Separation and properties of prestalk and prespore cells of Dictyostelium discoideum

We describe a method of separating prestalk and prespore cells of Dictyostelium discoideum slugs using a self-generating Percoll gradient. This method gives quantitative recovery of cells and good purity. Separated prestalk and prespore cells possess different levels of the enzymes UDP galactose :polysaccharide transferase, cAMP phosphodiesterase and glycogen phosphorylase. We have used this method, as well as mechanical dissection of slugs, to examine the fate of separated prestalk and prespore cells in Dictyostelium strains that are able to give rise to mature stalk and spore cells in cell monolayers. The results from such experiments provide direct evidence that prestalk and prespore cells from the migrating slug stage are programmed to differentiate into stalk and spore cells respectively.     

Decay and synthesis of ribosomal proteins during Dictyostelium discoideum development

Summary Ribosome turnover is a prominent process during cell differentiation in Dictyostelium discoideum. At the end of 24 h of development on filters, the cells contain only 30% of the ribosome content of vegetatively growing cells. We determined the relative rates of synthesis and decay of each of the ribosomal proteins during this period. Approximately 80% of the total vegetative cell ribosomal proteins were degraded during the course of fruiting body construction. Ribosomal RNA and protein degradation apparently occurred coordinately during development. Although all ribosomal proteins decayed during development, some were more stable and a few less stable than the average. In addition, all the ribosomal proteins were synthesized during this period. Most ribosomal proteins were synthesized at the same rate as other cellular proteins, although a number were made at lower or higher rates. It was estimated that about 35% of the ribosomes in developed cells represented those, that were made during cell differentiation. Differential decay and/or synthesis of ribosomal proteins could account for the observed difference in protein content of ribosomes from growing amoebae and late development cells and spores.Paper No. 4 in the series, Studies on Ribosomal Proteins in Dictyostelium discoideum. Paper No. 3 is Ramagopal and Ennis (1982)     

Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA

In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. The primary sequence of ms RNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25 000 × g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA. Received: 17 May 1997 / Accepted: 26 August 1997     

Genetic, biochemical, and developmental studies of nystatin resistant mutants in Dictyostelium discoideum

Summary Conditions are given for the isolation of nystatin resistant mutants of the cellular slime mold Dictyostelium discoideum. These mutants fall into three phenotypic groups; corresponding to three genes: nysA, nysB, and nysC. Mutants in nysB and nysC affect sterol metabolism since they have altered sterol compositions. Each group contains several unique, but as yet unidentified, sterols in place of the wild type sterol. The nysC strains are most nystatin resistant, display altered sensitivity to some drugs, and grow on nystatin from amoebae or spores. All other mutants are nystatin resistant only as amoebae. Although nysC mutants grow normally, they make small fruiting bodies which appear to result from the formation of smaller aggregates.Supported by N.I.H. grant GM 18476     

The effects of proteases on proteins and glycoproteins of Dictyostelium discoideum plasma membranes

Dictyostelium discoideum cells were incubated with proteases, the plasma membranes subsequently isolated and changes in proteins and glycoproteins examined with dodecylsulfate gel electrophoresis. Low papain concentrations gave rise to a protein band which apparently derived from actin. Since actin was the only protein attacked, the results suggest some part of the actin is exposed on the outer surface of the cell. Higher papain concentrations released a substantial portion of actin from the plasma membrane and partially digested some of the glycoproteins. Since the new actin-derived band was not further digested, the glycoproteins may be required to stabilize the actin polymer rather than anchor those actin molecules which are directly associated with the plasma membrane. Pronase treatment released the two myosin heavy chains from the plasma membrane, in particular the higher molecular weight chain. Actin was not affected. Some glycoproteins were digested. Trypsin attacked many of the plasma membrane proteins, and the myosin heavy chains were completely removed. Actin was only moderately affected. However, the glycoproteins were entirely resistant to trypsin. Apparently the myosin heavy chains are attacked either due to their partial exposure on the cell surface or the exposure of proteins which anchor them in the membrane. These anchoring proteins cannot be glycoproteins or actin. Proteins and glycoproteins were largely digested when isolated plasma membranes were incubated with papain and pronase. The effects of trypsin on whole cells and isolated plasma membranes were similar.     

Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum

An extrachromosomally replicating transformation vector for Dictyostelium discoideum has been constructed using sequences of the endogenous Dictyostelium plasmid Ddp2. This transformation vector pnDeI (9.6 kb) replicates as a high copy number plasmid in Dictyostelium and is located in the nucleus. It has been constructed as shuttle vector containing the Escherichia coli vector pUC19 for replication and selection in E. coli and a part of the Tn903 transposon which confers resistance to G418 for selection in Dictyostelium. In order to show that the vector can be used for cloning and stable propagation of Dictyostelium DNA, a fragment of the Dictyostelium alpha-actinin gene that was marked with a synthetic oligonucleotide was cloned into pnDeI and found to be stably maintained in the extrachromosomal vector without undergoing noticeable recombination with the endogenous gene.     

