Role of cAMP-dependent protein kinase during growth and early development of Dictyostelium discoideum

cAMP-dependent protein kinase (PKA) is an essential regulator of gene expression and cell differentiation during multicellular development of Dictyostelium discoideum. Here we show that PKA activity also regulates gene expression during the growth phase and at the transition from growth to development. Overexpression of PKA leads to overexpression of the discoidinIgamma promoter, while expression of the discoidinIgamma promoter is reduced when PKA activity is reduced, either by expression of a dominant negative mutant of the regulatory subunit or by disruption of the gene for the catalytic subunit (PKA-C). The discoidin phenotype of PKA-C null cells is cell autonomous. In particular, normal secretion of discoidin-inducing factors was demonstrated. In addition, PKA-C null cells are able to respond to media conditioned by PSF and CMF. We conclude that PKA is a major activator of discoidin expression. However, it is not required for production or transduction of the inducing extracellular signals. Therefore, PKA-dependent and PKA-independent pathways regulate the expression of the discoidin genes.     

Induction by folate and folate analogs of extracellular and membrane-bound phosphodiesterase from Dictyostelium discoideum

Folate stimulation is known to enhance Dictyostelium discoideum differentiation. During early differentiation, D. discoideum cells possess two classes of folate receptors which can be distinguished by their difference in specificity (R. J. W. de Wit, FEBS Lett. 150, 445-448, 1982). We investigated the type of receptor by which folate affects cell differentiation. Two independently regulated developmental markers were used: the extracellular phosphodiesterase-inhibitor system and cell-surface phosphodiesterase activity. Our results indicate that the major effect of folate on development is mediated by the folate-specific receptor. The nonspecific folate receptor was only involved in a minor, transient enhancement of the extracellular phosphodiesterase activity very early in development.     

Use of a transactive regulatory mutant of Dictyostelium discoideum in a eucaryotic expression system.

The discoidin proteins of Dictyostelium discoideum are highly expressed during development. The Disc I gamma promoter allows the regulation of heterologous protein expression by experimental conditions. We report conditions under which the promoter activity is efficiently repressed during growth in the wildtype strain AX2. In addition we show that a mutant which overexpresses the discoidins also overexpresses the reporter genes beta-galactosidase, luciferase and CAT 10- to 100-fold when these are placed under the control of a Disc I gamma promoter. This system may be generally useful for the overexpression of genes in Dictyostelium, both for functional studies in vivo and for the production of heterologous proteins for purification.     

Signaling molecules involved in the transition of growth to development of Dictyostelium discoideum

The social amoeba Dictyostelium discoideum, a powerful paradigm provides clear insights into the regulation of growth and development. In addition to possessing complex individual cellular functions like a unicellular eukaryote, D. discoideum cells face the challenge of multicellular development. D. discoideum undergoes a relatively simple differentiation process mainly by cAMP mediated pathway. Despite this relative simplicity, the regulatory signaling pathways are as complex as those seen in metazoan development. However, the introduction of restriction-enzyme-mediated integration (REMI) technique to produce developmental gene knockouts has provided novel insights into the discovery of signaling molecules and their role in D. discoideum development. Cell cycle phase is an important aspect for differentiation of D. discoideum, as cells must reach a specific stage to enter into developmental phase and specific cell cycle regulators are involved in arresting growth phase genes and inducing the developmental genes. In this review, we present an overview of the signaling molecules involved in the regulation of growth to differentiation transition (GDT), molecular mechanism of early developmental events leading to generation of cAMP signal and components of cAMP relay system that operate in this paradigm.     

Vacuole membrane protein 1 is an endoplasmic reticulum protein required for organelle biogenesis, protein secretion, and development

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1(-) Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1(-) cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.     

Role of phospholipase C in Dictyostelium: formation of inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity.

The micro-organism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphatidylinositol-specific phospholipase C (PLC) shows strong homology with the mammalian PLC-delta isoforms. To study the role of PLC in Dictyostelium, a plc- mutant was constructed by disruption of the PLC gene. No basal or stimulated PLC activity could be measured during the whole developmental programme of the plc- cells. Loss of PLC activity did not result in a visible alteration of growth or development. Further analysis showed that developmental gene regulation, cAMP-mediated chemotaxis and activation of guanylyl and adenylyl cyclase were normal. Although the cells lack PLC activity, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was present at only slightly lower concentrations compared with control cells. Mass analysis of inositol phosphates demonstrated the presence of a broad spectrum of inositol phosphates in Dictyostelium, which was unaltered in the plc- mutant. Cell labelling experiments with [3H]inositol indicated that [3H]Ins(1,4,5)P3 was formed in a different manner in the mutant than in control cells.     

