. The nuclear events of zygote differentiation involve nuclear swelling, migration, and fusion. The precise timing of these events has been detailed. Of particular interest for genetic analyses via the macrocyst is the presence of a small population of multinucleate cells (maximum level is 1.67% of the cell population) which usually possess 3 or 4 nuclei but may have as many as 10 or more. Although these multinucleate cells contain many nuclei, our evidence suggests that only one is a zygote nucleus. The genetic implications of these data and the potential value of using the mating system for the analysis of cell fusion are discussed.

Cell behavior in Dictyostelium discoideum: preaggregation response to localized cyclic AMP pulses.

The motion of cells in the aggregation phase of Dictyostelium discoideum development is complex. To probe its mechanisms we applied precisely timed (+/- 1 s) and positioned (+/-2 micrometers) pulses of cyclic AMP to fields of cells of moderate density using a micropipette. We recorded cell behavior by time lapse microcinematography and extracted cell motion data from the film with our Galatea computer system. Analysis of these data reveals: (a) Chemotaxis lasts only about as long as the cyclic AMP signal; in particular, brief pulses (approximately 5 s) do not induce chemotaxis. (b) Chemotactic competence increases gradually from within an hour after the initiation of development (starvation) to full competence at approximately 15 h when aggregation begins under our conditions. (c) Cell motion reverses rapidly (within 20 s) when the external gradient is reversed. There is no refractory period for motion. We present a new description of the process of aggregation consistent with our result and other recent findings. (d) The behavioral response to cyclic AMP includes a phenomenon we call "cringing." In a prototypical cringe the cell speed drops within 3 s after a brief cyclic AMP stimulus, and the cell stops and rounds and then resumes motion after 25 s. (e) The development of the speed response in cringing as the cells age closely parallels the development of the cyclic AMP-induced light-scattering response of cells in suspension. (f) Cringing occurs in natural populations during weak oriented movement. The computerized analysis of cell behavior proves to be a powerful technique which can reveal significant phenomena that are not apparent to the eye even after repeated examination of the film.

Vesicle-mediated transfer of phospholipids to plasma membrane during cell aggregation of Dictyostelium discoideum.

We have previously reported that synthesis of phospholipids increases 4-fold at the onset of chemotactic migration during development of Dictyostelium discoideum and that the newly synthesized phospholipids are preferentially incorporated into the plasma membrane (De Silva, N. S. and Siu, C-H. (1980) J. Biol. Chem. 255, 8489-8496). To test the hypothesis that the rapid transfer of phospholipids to the plasma membrane is mediated by vesicles, we isolated phospholipid-rich vesicles from cells at 6 h of development. These vesicles had an average size of 0.35 micrometer in diameter. They banded at a density of 1.097 g/cm3 and they had a phospholipid: protein (w/w) ratio of 2.25. The predominant classes of phospholipids in these vesicles were phosphatidylethanolamine and phosphatidylcholine. In pulse-labeling studies using [3H]glycerol, these low density vesicles had the highest phospholipid-specific activity, which was about 3 times higher than that of 6-h plasma membranes. Almost 80% of the incorporated radioactivity was found to be associated with phosphatidylethanolamine and phosphatidylcholine. When cells were chased with cold precursor after pulse labeling, the specific activity of these vesicles dropped by almost 20-fold in 90 min, while plasma membranes showed a 2.5-fold increase in 60 min. Addition of colchicine to 7-h cells inhibited the translocation of newly synthesized phospholipids to the plasma membrane. The low density vesicles were found in much reduced amounts in preaggregation stage cells or the aggregateless mutant WL3. These results indicate that transfer of newly synthesized phospholipids from their site of synthesis to the plasma membrane probably occurs through a special class of phospholipid-rich vesicles.

Role of Cell Sorting in Pattern Formation in Dictyostelium discoideum

To examine the relationship between cell sorting and cell differentiation in the development of Dictyostelium discoideum, labeled cells grown in the absence of glucose [G(-)cells] and unlabeled cells grown in its presence [G(+)cells] were mixed and either allowed to undergo normal morphogenesis or cultured under submerged conditions. Changes in the distributions within a cell aggregate of labeled cells and cells stained with the conjugated antispore serum (prespore cells) were followed on the same sections by the methods of autoradiography and immunohistochemistry. In normal morphogenesis, differentiation of prespore cells apparently initiated and proceeded coincident with sorting out between G(+) and G(-) cells, during formation of standing slug. By contrast, within an aggregate formed under submerged conditions, prespore cells began to differentiate long before G(+) and G(-) cells were sorted out, indicating that the cell sorting is not a prerequisite for the cell differentiation. The sorting out, however, brought about an accumulation of prespore cells in a hemisphere, thus producing a prestalk-prespore pattern within the aggregate.

