Developmental constraints enforce altruism and avert the tragedy of the commons in a social microbe.

Organisms often cooperate through the production of freely available public goods. This can greatly benefit the group but is vulnerable to the "tragedy of the commons" if individuals lack the motivation to make the necessary investment into public goods production. Relatedness to groupmates can motivate individual investment because group success ultimately benefits their genes' own self-interests. However, systems often lack mechanisms that can reliably ensure that relatedness is high enough to promote cooperation. Consequently, groups face a persistent threat from the tragedy unless they have a mechanism to enforce investment when relatedness fails to provide adequate motivation. To understand the real threat posed by the tragedy and whether groups can avert its impact, we determine how the social amoeba Dictyostelium discoideum responds as relatedness decreases to levels that should induce the tragedy. We find that, while investment in public goods declines as overall within-group relatedness declines, groups avert the expected catastrophic collapse of the commons by continuing to invest, even when relatedness should be too low to incentivize any contribution. We show that this is due to a developmental buffering system that generates enforcement because insufficient cooperation perturbs the balance of a negative feedback system controlling multicellular development. This developmental constraint enforces investment under the conditions expected to be most tragic, allowing groups to avert a collapse in cooperation. These results help explain how mechanisms that suppress selfishness and enforce cooperation can arise inadvertently as a by-product of constraints imposed by selection on different traits.

Inferring Adaptive Codon Preference to Understand Sources of Selection Shaping Codon Usage Bias.

Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify "adaptive codon preference," a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated "preference" largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome.

Mutant resources for functional genomics in Dictyostelium discoideum using REMI-seq technology.

BACKGROUND: Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. RESULTS: We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of 'barcoded' mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. CONCLUSIONS: We present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.

Strategic investment explains patterns of cooperation and cheating in a microbe.

Contributing to cooperation is typically costly, while its rewards are often available to all members of a social group. So why should individuals be willing to pay these costs, especially if they could cheat by exploiting the investments of others? Kin selection theory broadly predicts that individuals should invest more into cooperation if their relatedness to group members is high (assuming they can discriminate kin from nonkin). To better understand how relatedness affects cooperation, we derived the ?Collective Investment" game, which provides quantitative predictions for patterns of strategic investment depending on the level of relatedness. We then tested these predictions by experimentally manipulating relatedness (genotype frequencies) in mixed cooperative aggregations of the social amoeba Dictyostelium discoideum, which builds a stalk to facilitate spore dispersal. Measurements of stalk investment by natural strains correspond to the predicted patterns of relatedness-dependent strategic investment, wherein investment by a strain increases with its relatedness to the group. Furthermore, if overall group relatedness is relatively low (i.e., no strain is at high frequency in a group) strains face a scenario akin to the "Prisoner's Dilemma" and suffer from insufficient collective investment. We find that strains employ relatedness-dependent segregation to avoid these pernicious conditions. These findings demonstrate that simple organisms like D. discoideum are not restricted to being ?cheaters" or ?cooperators" but instead measure their relatedness to their group and strategically modulate their investment into cooperation accordingly. Consequently, all individuals will sometimes appear to cooperate and sometimes cheat due to the dynamics of strategic investing.

Coenzyme A and protein CoAlation levels are regulated in response to oxidative stress and during morphogenesis in Dictyostelium discoideum.

Dictyostelium discoideum (D. discoideum) is a simple eukaryote with a unique life cycle in which it differentiates from unicellular amoebae into a fruiting body upon starvation. Reactive oxygen species (ROS) have been associated with bacterial predation, as well as regulatory events during D. discoideum development and differentiation. Coenzyme A (CoA) is a key metabolic integrator in all living cells. A novel function of CoA in redox regulation, mediated by covalent attachment of CoA to cellular proteins in response to oxidative or metabolic stress, has been recently discovered and termed protein CoAlation. In this study, we report that the level of CoA and protein CoAlation in D. discoideum are developmentally regulated, and correlate with the temporal expression pattern of genes implicated in CoA biosynthesis during morphogenesis. Furthermore, treatment of growing D. discoideum cells with oxidising agents results in a dose-dependent increase of protein CoAlation. However, much higher concentrations were required when compared to mammalian cells and bacteria. Increased resistance of D. discoideum to oxidative stress induced by H2O2 has previously been attributed to high levels of catalase activity. In support of this notion, we found that H2O2-induced protein CoAlation is significantly increased in CatA-deficient D. discoideum cells. Collectively, this study provides insights into the role of CoA and protein CoAlation in the maintenance of redox homeostasis in amoeba and during D. discoideum morphogenesis.

