Influence of cyclic AMP and hydrolysis products on cell type regulation in Dictyostelium discoideum

Development ◽  
1985 ◽  
Vol 86 (1) ◽  
pp. 19-37
Author(s):  
Cornelis J. Weijer ◽  
Antony J. Durston
Keyword(s):  
Cyclic Amp ◽  
Suspension Culture ◽  
Dictyostelium Discoideum ◽  
Double Negative ◽  
Cell Type ◽  
Camp Phosphodiesterase ◽  
Feedback Model ◽  
Hydrolysis Products ◽  
Low Concentrations ◽  
Extracellular Camp

We describe the effect of cyclic AMP on regulation of the proportion of prespore and prestalk cells in Dictyostelium discoideum. Prespore and prestalk cells from slugs were enriched on Percoll density gradients and allowed to regulate in suspension culture under 100% oxygen. The transition of prespore to prestalk cells is blocked by cAMP, while cAMP phosphodiesterase and caffeine cause a decrease in the number of prespore cells. This suggests that extracellular cAMP plays a role in cell type proportioning by inhibiting the conversion of prespore to prestalk cells. Low concentrations of cAMP prevent the conversion of prestalk to prespore cells; the same effect is seen with hydrolysis products of cAMP, 5 AMP, adenosine and also adenine. We suggest that, when low concentrations of cAMP are added to regulating cells, the cAMP itself is quickly broken down and the breakdown products thereafter inhibit the prestalk-to-prespore conversion. The relevance of these findings is discussed in the context of an non-positional double-negative feedback model for cell type homeostasis.

Differentiation of Dictyostelium discoideum mutant cells in a shaken suspension culture and the effect of cyclic AMP

1981 ◽  
Vol 51 (1) ◽  
pp. 131-142
Author(s):  
K. Abe ◽  
Y. Saga ◽  
H. Okada ◽  
K. Yanagisawa
Keyword(s):  
Cyclic Amp ◽  
Suspension Culture ◽  
Dictyostelium Discoideum ◽  
Solid Surface ◽  
Enzyme Activities ◽  
Parental Strain ◽  
Mutant Cell ◽  
Specific Activities ◽  
Mutant Cells ◽  
Kinetics Of

In Dictyostelium discoideum, 16 mutants in which cells differentiate into spores and stalk cells without normal morphogenesis were isolated. All these mutants are rapidly developing and capable of differentiating in a shaken suspension of phosphate buffer.The developmental kinetics of specific activities of enzymes in one of the mutants, HTY 1851, cultured in the suspension was compared with that in the parental strain, X2, developed on a solid surface. Most of the enzyme activities appeared much earlier and the peaks of the activities were lower in HTY1851 than X2, but the order or appearance of the activities was the same in both the strains cultured under the conditions described above. These results suggest that the biochemical steps in the development of the mutant in a shaken suspension are essentially the same as those of the parental strain X2 on a solid surface. It was also found that addition of cyclic AMP (2.5 X 10-5 M to 1 X 10-4 M) to the mutant cell suspension 6–8 h after the initiation of development induced an increase in the number of spores and the specific activities of some enzymes to values twice as high as those of an untreated control.

Identification of the sequences controlling cyclic AMP regulation and cell-type-specific expression of a prestalk-specific gene in Dictyostelium discoideum

1987 ◽  
Vol 7 (1) ◽  
pp. 149-159
Author(s):  
S Datta ◽  
R A Firtel
Keyword(s):  
Amino Acids ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Base Pair ◽  
Regulatory Region ◽  
Specific Gene ◽  
Cell Type ◽  
Specific Expression ◽  
Endogenous Gene ◽  
Cell Type Specific Expression