Two distinct types of cell surface folic acid-binding proteins in Dictyostelium discoideum

Folic acid is degraded too fast by Dictyostelium discoideum to study binding of this ligand to cell surface binding proteins. Folate deaminase activity was inhibited in the presence of 3.3 × 10⁻⁴ M 8-azaguanine. This inhibitor enabled us to detect two folate binding proteins. One type bound folic acid and deamino-folic acid with the same affinity (K_0.5 = 3–6 × 10⁻⁷ M) and apparently negative cooperativity. Binding to only this type was observed if 8-azaguanine was omitted. The second type bound folic acid noncooperatively with K_d = 7 × 10⁻⁷ M. Deamino-folic acid did not compete even at a 1000-fold excess. This type may correspond to the chemotactic receptor.     

Characterization of a glycosyl-phosphatidylinositol degrading activity in Dictyostelium discoideum membranes

Antigen 117 is a glycolipid-anchored cell surface protein implicated in cell-cell cohesion of Dictyostelium discoideum amoebae. Previous studies have demonstrated that during cell aggregation some of the protein is released from the cell surface. Here we report the characterization of the enzymatic activity involved in the 117 antigen release. The data indicate that the releasing enzyme is a phosphatidylinositol phospholipase C. The data also indicate that structural features of glycolipid anchors are conserved in a variety of organisms.     

Quantitative studies on cell differentiation during morphogenesis of the cellular slime mold Dictyostelium discoideum

Taking advantage of the fact that differentiation of the prespore cell of Dictyostelium discoideum is characterized by synthesis of a prespore specific antigen, the process of its differentiation during the course of morphogenesis was quantitatively studied by determining the proportion of prespore cells and their cellular contents of the antigen, using the method of microfluorometry in combination with immunocytochemistry with antispore serum. The cells synthesizing the antigen became first detectable in the early aggregation center which was about to form a papilla. As the papilla elongated, the number of prespore cells rapidly increased up to the stationary level (70–80% of total cells) before completion of slug formation. During the process antigenic contents of prespore cells were gradually increased and leveled off in the early migration stage. When culmination was induced, antigenic contents were markedly increased to the maximum, which was followed by a sudden decrease immediately before spore formation. On the other hand, the proportions of prespore to total cells were kept constant at the stationary level all through the migration and culmination stages, in spite of a persistent decrease during culmination in the total number of cells due to continuous differentiation of the prestalk into the mature stalk cells. These results were discussed in relation to possible mechanisms of differentiation in this organism.     

Mass production of Dictyostelium discoideum in homogeneous and heterogeneous cultivation systems

The social amoeba Dictyostelium discoideum is a promising host for the expression of recombinant proteins requiring post-translational modifications. Limited maximal cell densities and slow growth rates, however, disfavor its application. Little attention has been paid to improve its cultivation. Here, some strategies are described, which allow the attainment of higher cell densities. This can be achieved by cultivation on an improved synthetic medium as well as in immobilized form. Two promising inorganic porous supports are presented—broken pumice and a ceramic catalyst carrier. Cell densities up to 4.5×10⁷ ml⁻¹ are obtained during suspension cultivation on the improved synthetic medium. This is about three times as much as can be expected for cultivations on conventional complex media. Cells in the pores of broken pumice and a ceramic support reach up to 15–20 times higher local cell densities compared with cells growing on conventional axenic media in suspension. Thus, the cell density of 3.5×10⁸ ml⁻¹ in the ceramic carrier (CeramTec^®) is the highest cell density for D. discoideum observed so far in cultivation systems. The immobilized cell density could be maintained for a long period of time by either repeated medium replacement or continuous cultivation. To minimize the external volume of the medium a trickle bed reactor was operated continuously.     

Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum

A 41,000 M_r cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-riboxylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and 3 the NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6., 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.     

Cyclic AMP, pattern formation and movement in the slime mould, Dictyostelium discoideum

It is important to determine whether a gradient of cyclic AMP exists along the migrating slime mould grex because such a gradient might be involved in pattern formation and the polarity of grex movement. Biochemical measurements provided results which could be consistent with Bonner's suggestion that 50% of the cyclic-AMP produced by the grex is produced by the anterior one-tenth. However, chemotactically sensitive cells cannot detect a gradient of cyclic-AMP emission along the grex. Migrating grex cells themselves are not chemotactically sensitive to cyclic-AMP. It seems unlikely that chemotaxis is involved in controlling the polarity of grex movement.     

Characteristics of DNA repair of a UV-sensitive mutant (radC) of Dictyostelium discoideum

Summary A radiation-sensitive mutant, TW8(radC), of Dictyostelium discoideum is more sensitive to ultraviolet light (UV) killing than the parental wild strain NC4(RAD ⁺), but is resistant to 4-nitroquinoline 1-oxide (4NQO) at almost the same level as NC4. In TW8 amoebae, single-strand breaks of DNA molecules were hardly detectable immediately after UV irradiation, and the removal of pyrimidine dimers was depressed during the postirradiation incubation when compared with that of NC4 amoebae. After treatment with 4NQO, however, single-strand breaks were detected in TW8 amoebae. The almost complete rejoining of these breaks was also detected after the removal of 4HAQO-adducts. The TW8 amoebae have an efficient repair capacity against DNA damage caused by 4NQO, MMS, MMC and MNNG but not UV.Abbreviations 4NQO 4-nitroquinoline 1-oxide - MMS methyl methanesulphonate - MMC mitomycin C - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

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 Ca²⁺-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.