Functional properties of five Dictyostelium discoideum P2X receptors.

The Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels (P2XA, P2XB, P2XD, and P2XE). At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.

Sociogenomics of self vs. non-self cooperation during development of Dictyostelium discoideum.

BACKGROUND: Dictyostelium discoideum, a microbial model for social evolution, is known to distinguish self from non-self and show genotype-dependent behavior during chimeric development. Aside from a small number of cell-cell recognition genes, however, little is known about the genetic basis of self/non-self recognition in this species. Based on the key hypothesis that there should be differential expression of genes if D. discoideum cells were interacting with non-clone mates, we performed transcriptomic profiling study in this species during clonal vs. chimeric development. The transcriptomic profiles of D. discoideum cells in clones vs. different chimeras were compared at five different developmental stages using a customized microarray. Effects of chimerism on global transcriptional patterns associated with social interactions were observed. RESULTS: We find 1,759 genes significantly different between chimera and clone, 1,144 genes associated significant strain differences, and 6,586 genes developmentally regulated over time. Principal component analysis showed a small amount of the transcriptional variance to chimerism-related factors (Chimerism: 0.18%, Chimerism × Timepoint: 0.03%). There are 162 genes specifically regulated under chimeric development, with continuous small differences between chimera vs. clone over development. Almost 60% of chimera-associated differential genes were differentially expressed at the 4 h aggregate stage, which corresponds to the initial transition of D. discoideum from solitary life to a multicellular phase. CONCLUSIONS: A relatively small proportion of over-all variation in gene expression is explained by differences between chimeric and clonal development. The relatively small modifications in gene expression associated with chimerism is compatible with the high level of cooperation observed among different strains of D. discoideum; cells of distinct genetic backgrounds will co-aggregate indiscriminately and co-develop into fruiting bodies. Chimeric development may involve re-programming of the transcriptome through small modifications of the developmental genetic network, which may also indicate that response to social interaction involves many genes with individually small transcriptional effect.

Cell type-specific filamin complex regulation by a novel class of HECT ubiquitin ligase is required for normal cell motility and patterning.

Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and 'salt and pepper' patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA(-) pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA(-) pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA(-) background rescues pstO cell localisation. hfnA(-) cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on 'salt and pepper' differentiation and sorting out.

A simple mechanism for complex social behavior.

The evolution of cooperation is a paradox because natural selection should favor exploitative individuals that avoid paying their fair share of any costs. Such conflict between the self-interests of cooperating individuals often results in the evolution of complex, opponent-specific, social strategies and counterstrategies. However, the genetic and biological mechanisms underlying complex social strategies, and therefore the evolution of cooperative behavior, are largely unknown. To address this dearth of empirical data, we combine mathematical modeling, molecular genetic, and developmental approaches to test whether variation in the production of and response to social signals is sufficient to generate the complex partner-specific social success seen in the social amoeba Dictyostelium discoideum. Firstly, we find that the simple model of production of and response to social signals can generate the sort of apparent complex changes in social behavior seen in this system, without the need for partner recognition. Secondly, measurements of signal production and response in a mutant with a change in a single gene that leads to a shift in social behavior provide support for this model. Finally, these simple measurements of social signaling can also explain complex patterns of variation in social behavior generated by the natural genetic diversity found in isolates collected from the wild. Our studies therefore demonstrate a novel and elegantly simple underlying mechanistic basis for natural variation in complex social strategies in D. discoideum. More generally, they suggest that simple rules governing interactions between individuals can be sufficient to generate a diverse array of outcomes that appear complex and unpredictable when those rules are unknown.

Facultative cheater mutants reveal the genetic complexity of cooperation in social amoebae.

Cooperation is central to many major transitions in evolution, including the emergence of eukaryotic cells, multicellularity and eusociality. Cooperation can be destroyed by the spread of cheater mutants that do not cooperate but gain the benefits of cooperation from others. However, cooperation can be preserved if cheaters are facultative, cheating others but cooperating among themselves. Several cheater mutants have been studied before, but no study has attempted a genome-scale investigation of the genetic opportunities for cheating. Here we describe such a screen in a social amoeba and show that cheating is multifaceted by revealing cheater mutations in well over 100 genes of diverse types. Many of these mutants cheat facultatively, producing more than their fair share of spores in chimaeras, but cooperating normally when clonal. These findings indicate that phenotypically stable cooperative systems may nevertheless harbour genetic conflicts. The opportunities for evolutionary moves and countermoves in such conflicts may select for the involvement of multiple pathways and numerous genes.