We have cloned and analyzed a developmentally and spatially regulated prestalk cell-specific gene from Dictyostelium discoideum. The gene encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. Amino acid comparisons between these enzymes showed that the active-site amino acids were conserved, as were amino acids known to be important for catalysis and residues which form the intramolecular cysteine bridges. We have constructed a series of internal deletions, duplications, and linker scanner mutations within the region 300 base pairs 5' to the cap site. Analysis of expression of the mutations in transformants identified a approximately 35-base pair GC-rich region containing a dAdC/dGdT palindromic repeat and a G-rich box which is homologous to the 3' GT half of the palindromic repeat. Deletion or disruption of the G box resulted in a approximately 50-fold drop in the level of expression of the gene fusion in transformants in response to cyclic AMP in single-cell culture but did not affect the temporal pattern of regulation or control by cyclic AMP. The expression of such constructs during normal multicellular differentiation paralleled that of the endogenous gene; however, the level of RNA from the constructs was only approximately 10-fold lower than that of constructs containing the G box. Deletion of the 3' half of the palindromic sequence and the G box region resulted in a dramatic decrease in the level of transcription, although the constructs still showed proper temporal expression. These results suggest that this 35-base-pair region acts as an important part of the regulatory region for cell type and cyclic AMP regulation.

Cyclic AMP-phosphodiesterase induces dedifferentiation of prespore cells in Dictyostelium discoideum slugs: evidence that cyclic AMP is the morphogenetic signal for prespore differentiation

Development ◽  
1988 ◽  
Vol 103 (3) ◽  
pp. 611-618 ◽  
Author(s):  
M. Wang ◽  
R. van Driel ◽  
P. Schaap
Keyword(s):  
Cyclic Amp ◽  
Hydrolysis Product ◽  
Camp Level ◽  
Camp Phosphodiesterase ◽  
Multicellular Development ◽  
Local Reduction ◽  
Coated Spheres ◽  
Extracellular Camp ◽  
Camp Levels ◽  
General Reduction

We investigated whether cyclic AMP is an essential extracellular stimulus for the differentiation of prespore cells in slugs of D. discoideum. A local reduction of the extracellular cAMP level inside the slug was induced by implantation of cAMP-phosphodiesterase (cAMP-PDE)-coated spheres in intact slugs. This treatment caused the disappearance of prespore antigen in the vicinity of the sphere. A general reduction of extracellular cAMP levels in slugs, induced by submerging slugs in 0.25i.u.ml-1 cAMP-PDE, reduced the proportion of prespore cells from 66% to 15%, without affecting slug morphology. The cAMP-PDE-induced dedifferentiation of prespore cells was counteracted by cAMP and was not due to the production of the hydrolysis product 5′AMP, but to the reduction of extracellular cAMP levels. We conclude that extracellular cAMP is the major morphogenetic signal for the differentiation of prespore cells in the multicellular stages of D. discoideum development and we present a working hypothesis for the generation of the prestalk/prespore pattern during multicellular development.

Spatial and temporal regulation of a foreign gene by a prestalk-specific promoter in transformed Dictyostelium discoideum

1986 ◽  
Vol 6 (3) ◽  
pp. 811-820
Author(s):  
S Datta ◽  
R H Gomer ◽  
R A Firtel
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Regulatory Elements ◽  
Foreign Gene ◽  
Specific Gene ◽  
Cell Type ◽  
Coding Region ◽  
Endogenous Gene ◽  
Temporal Regulation

We analyzed a developmentally regulated prestalk-specific gene from Dictyostelium discoideum encoding a cathepsin-like protease. A hybrid gene was constructed by fusing 2.5 kilobases of 5' flanking sequences and part of the coding region of the gene in-frame to the Escherichia coli beta-glucuronidase gene and was transformed into D. discoideum cells. In cells transformed with this vector, the gene fusion showed the same temporal regulation as the endogenous gene during multicellular development and, like endogenous prestalk genes, was highly inducible by cyclic AMP in in vitro cell cultures. Moreover, immunofluorescence studies showed that the fusion protein had the same spatial distribution within the migrating pseudoplasmodium as the endogenous gene. The results indicate that the regions of the D. discoideum prestalk-specific cathepsin gene contain all the necessary information for proper temporal, spatial, and cyclic AMP regulation of a prestalk cell-type gene in D. discoideum transformants and leads the way for experiments to identify the cell-type-specific regulatory elements.

