A new environmentally resistant cell type from Dictyostelium.
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ABSTRACT:
SUBMITTER: Serafimidis I
PROVIDER: S-EPMC2786962 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The mature fruiting body of Dictyostelium consists of stalk and spore cells but its construction, and the migration of the preceding slug stage, requires a number of specialized sub-types of prestalk cell whose nature and function are not well understood. The prototypic prestalk-specific gene, ecmA, is inducible by the polyketide DIF-1 in a monolayer assay and requires the DimB and MybE transcription factors for full inducibility. We perform genome-wide microarray analyses, on parental, mybE- and dimB- cells, and identify many additional genes that depend on MybE and DimB for their DIF-1 inducibility. Surprisingly, an even larger number of genes are only DIF inducible in mybE- cells, some genes are only inducible in DimB- cells and some are inducible when either transcription factor is absent. Thus in assay conditions where MybE and DimB function as inducers of ecmA these genes fall under negative control by the same two transcription factors. We have studied in detail rtaA, one of the MybE and DimB repressed genes. One especially enigmatic group of prestalk cells is the anterior-like cells (ALCs), which exist intermingled with prespore cells in the slug. A promoter fusion reporter gene, rtaA:gal(u), is expressed in a subset of the ALCs that is distinct from the ALC population detected by a reporter construct containing ecmA and ecmB promoter fragments. At culmination, when the ALC sort out from the prespore cells and differentiate to form three ancillary stalk cell structures: the upper cup, the lower cup and the outer basal disk, the rtaA:gal(u) expressing cells preferentially populate the upper cup region. This fact, and their virtual absence from the anterior and posterior regions of the slug, identifies them as a new prestalk sub-type: the pstU cells. PstU cell differentiation is, as expected, increased in a dimB- mutant during normal development but, surprisingly, they differentiate normally in a mutant lacking DIF. Thus genetic removal of MybE or DimB reveals an alternate DIF-1 activation pathway, for pstU differentiation, that functions under monolayer assay conditions but that is not essential during multicellular development.
| S-EPMC2845816 | biostudies-literature
Project description:Cell-type specific genes were recognized by interrogating microarrays carrying Dictyostelium gene fragments with probes prepared from fractions enriched in prestalk and prespore cells. Cell-type specific accumulation of mRNA from 17 newly identified genes was confirmed by Northern analyses. DNA microarrays carrying 690 targets were used to determine expression profiles during development. The profiles were fit to a biologically based kinetic equation to extract the times of transcription onset and cessation. Although the majority of the genes that were cell-type enriched at the slug stage were first expressed as the prespore and prestalk cells sorted out in aggregates, some were found to be expressed earlier before the cells had even aggregated. These early genes may have been initially expressed in all cells and then preferentially turned over in one or the other cell type. Alternatively, cell type divergence may start soon after the initiation of development.
| S-EPMC59697 | biostudies-literature
Project description:Starved Dictyostelium cells aggregate into groups of nearly 105 cells. AMPK is a highly conserved serine/threonine protein kinase consisting of a catalytic and two regulatory subunits. As multi-cellular development in Dictyostelium is initiated upon starvation, we explored the role of the energy sensor, AMPK, which shows significant similarity to human AMPK and is expressed throughout development. Deletion of the ampkα gene results in the formation of numerous small-sized aggregates that develop asynchronously to form few fruiting bodies with small sori and long stalks. On the other hand, ampkαOE cells form fruiting bodies with small stalks and large sori when compared with wild-type, Ax2. A minimum of 5% ampkα- cells in a chimaera with Ax2 cells was sufficient to reduce the aggregate size. Also, the conditioned media collected from ampkα- cells triggered Ax2 cells to form smaller aggregates. The starved ampkα- cells showed low glucose levels and formed large aggregates when glucose was supplied exogenously. Interestingly, ampkα- cells exhibit abnormal cell-type patterning with increased prestalk region and a concomitant reduction of prespore region. In addition, there was a loss of distinct prestalk/prespore boundary in the slugs.
