Production and activity of spore differentiation factors (SDFs) in Dictyostelium

SDF-1 and SDF-2 are peptides that promote terminal spore differentiation under submerged conditions. The present study shows that they accumulate differentially and are released during the development of wild-type cells and can promote spore formation in cells disaggregated from wild-type culminants. SDF-1 accumulates during the slug stage and is released in a single burst at the onset of culmination while SDF-2 accumulates during early culmination and is released in a single burst from mid-culminants. The effects of SDF-1 and SDF-2 on stalk cell formation in cell monolayers were investigated. SDF-1 by itself induces stalk cell formation in some strains and also synergizes with the stalk-cell-inducing factor, DIF-1. cAMP has an inhibitory effect on stalk cell formation when either DIF-1 or SDF-1 are present on their own but is almost not inhibitory when both are present. SDF-2 alone does not induce stalk cell formation and appears to inhibit the response to DIF-1. At the same time, it increases the extent of vacuolization of the stalk cells that are produced. We propose that the release of SDF-1 and then of SDF-2 may mark irreversible steps in the developmental programme associated, respectively, with culmination and spore maturation.     

Temperature-sensitive Gbeta mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors

Deletion of the single gene for the Dictyostelium G protein beta-subunit blocks development at an early stage. We have now isolated temperature-sensitive alleles of Gbeta to investigate its role in later development. We show that Gbeta is directly required for adenylyl cyclase A activation and for morphogenetic signaling during the entire developmental program. Gbeta was also essential for induction of aggregative gene expression by cAMP pulses, a process that is mediated by serpentine cAMP receptors (cARs). However, Gbeta was not required for cAR-mediated induction of prespore genes and repression of stalk genes, and neither was Gbeta needed for induction of prestalk genes by the differentiation inducing factor (DIF). cAMP induction of prespore genes and repression of stalk genes is mediated by the protein kinase GSK-3. GSK-3 also determines cell-type specification in insects and vertebrates and is regulated by the wingless/wnt morphogens that are detected by serpentine fz receptors. The G protein-dependent and -independent modes of cAR-mediated signaling reported here may also exist for the wingless/wnt signaling pathways in higher organisms.     

A Serum Response Factor homolog is required for spore differentiation in Dictyostelium

A homolog of the Serum Response Factor (SRF) has been isolated from Dictyostelium discoideum and its function studied by analyzing the consequences of its gene disruption. The MADS-box region of Dictyostelium SRF (DdSRF) is highly conserved with those of the human, Drosophila and yeast homologs. srfA is a developmentally regulated gene expressed in prespore and spore cells. This gene plays an essential role in sporulation as its disruption leads to abnormal spore morphology and loss of viability. The mutant spores were round and cellulose deposition seemed to be partially affected. Initial prestalk and prespore cell differentiation did not seem to be compromised in the mutant since the expression of several cell-type-specific markers were found to be unaffected. However, the mRNA level of the spore marker spiA was greatly reduced. Activation of the cAMP-dependent protein kinase (PKA) by 8-Br-cAMP was not able to fully bypass the morphological defects of srfA- mutant spores, although this treatment induced spiA mRNA expression. Our results suggest that DdSRF is required for full maturation of spores and participates in the regulation of the expression of the spore-coat marker spiA and probably other maturation genes necessary for proper spore cell differentiation.     

A deubiquitinating enzyme that disassembles free polyubiquitin chains is required for development but not growth in Dictyostelium

Although cell differentiation usually involves synthesis of new proteins, little is known about the role of protein degradation. In eukaryotes, conjugation to ubiquitin polymers often targets a protein for destruction. This process is regulated by deubiquitinating enzymes, which can disassemble ubiquitin polymers or ubiquitin-substrate conjugates. We find that a deubiquitinating enzyme, UbpA, is required for Dictyostelium development. ubpA cells have normal protein profiles on gels, grow normally, and show normal responses to starvation such as differentiation and secretion of conditioned medium factor. However, ubpA cells have defective aggregation, chemotaxis, cAMP relay, and cell adhesion. These defects result from low expression of cAMP pulse-induced genes such as those encoding the cAR1 cAMP receptor, phosphodiesterase, and the gp80 adhesion protein. Treatment of ubpA cells with pulses of exogenous cAMP allows them to aggregate and express these genes like wild-type cells, but they still fail to develop fruiting bodies. Unlike wild type, ubpA cells accumulate ubiquitin-containing species that comigrate with ubiquitin polymers, suggesting a defect in polyubiquitin metabolism. UbpA has sequence similarity with yeast Ubp14, which disassembles free ubiquitin chains. Yeast ubp14 cells have a defect in proteolysis, due to excess ubiquitin chains competing for substrate binding to proteasomes. Cross-species complementation and enzyme specificity assays indicate that UbpA and Ubp14 are functional homologs. We suggest that specific developmental transitions in Dictyostelium require the degradation of specific proteins and that this process in turn requires the disassembly of polyubiquitin chains by UbpA.     