scholarly journals EppA, a Putative Substrate of DdERK2, Regulates Cyclic AMP Relay and Chemotaxis in Dictyostelium discoideum

2006 ◽  
Vol 5 (7) ◽  
pp. 1136-1146 ◽  
Author(s):  
Songyang Chen ◽  
Jeffrey E. Segall
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Mitogen Activated Protein Kinase ◽  
Intracellular Camp ◽  
Mitogen Activated Protein ◽  
Delayed Development ◽  
Induced Signal ◽  
Extracellular Camp ◽  
Intracellular Camp Accumulation ◽  
Dimensional Electrophoresis

ABSTRACT The mitogen-activated protein kinase DdERK2 is critical for cyclic AMP (cAMP) relay and chemotaxis to cAMP and folate, but the details downstream of DdERK2 are unclear. To search for targets of DdERK2 in Dictyostelium discoideum,32PO4 3−-labeled protein samples from wild-type and Dderk2 − cells were resolved by 2-dimensional electrophoresis. Mass spectrometry was used to identify a novel 45-kDa protein, named EppA (ERK2-dependent phosphoprotein A), as a substrate of DdERK2 in Dictyostelium. Mutation of potential DdERK2 phosphorylation sites demonstrated that phosphorylation on serine 250 of EppA is DdERK2 dependent. Changing serine 250 to alanine delayed development of Dictyostelium and reduced Dictyostelium chemotaxis to cAMP. Although overexpression of EppA had no significant effect on the development or chemotaxis of Dictyostelium, disruption of the eppA gene led to delayed development and reduced chemotactic responses to both cAMP and folate. Both eppA gene disruption and overexpression of EppA carrying the serine 250-to-alanine mutation led to inhibition of intracellular cAMP accumulation in response to chemoattractant cAMP, a pivotal process in Dictyostelium chemotaxis and development. Our studies indicate that EppA regulates extracellular cAMP-induced signal relay and chemotaxis of Dictyostelium.

scholarly journals A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum.

1985 ◽  
Vol 5 (4) ◽  
pp. 705-713 ◽  
Author(s):  
M C Mehdy ◽  
R A Firtel
Keyword(s):  
Gene Expression ◽  
Cyclic Amp ◽  
Conditioned Medium ◽  
Dictyostelium Discoideum ◽  
Single Cells ◽  
Gene Induction ◽  
Specific Gene ◽  
Additional Parameter ◽  
Cell Type ◽  
Cell Type Specific

We are studying cell differentiation in Dictyostelium discoideum by examining the regulation of genes that are preferentially expressed in different cell types. A system has been established in which prestalk- and prespore-cell-specific genes are expressed in single cells in response to culture conditions. We confirm our previous results showing that cyclic AMP induces prestalk genes and now show that it is also required for prespore gene induction. The expression of both classes of genes is additionally dependent on the presence of a factor(s) secreted by developing cells which we call conditioned medium factor(s). An assay for conditioned medium factor(s) shows that it is detectable within 2.5 h after the onset of development. Conditioned medium factor(s) also promotes the expression of genes induced early in development, but has no detectable effect on the expression of actin genes and a gene expressed maximally in vegetative cells. In the presence of conditioned medium factor(s), exogenous cyclic AMP at the onset of starvation fails to induce the prespore and prestalk genes. The addition of cyclic AMP between 2 and 12 h of starvation results in rapid prestalk gene expression, whereas prespore genes are induced at an invarient time (approximately 18 h after the onset of starvation). These data suggest that cyclic AMP and conditioned medium factor(s) are sufficient for prestalk gene induction, whereas an additional parameter(s) is involved in the control of prespore gene induction. In contrast to several previous studies, we show that multicellularity is not essential for the expression of either prespore or prestalk genes. These data indicate that prespore and prestalk genes have cell-type-specific as well as shared regulatory factors.

scholarly journals Spatial and temporal regulation of a foreign gene by a prestalk-specific promoter in transformed Dictyostelium discoideum.