Identification of endogenous binding proteins for the lectin discoidin-I in Dictyostelium discoideum.

Recent biochemical and genetic evidence has shown that the endogenous lectin discoidin-I is involved in intercellular adhesion during development of the cellular slime mold Dictyostelium discoideum. We have prepared discoidin-I affinity columns and used them to isolate the lectin receptors. By using the cell surface radioiodination method, 11 discoidin-I binding proteins were identified in wild-type NC4 cells by gel electrophoresis, with apparent molecular weights of 95,000, 85,000, 78,000, 72,000, 60,000, 33,000, 31,000, 28,000, 25,000, 21,000, and 15,000. Only three (gp33, gp31, and gp28) were under developmental regulation. The amount of gp31 increased 8- to 10-fold during aggregation, and it was the predominant discoidin-I binding protein synthesized at the aggregation stage. Discoidin-I binding proteins derived from aggregation stage cells were potent inhibitors of discoidin-I in a hemagglutination assay in vitro. The same preparation was found to promote aggregation of cells bearing discoidin-I on the surface, suggesting a multivalent interaction.

Changes in the complexity of nuclear RNA during development of Dictyostelium discoideum

Saturation hybridization with a radioactive single-copy genomic DNA probe has been used to measure the complexity of the total whole cell polyadenylated RNA populations during development of the cellular slime mold Dictyostelium discoideum. The complexity of the polyadenylated nuclear RNA has been deduced by comparison of these numbers with our previous estimates of the complexity of the polyadenylated RNA in the cytoplasm and on polysomes. Assuming asymmetric transcription, 53 to 54% of the single-copy portion of the genome is expressed in polyadenylated RNA in growing and early aggregating cells. Of this only about one-third or 19% is expressed on polysomes as mRNA. By contrast, during late aggregation and culmination, after the time when tips have formed on the aggregates a substantially greater portion of the single-copy genome, 80%, is expressed in the total population of polyadenylated RNA. Again, however, only about one-third or 30% of the single-copy genome is expressed as mRNA. Thus throughout growth and development the complexity of the polyadenylated nuclear RNA is about one and one-half times the complexity of the corresponding mRNA populations. The RNA population from the late developing cells is composed of all those RNA species expressed during growth and early development plus additional RNA species whose expression is specific to late development. Sequences which comprise the majority of the mass of the new polyadenylated RNA transcripts which appear in the cytoplasm and on the polysomes late in aggregation are not detectable in nuclear RNA from growing cells. This suggests that their developmental expression is largely regulated at the level of transcription rather than by controls at the level of processing or transport from the nucleus.

The cascade of membrane events during development of Dictyostelium discoideum.

One of the basic goals of biology is to understand the processes of development, including cytodifferentiation, morphogenetic movement, pattern formation, and expression of specific gene products. Since there is considerable evidence that many of these events are effected initially at the surface of the cell, we are interested in the properties of the plasma membrane and its constituent proteins and glycoproteins. The plasma membrane proteins of Dictyostelium discoideum are among the most thoroughly studied, and thus this organism serves as an important model for changes in the plasma membrane during eukaryotic development.KeywordsAdenylate CyclasePlasma Membrane ProteinDictyostelium DiscoideumSlime MoldStalk CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Changes in the polyadenylated messenger RNA population during development of Dictyostelium discoideum

The program of gene expression during the life cycle of Dictyostelium discoideum has been assessed by molecular hybridization of cDNA probes with polysomal RNA extracted at the following different stages of development: vegetative growth, interphase (2.5 hr), aggregation (8 hr), postaggregation (12 hr), and preculmination (18 hr). Several different cDNA probes were used. Two probes were prepared from vegetative stage poly(A +) RNA, one representing all species present and the other enriched for abundant species. A third cDNA probe was prepared from preculmination stage polysomal RNA and a fourth probe consisted of the preculmination stage cDNA depleted in those species also present at the vegetative stage. Hybridization of the various probes with the different polysomal RNA preparations has revealed developmental changes in the mRNA populations. These changes were not detected in an aggregation less mutant under similar conditions of starvation. Abundant RNA species of vegetative cells were found to drop to low levels, especially during the aggregation period. Fifty percent by mass of the RNA present in polysomes at 18 hr is not present during vegetative growth. Some of the new RNA species appeared during interphase and the remaining during the postaggregation period. A gradual increase in the number of copies per cell of certain RNA species comprising both new species as well as some shared with vegetative cells was observed throughout development. Other results indicated that the composition of polysomal and cytoplasmic RNA is similar during vegetative growth but differs markedly at 18 hr of development. Also, cytoplasmic RNA at 18 hr contained, in addition to polysomal RNA, a large proportion by mass of nonpolysomal RNA similar to vegetative RNA. The number of polysomal RNA species detected by this analysis during vegetative growth and during the preculmination stage were estimated to be 3000 and 3700, respectively. The number of copies of these RNA species ranged between 30 and 2000 per cell during vegetative growth and 3 to 300 per cell in polysomes at 18 hr. Developmentally induced RNAs which were preferentially distributed among abundant and intermediate classes were estimated to number 700–900 species.