| S-EPMC5541345 | biostudies-literature
Project description:Cullins function as scaffolds that, along with F-box/WD40-repeat-containing proteins, mediate the ubiquitination of proteins to target them for degradation by the proteasome. We have identified a cullin CulA that is required at several stages during Dictyostelium development. culA null cells are defective in inducing cell-type-specific gene expression and exhibit defects during aggregation, including reduced chemotaxis. PKA is an important regulator of Dictyostelium development. The levels of intracellular cAMP and PKA activity are controlled by the rate of synthesis of cAMP and its degradation by the cAMP-specific phosphodiesterase RegA. We show that overexpression of the PKA catalytic subunit (PKAcat) rescues many of the culA null defects and those of cells lacking FbxA/ChtA, a previously described F-box/WD40-repeat-containing protein, suggesting CulA and FbxA proteins are involved in regulating PKA function. Whereas RegA protein levels drop as the multicellular organism forms in the wild-type strain, they remain high in culA null and fbxA null cells. Although PKA can suppress the culA and fbxA null developmental phenotypes, it does not suppress the altered RegA degradation, suggesting that PKA lies downstream of RegA, CulA, and FbxA. Finally, we show that CulA, FbxA, and RegA are found in a complex in vivo, and formation of this complex is dependent on the MAP kinase ERK2, which is also required for PKA function. We propose that CulA and FbxA regulate multicellular development by targeting RegA for degradation via a pathway that requires ERK2 function, leading to an increase in cAMP and PKA activity.
| S-EPMC312710 | biostudies-literature
Project description:The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100-insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.
| S-EPMC25161 | biostudies-literature
Project description:BackgroundCompetitive social interactions are ubiquitous in nature, but their genetic basis is difficult to determine. Much can be learned from single gene knockouts in a eukaryote microbe. The mutants can be competed with the parent to discern the social impact of that specific gene. Dictyostelium discoideum is a social amoeba that exhibits cooperative behavior in the construction of a multicellular fruiting body. It is a good model organism to study the genetic basis of cooperation since it has a sequenced genome and it is amenable to genetic manipulation. When two strains of D. discoideum are mixed, a cheater strain can exploit its social partner by differentiating more spore than its fair share relative to stalk cells. Cheater strains can be generated in the lab or found in the wild and genetic analyses have shown that cheating behavior can be achieved through many pathways.ResultsWe have characterized the knockout mutant chtB, which was isolated from a screen for cheater mutants that were also able to form normal fruiting bodies on their own. When mixed in equal proportions with parental strain cells, chtB mutants contributed almost 60% of the total number of spores. To do so, chtB cells inhibit wild type cells from becoming spores, as indicated by counts and by the wild type cells' reduced expression of the prespore gene, cotB. We found no obvious fitness costs (morphology, doubling time in liquid medium, spore production, and germination efficiency) associated with the cheating ability of the chtB knockout.ConclusionsIn this study we describe a new gene in D. discoideum, chtB, which when knocked out inhibits the parental strain from producing spores. Moreover, under lab conditions, we did not detect any fitness costs associated with this behavior.
| S-EPMC3559258 | biostudies-literature
Project description:Regulated cell adhesion and motility have important roles during growth, development and tissue homeostasis. Consequently, great efforts have been made to identify genes that control these processes. One candidate is Rap1, as it has been implicated in the regulation of adhesion and motility in cell culture. To further study the role of Rap1 during multicellular development, we generated a mutant in a potential Rap1 GTPase activating protein (RapGAPB) in Dictyostelium. rapGAPB(-) cells have increased levels of active Rap1 compared with wild-type cells, indicating that RapGAPB regulates Rap1 activity. Furthermore, rapGAPB(-) cells exhibit hallmark phenotypes of other known mutants with hyperactivated Rap1, including increased substrate adhesion and abnormal F-actin distribution. However, unlike these other mutants, rapGAPB(-) cells do not exhibit impaired motility or chemotaxis, indicating that RapGAPB might only regulate specific roles of Rap1. Importantly, we also found that RapGAPB regulates Rap1 activity during multicellular development and is required for normal morphogenesis. First, streams of aggregating rapGAPB(-) cells break up as a result of decreased cell-cell adhesion. Second, rapGAPB(-) cells exhibit cell-autonomous defects in prestalk cell patterning. Using cell-type-specific markers, we demonstrate that RapGAPB is required for the correct sorting behaviour of different cell types. Finally, we show that inactivation of RapGAPB affects prestalk and prespore cell adhesion. We therefore propose that a possible mechanism for RapGAPB-regulated cell sorting is through differential adhesion.
