Fig 7 - uploaded by Christophe Anjard
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Involvement of GABA transaminase in SDF-2 production. (A) Low-density KP cells were incubated overnight (~18 hours) before the addition of the indicated compounds. The following concentrations were used: 1 μM vigabatrin, 10 units of SDF-3, 10 nM CGP55845, 1/5000 anti-GABA antibodies (final dilution), 1/500 anti-AcbA antibodies (final dilution). The number of spores was scored 1 hour after the addition of SDF-3 or 2 hours after the addition of vigabatrin. An aliquot of the cell supernatants was harvested and SDF-2 purified using anion-exchange resin before quantification on fresh KP cells. (B) Low-density gabT-null KP cells lacking functional GABA transaminase were incubated for 17 hours in cAMP buffer. The number of spores was scored before and again 3 hours after addition of 1/5000 anti-GABA antibodies (final dilution), 1/500 anti-AcbA antibodies (final dilution), or 10 nM CGP55845. Aliquots of the cell supernatants were harvested and SDF-2 purified using anion-exchange resin before quantification on fresh KP cells. Minus indicates production of less than 0.2 units of SDF-2/10 3 cells, whereas plus indicates more than 5000 units of SDF-2/10 3 cells.
Source publication
Encapsulation of prespore cells of Dictyostelium discoideum is controlled by several intercellular signals to ensure appropriate timing during fruiting body formation. Acyl-CoA-binding protein, AcbA, is secreted by prespore cells and processed by the prestalk protease TagC to form the 34 amino acid peptide SDF-2 that triggers rapid encapsulation. A...
Contexts in source publication
Context 1
... (gabT). The predicted protein is highly similar to mammalian homologs. The role of gabT was investigated by inactivation using the non-reversible GABA transaminase inhibitor vigabatrin or by disruption of gabT. When vigabatrin was added to low-density KP cells, it induced SDF-2 release after 1 hour, followed by an induction of spore formation (Fig. 7A). This induction was prevented by anti-GABA or anti-AcbA antibodies, as well as by the GABA antagonist CGP 55845. The inactivation of gabT in wild- type cells has little consequence for the development cycle. The gabT-null strain finished development 1-2 hours earlier than the wild-type strain and was less synchronous (not shown). ...
Context 2
... strain finished development 1-2 hours earlier than the wild-type strain and was less synchronous (not shown). Inactivation of gabT in KP cells resulted in a strain that secretes SDF-2 spontaneously. At a cell density of 210 4 /cm 2 , SDF-2 appeared within 16 hours (data not shown), whereas it took 19-20 hours at a density of 110 3 cells/cm 2 (Fig. 7B). Addition of CGP 55845, anti- GABA or anti-AcbA antibodies to the gabT-null KP cells after 17 hours of incubation prevented SDF-2 production and the resulting induction of spore ...
Context 3
... decarboxylase GadA, in which GABA levels are less than 20% of those in KP cells. SDF-3 signaling might stimulate GadA or could inhibit the GABA-degrading enzyme GABA transaminase, or both. We found that low-density cultures of a KP strain lacking GABA transaminase spontaneously produce SDF-2 and form many more spores than the parental KP strain (Fig. 7). This spontaneous sporulation was inhibited by addition of antibodies to GABA or the GABA antagonist CGP55845 to the low-density cultures. The levels of intercellular GABA appear to be in kinetic equilibrium determined by the relative rates of secretion and degradation. It is possible that GrlE only requires transient binding of GABA ...
Citations
... This regulation is principally dictated by the StAR protein, a rapidly synthesized mitochondrial phosphoprotein, influenced by both cAMP/PKA-dependent and cAMP/PKA-independent signaling pathways [126]. In Dictyostelium, steroids initiate a signaling cascade that triggers rapid sporulation [127]. In this work we have identified FdfT, lathosterol oxidase and Smt1 downregulated in response to cAMP. ...