Synthesis of spore- and stalk-specific proteins during differentiation of Dictyostelium discoideum

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.

Surface labeling of membrane glycoproteins and their drastic changes during development of Dictyostelium discoideum.

Surface glycoproteins on plasma membranes from Dictyostelium discoideum were labeled by sodium metaperiodate oxidation and sodium boro[3H]hydride reduction. The amount of incorporation of tritium from NaB3H4 reached a plateau after 10 min at a periodate concentration of 20 mM. The density analysis carried out by sucrose density-gradient centrifugation showed that the plasma membranes were selectively labeled by this technique. About 84% of the radioactivity incorporated was released by hydrolysis with 0.25 M H2SO4 for 3 h at 100 degrees C. The released materials were eluted at a bed volume after chromatography using a Sephadex G-50 column. From the paper chromatographic analysis of the eluate, four radioactive spots were detected. Two of them were glycerol and glyceraldehyde and the other two spots seemed to be oligosaccharides. Using the above method, the plasma membranes from aggregation-phase cells were labeled four times more than those from growth-phase cells. The labeled plasma membrane fraction during the aggregation phase was separated into at least 39 distinct glycoprotein bands on a one-dimensional dodecylsulfate/polyacrylamide gel. Six of the 39 bands increased markedly in density and two new bands appeared. In contrast, four bands decreased in density in the aggregation phase. Using this method, the surface glycoproteins can be analyzed directly be gel electrophoresis of the lysate of labeled whole cells without preparing the plasma membranes. Changes during development of glycoproteins on outer cell surfaces were also confirmed by O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis. Utilizing this technique, glycoproteins on plasma membranes of D. discoideum were separated into 63 individual spots; 45 of these 63 spots changed during the early developmental course of D. discoideum. This is in contrast to the slight change in the soluble and membrane proteins during this phase. This fact also suggests that the glycoproteins have important roles in the process of aggregation.

Base composition of transfer RNAs from vegetative and developing cells of Dictyostelium discoideum

In a previous comparison of tRNAs from vegetative and developing cells of Dictyostelium discoideum, displacements of chromatographic peaks were observed for a number of aminoacyl-tRNAs (Palatnik, C.M., Katz, E.R. and Brenner, M. (1977) J. Biol. Chem. 252, 694–703). Since the development peak eluted ahead of the vegetative peak in all but one case, and since levels of amino acid acceptance and tRNA chromatographic profiles were otherwise quite similar, it was suggested that a common change or a very small number of changes in base modification were occurring during development. We have therefore compared tRNAs from both stages with respect particularly to their minor base composition. Of the minor bases which we have analyzed no reproducible differences between the two stages have been detected. Moreover, we have been unable to confirm a recent report (Dingermann, T., Schmidt, W. and Kersten, H. (1977) FEBS Lett. 80, 205–208) that a significant change occurs in the ribothymidine content of tRNA during Dictyostelium discoideum development.

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[ 32P]monophosphate (8- N 3-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.

Protein-bound mono(ADP-ribose) residues in differentiating cells of Dictyostelium discoideum

Changes in protein-bound mono(ADP-ribose) residues during development of Dictyostelium discoideum were determined. NAD(H) levels and the amounts of the NH 2OH resistant and sensitive subfractions of mono(ADPR) were found not to be different between exponentially growing and aggregation-competent cells in which mitosis has ceased. Divergent changes occured at the differentiation stages following aggregation as indicated by an increase in the ratio of these subfractions from approx. 1 during the growth phase and aggregation competent stage to 2 in the grex, a stage which follows cell aggregation. The fraction of NH 2OH sensitive conjugates closely followed the changes in total cellular protein, while the NH 2OH resistant ADPR protein conjugates, when based on protein, increased during the stages following aggregation. NAD(H) and NADP(H) levels per unit DNA decreased significantly during this period. The mono(ADPR) to DNA ratio in D. discoideum is comparable to that in proliferating Physarum polycephalum and to non-proliferating adult rat liver. The total amount of mono(ADPR) residues per nucleus is, however, approximately 70-fold higher in the liver, indicating that the quantity of mono(ADPR) residues is more closely related to the size of the eukaryotic genome than to cell proliferation.