| S-EPMC2724730 | biostudies-literature
Project description:BackgroundA 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.ResultsRNA-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.ConclusionsThis 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.
| S-EPMC6198379 | biostudies-literature
Project description:Mucolipidosis type IV, a devastating neurological lysosomal disease linked to mutations in the transient receptor potential channel mucolipin 1, TRPML1, a calcium permeable channel in the membranes of vesicles in endolysosomal system. TRPML1 function is still being elucidated and a better understanding of the molecular pathogenesis of Mucolipidosis type IV, may facilitate development of potential treatments. We have created a model to study mucolipin function in the eukaryotic slime mould Dictyostelium discoideum by altering expression of its single mucolipin homologue, mcln. We show that in Dictyostelium mucolipin overexpression contributes significantly to global chemotactic calcium responses in vegetative and differentiated cells. Knockdown of mucolipin also enhances calcium responses in vegetative cells but does not affect responses in 6-7 h developed cells, suggesting that in developed cells mucolipin may help regulate local calcium signals rather than global calcium waves. We found that both knocking down and overexpressing mucolipin often, but not always, presented the same phenotypes. Altering mucolipin expression levels caused an accumulation or increased acidification of Lysosensor Blue stained vesicles in vegetative cells. Nutrient uptake by phagocytosis and macropinocytosis were increased but growth rates were not, suggesting defects in catabolism. Both increasing and decreasing mucolipin expression caused the formation of smaller slugs and larger numbers of fruiting bodies during multicellular development, suggesting that mucolipin is involved in initiation of aggregation centers. The fruiting bodies that formed from these smaller aggregates had proportionately larger basal discs and thickened stalks, consistent with a regulatory role for mucolipin-dependent Ca2+ signalling in the autophagic cell death pathways involved in stalk and basal disk differentiation in Dictyostelium. Thus, we have provided evidence that mucolipin contributes to chemotactic calcium signalling and that Dictyostelium is a useful model to study the molecular mechanisms involved in the cytopathogenesis of Mucolipidosis type IV.
| S-EPMC9043695 | biostudies-literature
Project description:Cell differentiation is traditionally monitored with a few marker genes, which may bias results. To understand the evolution and regulation of the spore, stalk, cup and basal disc cells in Dictyostelia, we previously performed RNAseq on purified cell-types of taxon-group representative dictyostelids. Using promoter-lacZ constructs in D. discoideum, we here investigate the spatio-temporal expression pattern of 29 cell-type specific genes. Genes selected for spore- or cup-specificity in RNAseq were validated as such by lacZ expression, but genes selected for stalk-specificity showed variable additional expression in basal disc, early cup or prestalk populations. We measured responses of 25 genes to 15 single or combined regimes of induction by stimuli known to regulate cell differentiation. The outcomes of these experiments were subjected to hierarchical clustering to identify whether common modes of regulation were correlated with specific expression patterns. The analysis identified a cluster combining the spore and cup genes, which shared upregulation by 8-bromo cyclic AMP and down-regulation by Differentiation Inducing Factor 1 (DIF-1). Most stalk-expressed genes combined into a single cluster and shared strong upregulation by cyclic di-guanylate (c-di-GMP), and synergistic upregulation by combined DIF-1 and c-di-GMP. There was no clustering of genes expressed in other soma besides the stalk, but two genes that were only expressed in the stalk did not respond to any stimuli. In contrast to current models, the study indicates the existence of a stem-cell like soma population in slugs, whose members only acquire ultimate cell fate after progressing to their terminal location during fruiting body morphogenesis.
| S-EPMC9117722 | biostudies-literature