Cyclic AMP acts as a secondary messenger involving different cellular functions in eukaryotes. Here, proteomic and transcriptomic profiling has been combined to identify novel early developmentally regulated proteins in eukaryote cells. These proteomic and transcriptomic experiments were performed in Dictyostelium discoideum given the unique advantages that this organism offers as a eukaryotic model for cell motility and as a nonmammalian model of human disease. By comparing whole-cell proteome analysis of developed (cAMP-pulsed) wild-type AX2 cells and an independent transcriptomic analysis of developed wild-type AX4 cells, our results show that up to 70% of the identified proteins overlap in the two independent studies. Among them, we have found 26 proteins previously related to cAMP signaling and identified 110 novel proteins involved in calcium signaling, adhesion, actin cytoskeleton, the ubiquitin-proteasome pathway, metabolism, and proteins that previously lacked any annotation. Our study validates previous findings, mostly for the canonical cAMP-pathway, and also generates further insight into the complexity of the transcriptomic changes during early development. This article also compares proteomic data between parental and cells lacking glkA, a GSK-3 kinase implicated in substrate adhesion and chemotaxis in Dictyostelium. This analysis reveals a set of proteins that show differences in expression in the two strains as well as overlapping protein level changes independent of GlkA.
... GABA induces the release of the SDF-2 precursor AcbA from prespore cells and induces the expression of the protease TagC on the surface of prestalk cells (Anjard and Loomis, 2006). SDF-3 is a steroid which induces the release of GABA (Anjard et al., 2009). This complex series of signals that appear to activate each other thus appear to form a series of checks where the successful completion of one developmental stage permits the next stage to occur. ...
In the last few decades, we have learned a considerable amount about how eukaryotic cells communicate with each other, and what it is the cells are telling each other. The simplicity of Dictyostelium discoideum, and the wide variety of available tools to study this organism, makes it the equivalent of a hydrogen atom for cell and developmental biology. Studies using Dictyostelium have pioneered a good deal of our understanding of eukaryotic cell communication. In this review, we will present a brief overview of how Dictyostelium cells use extracellular signals to attract each other, repel each other, sense their local cell density, sense whether the nearby cells are starving or stressed, count themselves to organize the formation of structures containing a regulated number of cells, sense the volume they are in, and organize their multicellular development. Although we are probably just beginning to learn what the cells are telling each other, the elucidation of Dictyostelium extracellular signals has already led to the development of possible therapeutics for human diseases.
... Finally, after 5 years, a genetic hint showed that SDF-2 was a peptide cut out of the well-conserved metabolic protein AcbA. Christophe spent the next 5 years working out the surprisingly complex set of interacting pathways involving steroids, GABA and glutamate that fine-tuned the temporal control of sporulation (Anjard et al., 1998;Anjard and Loomis 2005, 2006, 2008Anjard et al., 2009Anjard et al., , 2011." "Using his highly sensitive SDF-2 bioassay, Christophe was able to show that GRASP mediates unconventional secretion by using components of the autophagy pathway to position AcbA within vesicles that subsequently fuse with the plasma membrane to release their cargo. Together with Vivek Malhotra and Suresh Subramani he was able to show that this pathway is also used in the yeasts Saccharomyces cerevisiae and Pichia pastoris. ...
William Farnsworth Loomis studied the social amoeba Dictyostelium discoideum for more than fifty years as a professor of biology at the University of California, San Diego, USA. This biographical reflection describes Dr. Loomis' major scientific contributions to the field within a career arc that spanned the early days of molecular biology up to the present day where the acquisition of high-dimensional datasets drive research. Dr. Loomis explored the genetic control of social amoeba development, delineated mechanisms of cell differentiation, and significantly advanced genetic and genomic technology for the field. The details of Dr. Loomis' multifaceted career are drawn from his published work, from an autobiographical essay that he wrote near the end of his career and from extensive conversations between him and the two authors, many of which took place on the deck of his beachfront home in Del Mar, California.