1986 ◽  
Vol 6 (3) ◽  
pp. 811-820 ◽  
Author(s):  
S Datta ◽  
R H Gomer ◽  
R A Firtel
Keyword(s):  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Regulatory Elements ◽  
Foreign Gene ◽  
Specific Gene ◽  
Cell Type ◽  
Coding Region ◽  
Endogenous Gene ◽  
Temporal Regulation

We analyzed a developmentally regulated prestalk-specific gene from Dictyostelium discoideum encoding a cathepsin-like protease. A hybrid gene was constructed by fusing 2.5 kilobases of 5' flanking sequences and part of the coding region of the gene in-frame to the Escherichia coli beta-glucuronidase gene and was transformed into D. discoideum cells. In cells transformed with this vector, the gene fusion showed the same temporal regulation as the endogenous gene during multicellular development and, like endogenous prestalk genes, was highly inducible by cyclic AMP in in vitro cell cultures. Moreover, immunofluorescence studies showed that the fusion protein had the same spatial distribution within the migrating pseudoplasmodium as the endogenous gene. The results indicate that the regions of the D. discoideum prestalk-specific cathepsin gene contain all the necessary information for proper temporal, spatial, and cyclic AMP regulation of a prestalk cell-type gene in D. discoideum transformants and leads the way for experiments to identify the cell-type-specific regulatory elements.

scholarly journals Genetic Interactions of the E3 Ubiquitin Ligase Component FbxA with Cyclic AMP Metabolism and a Histidine Kinase Signaling Pathway during Dictyostelium discoideum Development

2003 ◽  
Vol 2 (3) ◽  
pp. 618-626 ◽  
Author(s):  
Turgay Tekinay ◽  
Herbert L. Ennis ◽  
Mary Y. Wu ◽  
Margaret Nelson ◽  
Richard H. Kessin ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Signaling Pathway ◽  
Dictyostelium Discoideum ◽  
Ubiquitin Ligase ◽  
Histidine Kinase ◽  
Double Mutant ◽  
E3 Ubiquitin Ligase ◽  
Specific Gene ◽  
Wild Type ◽  
Camp Phosphodiesterase

ABSTRACT Dictyostelium discoideum amoebae with an altered fbxA gene, which is thought to encode a component of an SCF E3 ubiquitin ligase, have defective regulation of cell type proportionality. In chimeras with wild-type cells, the mutant amoebae form mainly spores, leaving the construction of stalks to wild-type cells. To examine the role of fbxA and regulated proteolysis, we have recovered the promoter of fbxA and shown that it is expressed in a pattern resembling that of a prestalk-specific gene until late in development, when it is also expressed in developing spore cells. Because fbxA cells are developmentally deficient in pure culture, we were able to select suppressor mutations that promote sporulation of the original mutant. One suppressor mutation resides within the gene regA, which encodes a cyclic AMP (cAMP) phosphodiesterase linked to an activating response regulator domain. In another suppressor, there has been a disruption of dhkA, a gene encoding a two-component histidine kinase known to influence Dictyostelium development. RegA appears precociously and in greater amounts in the fbxA mutant than in the wild type, but in an fbxA/dhkA double mutant, RegA is restored to wild-type levels. Because the basis of regA suppression might involve alterations in cAMP levels during development, the concentrations of cAMP in all strains were determined. The levels of cAMP are relatively constant during multicellular development in all strains except the dhkA mutant, in which it is reduced at least sixfold. The level of cAMP in the double mutant dhkA/fbxA is relatively normal. The levels of cAMP in the various mutants do not correlate with spore formation, as would be expected on the basis of our present understanding of the signaling pathway leading to the induction of spores. Altered amounts of RegA and cAMP early in the development of the mutants suggest that both fbxA and dhkA genes act earlier than previously thought.

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