Analysis of the prestarvation response in growing cells of Dictyostelium discoideum

We have previously shown that growing cells of Dictyostelium discoideum (strains NC4 and AX3) produce a soluble substance that accumulates in the medium in proportion to cell density; this substance regulates the production of certain proteins previously thought to be induced by starvation [Clarke et al., 1987]. We suggest the name PSF (prestarvation factor) for this substance. During growth, Dictyostelium cells monitor the relative concentrations of PSF and food bacteria. When PSF reaches a sufficiently high level relative to the concentration of bacteria, synthesis of PSF-regulated proteins is induced. We propose the name prestarvation response for this induction, which takes place in exponentially growing cells several generations before the food bacteria are depleted. We have explored the mechanism by which the food bacteria inhibit the response of Dictyostelium cells to PSF. We find that the bacteria do not inactivate PSF or inhibit its production; instead, they affect the ability of NC4 cells to detect PSF, possibly by binding to the same cell surface receptor. In the absence of bacteria, as during axenic growth of AX3 cells, the prestarvation response occurs at much lower cell densities, probably accounting for the presence of certain developmentally regulated mRNAs and proteins in axenic cultures.     

Dictyostelium annexin VII (synexin). cDNA sequence and isolation of a gene disruption mutant.

By cloning the cDNA coding for the membrane associated actin-binding protein p24, we identified a repetitive sequence motif consisting of the amino acids Gly, Tyr, Pro, Gln which is characteristic for a gene family in Dictyostelium discoideum. Using a cDNA probe corresponding to this motif, we isolated cDNA clones coding for a protein of the annexin family. On the basis of a long NH2-terminal sequence encompassing the Gly/Tyr/Pro/Gln motifs, the Dictyostelium annexin was identified as a homolog of vertebrate annexin VII (synexin). The mRNA coding for the Dictyostelium annexin VII has a size of 1.6 kilobases and is present during all developmental stages. Annexin VII is coded for by a single gene in Dictyostelium. A mutant deficient in annexin VII was isolated using a vector which carried the amino-terminal third of the Dictyostelium annexin VII cDNA followed by a viral epitope specific for a monoclonal antibody and a stop codon. Using this approach, homologous recombination in the annexin VII gene led to an expression of the viral epitope under the control of the endogenous annexin VII promoter. Lack of annexin VII is not a lethal event for D. discoideum, and the cells are able to undergo development on agar plates.     

Expression of early developmental genes in Dictyostelium discoideum is initiated during exponential growth by an autocrine-dependent mechanism.

Throughout growth, Dictyostelium cells continuously produce an autocrine factor, PSF, that accumulates in proportion to cell density. Production of PSF declines rapidly when cells are shifted to starvation conditions, and the properties of PSF are distinct from those of regulatory factors produced by starving cells. During late exponential growth, PSF induces expression of several early developmental genes, including those for proteins important in cAMP signaling and cell aggregation. Examples are the aggregation stage cAMP receptor (cAR1), the aggregation-specific form of cyclic nucleotide phosphodiesterase, and gp24 (contact sites B). Through PSF, growing cells detect environmental conditions (cell number high, food approaching depletion) that are appropriate for production of the gene products needed to initiate aggregation and development.     

A pleiotropic defect in cAMP-regulated gene expression in the Dictyostelium agg- mutant synag 7

During differentiation of Dictyostelium discoideum, cAMP functions as a diffusible, extracellular signal to direct chemotaxis and regulate developmental gene expression. The availability of signal-transduction mutants of Dictyostelium now makes it feasible to pursue a genetic analysis of cAMP signaling. The synag 7 mutant is defective in receptor-mediated adenylate cyclase stimulation and cannot relay a cAMP signal. To further characterize this mutant, mRNA levels of several cAMP-regulated genes were measured during development. cAMP-regulated gene expression was found to be dramatically altered in synag 7:several different genes which require cAMP for expression in wild-type cells were induced in synag 7 in the absence of cAMP. In addition, the gene-encoding discoidin I, which is normally expressed in starved cells and repressed by cAMP, is expressed at very low levels in starved synag 7 cells, possibly due to precocious repression. These results suggest that a pleiotropic regulator of cAMP-regulated gene expression is uncoupled from its normal controls during development in synag 7.     

CbfA, the C-module DNA-binding factor, plays an essential role in the initiation of Dictyostelium discoideum development

We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.     

Induction of gene expression in Dictyostelium by prestarvation factor, a factor secreted by growing cells

During growth, Dictyostelium cells continuously secrete a factor, PSF, that accumulates in proportion to cell density. At sufficient concentration, it triggers the production of discoidin I and certain lysosomal enzymes. Our earlier studies demonstrated these effects of PSF on protein and enzyme levels [Clarke et al., Differentiation 34:79-87, 1987; Clarke et al., Dev Genet 9: 315-326, 1988]. In the present study, we have examined whether PSF induces increased mRNA levels. By Northern blot analysis, we have found that discoidin I mRNA accumulates in exponentially growing NC4 cells as the cells reach high density; significant levels of mRNA are detectable in cells growing either on plates or in suspension, beginning about four generations before the end of exponential growth. High levels of discoidin I mRNA are also found in low-density cells grown in the presence of buffer conditioned by high-density cells. These results indicate that PSF induces the accumulation of discoidin I mRNA. Other "early developmental" genes, pCZ22 and the early I genes (16, 18, and 111), are also expressed in exponentially growing cells at high density or in the presence of conditioned buffer. We conclude that several genes previously found to be preferentially expressed very early in development are actually induced during late exponential growth by PSF.     

Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium

To investigate the intracellular role of the clathrin heavy chain in living cells, we have used "antisense" RNA to engineer mutant Dictyostelium discoideum cells that are severely deficient in clathrin heavy chain expression. Immunoblots stained with an anti-clathrin heavy chain antiserum revealed that mutant cells contained undetectable amounts of clathrin heavy chain protein. Similarly, Northern blots showed an absence of clathrin heavy chain mRNA. Clathrin heavy chain-deficient Dictyostelium cells were viable, but exhibited growth rates twofold slower than parental cells. Whereas many morphological features of the mutant cells were normal, mutant cells lacked coated pits and coated vesicles. Clathrin-deficient cells were also missing large translucent vacuoles that serve as endosomes and contractile vacuoles. In the absence of clathrin heavy chain, mutant cells displayed three distinct functional defects: (a) impairment in endocytosis of fluid phase markers, but competence in another endocytic pathway, the phagocytosis of solid particles; (b) defects in osmoregulation; and (c) inability to complete the starvation-induced development cycle.     

Absence of complementation in non-allelic mutants of Dictyostelium discoideum with defects in the transition from growth to development

In Dictyostelium discoideum, growth and development are mutually exclusive and the transition between the two phases of the life cycle is regulated by the environment. This regulation is disturbed in HBW3, a chemically induced mutant with an unknown molecular defect. The mutant develops rapidly and expresses developmental markers during growth. Here we show that HBW3 fails to complement another mutant which has a similar phenotype: a targeted knock-out of the gdt1 gene. We further show that both mutations are recessive and that both are located on chromosome III, suggesting that the two mutations might be allelic. Molecular analysis, however, demonstrates that the gdt1 gene is not mutated in HBW3. Thus, although a wild-type copy of each gene is present in diploid cell lines, the defects due to the recessive mutations synergize to produce a detectable phenotype. The phenotypic similarities and differences between the two mutants are discussed.     

新型重组糖蛋白表达载体--盘基网柄菌

近年来,盘基网柄菌作为异源重组糖蛋白表达载体的研究受到了学术界的重视,已经有多种具有生物活性的复杂糖蛋白成功地得到了表达。通过对表达产物的研究发现,盘基网柄菌具有各种翻译后加工机制,例如磷酸化、酰基化及形成GPI(糖基磷脂酰基醇)锚点等,具有类似于高等动物的糖基化修饰能力。与哺乳动物细胞表达载体相比较,盘基网柄菌具有培养成本低廉、细胞生长迅速及易于大规模培养的优势。盘基网柄菌有可能发展成为一种有重要应用前景的糖蛋白表达载体系统。     

A mutation in the cAMP signaling pathway affects sexual development of Dictyostelium discoideum

Amoebae of cellular slime molds have two developmental modes, asexual fruiting body formation and sexual macrocyst formation. How developmental choice is made is an interesting subject of wide importance. Light exposure and dry conditions are favorable for asexual development, while conditions of darkness and high humidity are so for sexual development. In Dictyostelium discoideum , the latter conditions enhance zygote formation, which determines the fate of surrounding cells for sexual development. Here, a mutant (TMC1) defective in the post-fusion aggregation of cells during sexual development is described. This mutant is also aggregationless in asexual development, and the level of cyclic adenosine monophosphate (cAMP) receptor is reduced. Correspondingly, a series of existing mutants with defects in cAMP signaling pathways showed the same sexual phenotype as TMC1. These results suggest that molecular mechanisms of development are shared by the two alternative developmental modes.     

Altered prestarvation response in a nystatin resistant Dictyostelium discoideum mutant

Wild-type Dictyostelium amoebae secrete an autocrine, prestarvation factor (PSF) that allows them to measure the amount of food bacteria compared to their cell density. When the ratio of PSF to bacteria reaches a threshold, the cells are signaled to prepare for eventual starvation. This prestarvation response (PSR) usually starts three to four generations before the end of exponential growth, leading to the accumulation of several aggregation specific genes during growth. We characterize a nystatin-resistant mutant, HK19, that expresses the PSR genes three generations earlier than wild type but has an otherwise wild-type PSR. Although HK19 has a full PSR during growth, HK19 continues to grow at the wild-type rate and reaches normal cell densities. Because HK19 temporally separates the PSR from starvation, it became possible to test whether starvation is required for development. Since HK19 growing at low density can be induced to clump with either cAMP or folate, it appears that the PSR and an external signal are sufficient for entry into development. These data suggest that the PSR is a complex genetic pathway that induces genes involved in the exit from growth and the entry into development.