... The signalling receptors include the histidine kinases dhkF, dhkJ and dhkH, as well as the endosomal transmembrane proteins phg1A, phg1B and phg1C, and the glutamate receptor like proteins grlA and grlC (Additional file 1: Figure S1c). GrlA is known to be required for spore maturation [20], but the roles of the other "signalling receptors" are unclear. GO CC terms showed slight enrichment for spore genes in "Golgi apparatus" (GO:0005794) and significant enrichment in "actin cytoskeleton" (GO:0015629), possibly related to the formation of actin rods in spores [21]. ...
Background
A major hallmark of multicellular evolution is increasing complexity by the evolution of new specialized cell types. During Dictyostelid evolution novel specialization occurred within taxon group 4. We here aim to retrace the nature and ancestry of the novel “cup” cells by comparing their transcriptome to that of other cell types.
Results
RNA-Seq was performed on purified mature spore, stalk and cup cells and on vegetative amoebas. Clustering and phylogenetic analyses showed that cup cells were most similar to stalk cells, suggesting that they share a common ancestor. The affinity between cup and stalk cells was also evident from promoter-reporter studies of newly identified cell-type genes, which revealed late expression in cups of many stalk genes. However, GO enrichment analysis reveal the unexpected prominence of GTPase mediated signalling in cup cells, in contrast to enrichment of autophagy and cell wall synthesis related transcripts in stalk cells. Combining the cell type RNA-Seq data with developmental expression profiles revealed complex expression dynamics in each cell type as well as genes exclusively expressed during terminal differentiation. Most notable were nine related hssA-like genes that were highly and exclusively expressed in cup cells.
Conclusions
This study reveals the unique transcriptomes of the mature cup, stalk and spore cells of D. discoideum and provides insight into the ancestry of cup cells and roles in signalling that were not previously realized. The data presented in this study will serve as an important resource for future studies into the regulation and evolution of cell type specialization.
Electronic supplementary material
The online version of this article (10.1186/s12864-018-5146-3) contains supplementary material, which is available to authorized users.
... Four cAMP receptors have been identified and two of these play a role in the cAMP chemotaxis involved with aggregated formation (Ginsburg et al. 1995). Several other receptors have been genetically analyzed, including some close paralogs of the cAMP receptors, and recently a receptor responsible for folate chemotaxis has been identified (Anjard et al. 2009;Pan et al. 2016;Prabhu et al. 2007;Raisley et al. 2004). In regards to G proteins, folate responses require the G␣4 G protein subunit and cAMP responses require the G␣2 subunit (Hadwiger et al. 1994;Hadwiger and Srinivasan 1999;Kumagai et al. 1989). ...
Amoeba often use cell movement as a mechanism to find food, such as bacteria, in their environment. The chemotactic movement of the soil amoeba Dictyostelium to folate or other pterin compounds released by bacteria is a well-documented foraging mechanism. Acanthamoeba can also feed on bacteria but relatively little is known about the mechanism(s) by which this amoeba locates bacteria. Acanthamoeba movement in the presence of folate or bacteria was analyzed in above agar assays and compared to that observed for Dictyostelium. The overall mobility of Acanthamoeba was robust like that of Dictyostelium but Acanthamoeba did not display a chemotactic response to folate. In the presence of bacteria, Acanthamoeba only showed a marginal bias in directed movement whereas Dictyostelium displayed a strong chemotactic response. A comparison of genomes revealed that Acanthamoeba and Dictyostelium share some similarities in G protein signaling components but that specific G proteins used in Dictyostelium chemotactic responses were not present in current Acanthamoeba genome sequence data. The results of this study suggest that Acanthamoeba does not use chemotaxis as the primary mechanism to find bacterial food sources and that the chemotactic responses of Dictyostelium to bacteria may have co-evolved with chemotactic responses that facilitate multicellular development.
