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 expression of glycoproteins in the extracellular matrix of the cellular slime mold Dictyostelium discoideum

In this report we examine the accumulation of glycoconjugates in the extracellular medium and insoluble matrices surrounding developing cells of the cellular slime mold Dictyostelium discoideum. Conditions were employed which permitted advanced development (slug stage and beyond) in suspension culture. Under these conditions, up to one-third of the total culture protein appeared as non-sedimentable, extracellular material over the course of 48 h of incubation. Most of the secreted molecules expressed carbohydrate antigens (glycoantigens) as detected by Western blotting, using a panel of six monoclonal antibodies. Since the glycoantigens are secreted, immunoelectron microscopy was used to localize the glycoantigens in the extracellular matrices surrounding normally developing cells, including the slime sheath, stalk tube, inner spore coat, outer spore coat, and intercellular fluid between spores. Each glycoantigen had a characteristic distribution, and each extracellular matrix space contained a unique combination of glycoantigens. Thus, although each of these matrices (except inter-spore fluid) contains cellulose as a primary component, they could be distinguished on the basis of their glycoantigen and, by inference, glycoprotein compositions. Furthermore, there were differences between anterior and posterior regions of both slime sheaths and stalk tubes. These observations show that secretion as detected in suspension culture occurs under normal conditions as a part of the process of depositing extracellular matrices around the cells. The distributions show that the cell aggregate positionally regulates the expression and deposition of secretory glycoproteins; the resultant patterns of expression of unique protein-linked carbohydrate structures imply a functional role in matrix organization and possibly cell activity which can now be explored.     

Sequence and expression of annexin VII of Dictyostelium discoideum.

Sequence analysis reveals that a gene expressed during growth and early development of Dictyostelium discoideum encodes a polypeptide which exhibits extensive similarity with annexins, a family of calcium/phospholipid binding proteins. Comparison of the amino acid composition of the N-termini suggests that the Dictyostelium annexin is a homologue of human synexin, also referred to as annexin VII.     

Establishment of a transient expression system for Dictyostelium discoideum.

We have established a rapid and sensitive transient expression system for Dictyostelium discoideum. We constructed a gene fusion containing the promoter from the Dictyostelium Actin 15 gene fused to the firefly luciferase gene. The enzymatic activity of this gene fusion, expressed at very high levels in stable transformants, was measured to determine optimum conditions for transient expression using electroporation to introduce the DNA into cells. With these conditions, we show that a luciferase gene fusion driven by a prestalk, cell-type specific promoter from the pst-cathepsin gene expresses luciferase at the appropriate developmental stage. In addition, we present results suggesting that the system will be useful for expressing genes in non-axenic cell lines. Finally, we observe that electroporation is more efficient for obtaining stable transformations than the standard calcium phosphate procedure using extrachromosomally replicating shuttle vectors but less efficient for vectors that integrate into the Dictyostelium chromosomes.     

Phosphotyrosine-containing proteins in Dictyostelium discoideum.

Phosphotyrosine-containing proteins in Dictyostelium discoideum were detected by immunoblot analysis and immunoprecipitation using a monoclonal anti-phosphotyrosine antibody. The iodinated antibody recognized on bots a cluster of 205-220 kDa polypeptides and bands of 107 and 60 kDa. The 107 and 60 kDa polypeptides and, in addition, a 82 kDa one became phosphorylated on tyrosine when the immunoprecipitate was incubated with [gamma-32P]ATP. In preparations from differentiating cells the intensity of the label was increased in the 60 kDa band and decreased in the 107 and 205-220 kDa bands.     

Developmental decisions in Dictyostelium discoideum.

A few hours after the onset of starvation, amoebae of Dictyostelium discoideum start to form multicellular aggregates by chemotaxis to centers that emit periodic cyclic AMP signals. There are two major developmental decisions: first, the aggregates either construct fruiting bodies directly, in a process known as culmination, or they migrate for a period as slugs. Second, the amoebae differentiate into either prestalk or prespore cells. These are at first randomly distributed within aggregates and then sort out from each other to form polarized structures with the prestalk cells at the apex, before eventually maturing into the stalk cells and spores of fruiting bodies. Developmental gene expression seems to be driven primarily by cyclic AMP signaling between cells, and this review summarizes what is known of the cyclic AMP-based signaling mechanism and of the signal transduction pathways leading from cell surface cyclic AMP receptors to gene expression. Current understanding of the factors controlling the two major developmental choices is emphasized. The weak base ammonia appears to play a key role in preventing culmination by inhibiting activation of cyclic AMP-dependent protein kinase, whereas the prestalk cell-inducing factor DIF-1 is central to the choice of cell differentiation pathway. The mode of action of DIF-1 and of ammonia in the developmental choices is discussed.     

Oligosaccharide heterogeneity of glycoproteins sulfated during the vegetative growth of Dictyostelium discoideum

Macromolecules are sulfated during the vegetative growth of Dictyostelium discoideum. A characterisation of the structures of sulfated oligosaccharides associated with these macromolecules indicates that the oligosaccharides are heterogeneous. Endoglycosidase and pronase digestion were used with gel-filtration chromatography to obtain two different oligosaccharide fractions and a glycopeptide fraction; these were further characterised by ion-exchange and lectin-affinity chromatography and by acid hydrolysis. The data indicate that up to 43% of the sulfate is associated with typical N-linked oligosaccharides, that up to 5% is associated with N-linked oligosaccharides that are either very large or extremely highly charged, and that the remaining sulfate is associated with oligosaccharides non-N-linked to protein. Each fraction was also shown to be heterogeneous at most other structural levels. Electrophoretic analyses following the endoglycosidase and pronase treatments indicated that all of the macromolecules are glycoproteins and suggested further that at least two of the oligosaccharide fractions are located on different groups of glycoproteins.     