Two transmembrane signaling mechanisms control expression of the cAMP receptor gene CAR1 during Dictyostelium development

Dictyostelium discoideum is among the best characterized organisms for the study of receptor/guanine nucleotide binding protein-mediated control of differentiation. Dictyostelium grow unicellularly but form fully differentiated multicellular organisms through a developmental program regulated by secreted cAMP activating specific cell-surface receptors. Dictyostelium respond differentially to cAMP at different developmental stages. During early development, expression of certain genes is induced by low-level oscillations of extracellular cAMP. Later, continuous, high cAMP concentrations will promote expression of specific genes in multicellular structures. Here, we show that the cAMP receptor gene CAR1, which is essential for development, utilizes two promoters that are activated at distinct stages of development and respond to different extracellular cAMP conditions. One promoter is active with low-level oscillations of cAMP; exposure to high cAMP concentrations will repress this promoter and induce a second promoter. The CAR1 mRNAs are alternatively spliced but encode identical proteins. Thus, through differential sensitivity to its own ligand, cAMP, two promoters and alternative splicing regulate CAR1 expression during Dictyostelium development.     

An intersection of the cAMP/PKA and two-component signal transduction systems in Dictyostelium

Terminal differentiation of both stalk and spore cells in Dictyostelium can be triggered by activation of cAMP-dependent protein kinase (PKA). A screen for mutants where stalk and spore cells mature in isolation produced three genes which may act as negative regulators of PKA: rdeC (encoding the PKA regulatory subunit), regA and rdeA. The biochemical properties of RegA were studied in detail. One domain is a cAMP phosphodiesterase (Km approximately 5 microM); the other is homologous to response regulators (RRs) of two-component signal transduction systems. It can accept phosphate from acetyl phosphate in a reaction typical of RRs, with transfer dependent on Asp212, the predicted phosphoacceptor. RegA phosphodiesterase activity is stimulated up to 8-fold by the phosphodonor phosphoramidate, with stimulation again dependent on Asp212. This indicates that phosphorylation of the RR domain activates the phosphodiesterase domain. Overexpression of the RR domain in wild-type cells phenocopies a regA null. We interpret this dominant-negative effect as due to a diversion of the normal flow of phosphates from RegA, thus preventing its activation. Mutation of rdeA is known to produce elevated cAMP levels. We propose that cAMP breakdown is controlled by a phosphorelay system which activates RegA, and may include RdeA. Cell maturation should be triggered when this system is inhibited.     

Sequence-specific protein interaction with a transcriptional enhancer involved in the autoregulated expression of cAMP receptor 1 in Dictyostelium

Major stages of Dictyostelium development are regulated by secreted, extracellular cAMP through activation of a serpentine receptor family. During early development, oscillations of extracellular cAMP mobilize cells for aggregation; later, continuous exposure to higher extracellular cAMP concentrations downregulates early gene expression and promotes cytodifferentiation and cell-specific gene expression. The cAMP receptor 1 gene CAR1 has two promoters that are differentially responsive to these extracellular cAMP stimuli. The early CAR1 promoter is induced by nM pulses of cAMP, which in turn are generated by CAR1-dependent activation of adenylyl cyclase (AC). Higher, non-fluctuating concentrations of cAMP will adapt this AC stimulus-response, repress the activated early promoter and induce the dormant late promoter. We now identify a critical element of the pulse-induced CAR1 promoter and a nuclear factor with sequence-specific interaction. Mutation of four nucleotides within the element prevents both in vitro protein binding and in vivo expression of an otherwise fully active early CAR1 promoter and multimerization of the wild-type, but not mutant, sequence will confer cAMP regulation to a quiescent heterologous promoter. These cis and trans elements, thus, constitute a part of the molecular response to the cAMP transmembrane signal cascade that regulates early development of Dictyostelium.     

Detection of subtle phenotypes: the case of the cell adhesion molecule csA in Dictyostelium

Dictyostelium amoebae aggregate into a multicellular organism by cAMP-driven chemotaxis and cell-cell adhesion. Cell adhesion is mediated by an EDTA-sensitive and an EDTA-resistant adhesion system. The latter is developmentally regulated and triggered by homophilic interactions of the membrane glycoprotein csA; on disruption of the encoding gene, EDTA-resistant contacts fail to form. Nevertheless, csA-null cells under usual laboratory conditions aggregate normally and complete development. By using experimental conditions that reproduce more closely the habitat of Dictyostelium amoebae, evidence is provided that csA is required for development and that its expression confers a selective advantage to populations of wild-type cells over csA-null mutants. The latter display reduced cell-cell adhesion, increased adhesiveness to the substratum, and slower motility, which lead to their sorting out from aggregating wild-type cells. It is proposed that the experimental conditions commonly used in the laboratory are not stringent enough to assess the developmental role of csA and other proteins. The assay described can be used to detect subtle phenotypes, to reexamine the developmental role of apparently nonessential genes, and to test the validity of recent models on emergence and maintenance of apparent genetic redundancy.     

A developmentally regulated kinesin-related motor protein from Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive alpha-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes in Dictyostelium. K7, expressed in Escherichia coli, supports plus end-directed microtubule motility in vitro at a speed of 0.14 micron/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.