... 11 Cytokinins and steroids can initiate sporulation in the protist Dictyostelium sp. 12 In previous work, experimental evolution created a population of Trichoderma citrinoviride with an accelerated life cycle. Daily transfer of an aliquot of liquid culture to fresh media selected for more efficient transfer. ...
In previous work, we evolved a population of Trichoderma citrinoviride in liquid cultures to speed up its asexual development cycle. The evolved population, called T-6, formed conidia 3 times sooner and in >1000-fold greater numbers. Here we identify the steroid pregnenolone as a molecular signal for this different behavior. Media in which the ancestral T. citrinoviride population was grown (called ancestral spent media) contained a submerged conidiation inhibitor. Growing the evolved population T-6 in ancestral spent media eliminated the abundant formation of conidia. This inhibition depended on the amount and age of the ancestral spent medium and the time that the ancestral spent medium was added to the T-6 culture. Fractionation of the ancestral spent medium identified a hydrophobic inhibiting compound with a molecular weight less than 2000 g/mol. A combination of GC-MS, ELISA and reaction with cholesterol oxidase identified it as pregnenolone. Addition of pregnenolone to cultures of T-6 inhibited submerged conidiation by inhibiting formation of conidiophores, while ten other analogous steroids did not. Pregnenolone also inhibited submerged conidiation of Fusarium graminearum PH-1, a plant pathogen that causes head blight in wheat and barley. This identification of steroids as signal molecules in fungi creates opportunities to disrupt this signaling to control fungal behavior.
... DIF-1, Spore Differentiation Factor 1 (SDF-1) and SDF-2 [66,67]. Additional signals are relayed by gamma-aminobutyric acid (GABA), glutamate, and steroids [68][69][70]. ...
Background
Development of the soil amoeba Dictyostelium discoideum is triggered by starvation. When placed on a solid substrate, the starving solitary amoebae cease growth, communicate via extracellular cAMP, aggregate by tens of thousands and develop into multicellular organisms. Early phases of the developmental program are often studied in cells starved in suspension while cAMP is provided exogenously. Previous studies revealed massive shifts in the transcriptome under both developmental conditions and a close relationship between gene expression and morphogenesis, but were limited by the sampling frequency and the resolution of the methods.
Results
Here, we combine the superior depth and specificity of RNA-seq-based analysis of mRNA abundance with high frequency sampling during filter development and cAMP pulsing in suspension. We found that the developmental transcriptome exhibits mostly gradual changes interspersed by a few instances of large shifts. For each time point we treated the entire transcriptome as single phenotype, and were able to characterize development as groups of similar time points separated by gaps. The grouped time points represented gradual changes in mRNA abundance, or molecular phenotype, and the gaps represented times during which many genes are differentially expressed rapidly, and thus the phenotype changes dramatically. Comparing developmental experiments revealed that gene expression in filter developed cells lagged behind those treated with exogenous cAMP in suspension. The high sampling frequency revealed many genes whose regulation is reproducibly more complex than indicated by previous studies. Gene Ontology enrichment analysis suggested that the transition to multicellularity coincided with rapid accumulation of transcripts associated with DNA processes and mitosis. Later development included the up-regulation of organic signaling molecules and co-factor biosynthesis. Our analysis also demonstrated a high level of synchrony among the developing structures throughout development.
Conclusions
Our data describe D. discoideum development as a series of coordinated cellular and multicellular activities. Coordination occurred within fields of aggregating cells and among multicellular bodies, such as mounds or migratory slugs that experience both cell-cell contact and various soluble signaling regimes. These time courses, sampled at the highest temporal resolution to date in this system, provide a comprehensive resource for studies of developmental gene expression.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1491-7) contains supplementary material, which is available to authorized users.