Codon preference in Dictyostelium discoideum.

Dictyostelium discoideum is of increasing interest as a model eukaryotic cell because its many attributes have recently been expanded to include improved genetic and biochemical manipulability. The ability to transform Dictyostelium using drug resistance as a selectable marker (1) and to gene target by high frequency homologous integration (2) makes this organism particularly useful for molecular genetic approaches to cell structure and function. Given this background, it becomes important to analyze the codon preference used in this organism. Dictyostelium displays a strong and unique overall codon preference. This preference varies between different coding regions and even varies between coding regions from the same gene family. The degree of codon preference may be correlated with expression levels but not with the developmental time of expression of the gene product. The strong codon preference can be applied to identify coding regions in Dictyostelium DNA and aid in the design of oligonucleotide probes for cloning Dictyostelium genes.     

Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants

myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on inositol-3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mould Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid medium, the mutants lose their viability when deprived of inositol for longer than 24 h. Here, we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol-polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-BPG is presented.     

Membrane sites regulating developmental gene expression in Dictyostelium discoideum

Postaggregative gene expression in Dictyostelium discoideum requires cell contact. Polyspecific monovalent antibodies (Fab) prepared from sera raised against membranes of aggregation- and postaggregation-stage cells were used to probe the cell interactions that induce rapid postaggregative synthesis of UDP-glucose pyrophosphorylase. When cells of strain V12M2 were dissociated after 8 hr of development and replated in the presence of immune Fab, both reaggregation and pyrophosphorylase synthesis were blocked. Fab neutralized by incubation with EDTA-high salt extracts of cells developed for 3 hr blocked pyrophosphorylase synthesis but not reaggregation. Therefore, some cell-surface components that regulate pyrophosphorylase synthesis (called E sites) are antigenically distinct from those required for reaggregation. The Fab provides a means to assay E sites during their purification. Addition of 10(-3) M cyclic AMP or cyclic GMP enabled the cells to bypass the blocking of E sites by Fab; pyrophosphorylase was synthesized in the absence of reaggregation. We hypothesize that E sites function by raising the level of intracellular cyclic AMP.     

Cyclic AMP and the control of aggregative phase gene expression in Dictyostelium discoideum.

The induction of aggregative phase functions and the acceleration of the onset of aggregation competence by nanomolar pulses of cyclic AMP can be mimicked by exposing developing cells to a high extracellular concentration of either cyclic AMP or cyclic GMP (5 × 10^?4M) during the first 1–2 hr of development. Pulses of cyclic AMP have previously been shown to result in oscillations of intracellular cyclic AMP concentration; we show that high extracellular concentrations of cyclic AMP and cyclic GMP cause intracellular cyclic AMP levels to increase. We describe a mutant, HM11, which has elevated levels of intracellular cyclic AMP from the beginning of development and which begins to accumulate cell-associated phosphodiesterase, an aggregative phase enzyme, within an hour of starvation. Our data suggest that the expression of aggregative phase functions is controlled by an elevation of intracellular cyclic AMP which may be either continuous or periodic.     

Acetylated and nonacetylated actins in Dictyostelium discoideum.

We have carried out a two-dimensional gel analysis of the actin system of Dictyostelium discoideum. Our results show that on the basis of isoelectric focusing, there is a single major [35S]methionine-labeled species which corresponds both to the actin purified by Uyemura et al. (Uyemura, D., Brown, S.S., and Spudich, J.A. (1978) J. Biol. Chem. 253, 9088-9095) and to the Coomassie Blue staining species seen in whole cell lysates of the organism. We also detect a minor labeled actin species, x, which has no corresponding Coomassie Blue staining counterpart. This species turns over much more rapidly than the major actin and has one more positive charge. It is not labeled with [3H]acetate, whereas the major actin is. When D. discoideum RNA is added to a mRNA-dependent rabbit reticulocyte lysate protein translation system, only one major actin is seen, and this species corresponds to the major actin observed in vivo. If endogenous acetyl coenzyme A is removed from the translation system, a second major actin appears corresponding in position to x. These results indicate that in D. discoideum, there is present a single major actin species in addition to a small amount of a rapidly turning over actin which is a nonacetylated form of the major actin. Additional experiments examining these actins through the developmental cycle of the organism show no consistent differences with the results obtained using vegetative cells.     

Endogenous biotinylated proteins in Dictyostelium discoideum.

Biotin-dependent enzymes are involved in carboxylation, decarboxylation and transcarboxylation reactions. Here, we have used sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotting followed by probing with avidin to identify biotin-containing polypeptides in Dictyostelium discoideum. Twenty biotinyl polypeptides were visualized, with a 23 kDa protein appearing transiently. Based upon the molecular mobility of the biotinyl polypeptides, D. discoideum may contain the biotin-dependent enzymes acetyl CoA carboxylase, proprionyl CoA carboxylase, pyruvate carboxylase, and 3-methylcrotonyl CoA carboxylase.