The regulation of the synthesis of actin and two other proteins induced early in Dictyostelium discoideum development

There is a large increase in the relative rate of synthesis of actin, and in the levels of actin mRNA, during the first hour of differentiation of Dictyostelium discoideum. We have measured the relative rate of actin synthesis and the levels and the half-lives of actin mRNA during growth and at various times during development of wild-type Dictyostelium and of 12 developmental mutants. From these values we could calculate the rate of synthesis—presumably transcription—of functional actin mRNA during differentiation. During differentiation of wild-type cells the relative rate of actin message synthesis showed four distinct phases: (1) a 6.5- to 13.5-fold increase between 0 and 90 min; (2) a precipitous decrease after 90 min; (3) a return to an intermediate level of synthesis after 4 to 8 hr; and (4) a precipitous decrease again after 16 hr. All four mutant strains exhibited the first three changes, but only the mutant which aggregated and formed tips showed the final decrease in actin mRNA synthesis at 16 hr. The other three, which do not aggregate, continued actin mRNA synthesis at a high level. They always maintained a high level of actin mRNA with a normal half-life, and a high rate of actin synthesis. These experiments suggested that the late decrease in actin message synthesis is dependent on a “late” controlling event and may require aggregation or tip formation. We also describe the properties of two proteins synthesized only during the first 90 min of development. The synthesis of these proteins increases at least 8-fold by 45 min and then decreases equally rapidly. These changes were explained by a parallel increase and decrease in the levels of the cytoplasmic messages for the two proteins. Therefore, the synthesis of these proteins is regulated by the transcription and degradation of their messages rather than by any sort of translational control. Moreover, the messages for these proteins have particularly short half-lives, less than 13 min, compared to 200−300 min for other Dictyostelium mRNAs. We calculated that the rates of synthesis of these messages also increased rapidly during the first 45 min of development and subsequently decreased equally rapidly. One of twelve developmental mutants examined does not synthesize these proteins during early development. This result suggested that a regulatory event controlling early development may also control the synthesis of these proteins.

Developmental changes in soluble proteins during the early development of Dictyostelium discoideum

Developmental changes in soluble proteins during the early development of Dictyostelium discoideum

Adenylate cyclase activity and cyclic AMP levels during the development of Dictyostelium discoideum.

Adenylate cyclase activity and endogenous cyclic AMP levels were measured using a highly sensitive radioimmunoassay and protein binding assay during 24 h of development of Dictyostelium discoideum. Adenylate cyclase activity was not detected until the aggregation stage of development (10 h) when a sudden peak of activity was found. The enzyme was active at all subsequent stages, although a slow decline in activity was observed. Similarly, cyclic AMP levels were not detectable through the first 7 h of development and then showed a sudden peak at aggregation. Following aggregation the cyclic AMP levels decreased to approximately 1/2 the peak value and maintained that level throughout the remainder of the developmental cycle. Adenylate cyclase had a narrow range of substrate saturation with a maximum velocity at 1 to 4 mM ATP at both the aggregation stage (10 h) and the sorocarp stage (24 h). At levels of ATP higher than 6 mM the enzyme from both stages was strongly inhibited. No activity was observed in the absence of Mg2+ or dithiothreitol. The activity from 10-, 14-, and 20-h stages was found bound to a 25,000 x g pellet fraction. The sudden appearance of adenylate cyclase and its product cyclic AMP at aggregation provides additional evidence of a role for this nucleotide in chemotaxis, and the retention of enzyme activity and nucleotide level during the subsequent stages may reflect a further function of cyclic AMP during formation of the two cell types.

Cyclic GMP and cyclic AMP changes in response to folic acid pulses during cell development of Dictyostelium discoideum

Folic acid pulses induced developmental processes in agip 71, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. Cells that had received folic acid pulses were able to form EDTA-stable cell aggregates and to complete full differentiation to fruiting bodies. In these cells no autonomous periodic activities were observed by light scattering. Folic acid pulses elicited increases in the concentrations of cyclic GMP and cyclic AMP. In undifferentiated cells, folic acid caused a rapid increase in the level of cyclic GMP without a significant change in the level of cyclic AMP. In an advanced developmental state folic acid caused an increase in cyclic AMP in addition to two successive peaks of cyclic GMP. Experiments performed with the parent strain, Ax-2, also showed that during the development towards aggregation competence, cells acquired the ability to produce a cyclic AMP peak in response to folic acid.

A cinematographical study of the development of vitally stained Dictyostelium discoideum.

We report observations from time-lapse films of the development of Dictyostelium discoideum (Dd) stained with the vital dye neutral red. We have used this dye to enable us to track individual cells, as they move through multicellular tissues in later Dd stages. Our observations lead us to new conclusions about the control of cell movement and cell contact during Dd development, including the tentative conclusion that the aggregation control competences regulate cell behaviour throughout the whole of Dd. development. We are led to specific hypotheses for the mechanisms of later morphogenetic movements and pattern formation.