... These strains were shown to initiate culmination under conditions where wild type cells were still waiting for an environmental signal. They also constitutively released intercellular signals that triggered terminal differentiation of spores and stalk cells (Anjard and Loomis, 2005;2006;Anjard, Su and Loomis, 2009). Many of these signals also activated the cAMP/PKA pathway (Loomis, 2014). ...
Cells grow, move, expand, shrink and die in the process of generating the characteristic shapes of organisms. Although the structures generated during development of the social amoeba Dictyostelium discoideum look nothing like the structures seen in metazoan embryogenesis, some of the morphogenetic processes used in their making are surprisingly similar. Recent advances in understanding the molecular basis for directed cell migration, cell type specific sorting, differential adhesion, secretion of matrix components, pattern formation, regulation and terminal differentiation are reviewed. Genes involved in Dictyostelium aggregation, slug formation, and culmination of fruiting bodies are discussed.
Copyright © 2015. Published by Elsevier Inc.
... In addition, high solute levels in the spore head also act directly on adenylate cyclase G to increase cAMP levels and to prevent precocious germination. Abbreviations: AmtA, AmtC ammonia transporters A and C; DhkA, DhkB, DhkC, DokA histidine kinases A, B, C and DokA; ACR and ACG: adenylate cyclases A and G; RdeA phosphoryl transfer protein; RegA cAMP phosphodiesterase with response regulator; PKA cAMP-dependent protein kinase; TagC ABC transporter with intrinsic serine protease moiety; GadA glutamate decarboxylase A; GABA gamma-amino butyric acid; AcbA acetyl coenzyme binding protein A; SDF-2, SDF-3 spore differentiation factors 2 and 3 (Anjard et al. 2009). GABA induces secretion of AcbA (Acyl-CoA binding protein) from prespore cells. ...
Dictyostelid social amoebas represent one of several groups of genetically divergent lineages that display aggregative multicellularity. In this chapter, we describe the evolution of developmental complexity in Dictyostelia and discuss the signalling mechanisms that control the developmental programme of the model organism Dictyostelium discoideum. We also reconstruct the evolutionary history of these developmental control mechanisms from environmental sensing in the unicellular ancestors of Dictyostelia. Finally, we explore the parameters that define the boundary between uni- and multicellularity.
... They employ peptides called spore differentiation factor 1 (SDF-1) and SDF-2, which are secreted as precursors from prespore cells and processed by the serine protease-ABC transporters TagB and TagC, respectively [39,40]. The whole process is accelerated by GABA-and MPBD-mediated cascades, the former being triggered by a steroid type SDF (SDF-3) [41]. The accumulation of SDF precursors in the prespore cells is controlled by intracellular cAMP levels via protein kinase A [42]. ...
Social amoebae are lower eukaryotes that inhabit the soil. They are characterized by the construction of a starvation-induced multicellular fruiting body with a spore ball and supportive stalk. In most species, the stalk is filled with motile stalk cells, as represented by the model organism Dictyostelium discoideum, whose developmental mechanisms have been well characterized. However, in the genus Acytostelium, the stalk is acellular and all aggregated cells become spores. Phylogenetic analyses have shown that it is not an ancestral genus but has lost the ability to undergo cell differentiation.
We performed genome and transcriptome analyses of Acytostelium subglobosum and compared our findings to other available dictyostelid genome data. Although A. subglobosum adopts a qualitatively different developmental program from other dictyostelids, its gene repertoire was largely conserved. Yet, families of polyketide synthase and extracellular matrix proteins have not expanded and a serine protease and ABC transporter B family gene, tagA, and a few other developmental genes are missing in the A. subglobosum lineage. Temporal gene expression patterns are astonishingly dissimilar from those of D. discoideum, and only a limited fraction of the ortholog pairs shared the same expression patterns, so that some signaling cascades for development seem to be disabled in A. subglobosum.
The absence of the ability to undergo cell differentiation in Acytostelium is accompanied by a small change in coding potential and extensive alterations in gene expression patterns.