Functional mechanism study of the allelochemical myrigalone A identifies a group of ultrapotent inhibitors of ethylene biosynthesis in plants.

Allelochemicals represent a class of natural products released by plants as root, leaf, and fruit exudates that interfere with the growth and survival of neighboring plants. Understanding how allelochemicals function to regulate plant responses may provide valuable new approaches to better control plant function. One such allelochemical, Myrigalone A (MyA) produced by Myrica gale, inhibits seed germination and seedling growth through an unknown mechanism. Here, we investigate MyA using the tractable model Dictyostelium discoideum and reveal that its activity depends on the conserved homolog of the plant ethylene synthesis protein 1-aminocyclopropane-1-carboxylic acid oxidase (ACO). Furthermore, in silico modeling predicts the direct binding of MyA to ACO within the catalytic pocket. In D. discoideum, ablation of ACO mimics the MyA-dependent developmental delay, which is partially restored by exogenous ethylene, and MyA reduces ethylene production. In Arabidopsis thaliana, MyA treatment delays seed germination, and this effect is rescued by exogenous ethylene. It also mimics the effect of established ACO inhibitors on root and hypocotyl extension, blocks ethylene-dependent root hair production, and reduces ethylene production. Finally, in silico binding analyses identify a range of highly potent ethylene inhibitors that block ethylene-dependent response and reduce ethylene production in Arabidopsis. Thus, we demonstrate a molecular mechanism by which the allelochemical MyA reduces ethylene biosynthesis and identify a range of ultrapotent inhibitors of ethylene-regulated responses.

An intracellular P2X receptor required for osmoregulation in Dictyostelium discoideum.

P2X receptors are membrane ion channels gated by extracellular ATP that are found widely in vertebrates, but not previously in microbes. Here we identify a weakly related gene in the genome of the social amoeba Dictyostelium discoideum, and show, with the use of heterologous expression in human embryonic kidney cells, that it encodes a membrane ion channel activated by ATP (30-100 muM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists but was blocked by nanomolar concentrations of Cu2+ ions. In D. discoideum, the receptor was found on intracellular membranes, with prominent localization to an osmoregulatory organelle, the contractile vacuole. Targeted disruption of the gene in D. discoideum resulted in cells that were unable to regulate cell volume in hypotonic conditions. Cell swelling in these mutant cells was accompanied by a marked inhibition of contractile vacuole emptying. These findings demonstrate a new functional role for P2X receptors on intracellular organelles, in this case in osmoregulation.

Structured growth and genetic drift raise relatedness in the social amoeba Dictyostelium discoideum.

One condition for the evolution of altruism is genetic relatedness between altruist and beneficiary, often achieved through active kin recognition. Here, we investigate the power of a passive process resulting from genetic drift during population growth in the social amoeba Dictyostelium discoideum. We put labelled and unlabelled cells of the same clone in the centre of a plate, and allowed them to proliferate outward. Zones formed by genetic drift owing to the small population of actively growing cells at the colony edge. We also found that single cells could form zones of high relatedness. Relatedness increased at a significantly higher rate when food was in short supply. This study shows that relatedness can be significantly elevated before the social stage without a small founding population size or recognition mechanism.

The genetic architecture underlying prey-dependent performance in a microbial predator.

Natural selection should favour generalist predators that outperform specialists across all prey types. Two genetic solutions could explain why intraspecific variation in predatory performance is, nonetheless, widespread: mutations beneficial on one prey type are costly on another (antagonistic pleiotropy), or mutational effects are prey-specific, which weakens selection, allowing variation to persist (relaxed selection). To understand the relative importance of these alternatives, we characterised natural variation in predatory performance in the microbial predator Dictyostelium discoideum. We found widespread nontransitive differences among strains in predatory success across different bacterial prey, which can facilitate stain coexistence in multi-prey environments. To understand the genetic basis, we developed methods for high throughput experimental evolution on different prey (REMI-seq). Most mutations (~77%) had prey-specific effects, with very few (~4%) showing antagonistic pleiotropy. This highlights the potential for prey-specific effects to dilute selection, which would inhibit the purging of variation and prevent the emergence of an optimal generalist predator.

Non-genetic heterogeneity and cell fate choice in Dictyostelium discoideum.

From microbes to metazoans, it is now clear that fluctuations in the abundance of mRNA transcripts and protein molecules enable genetically identical cells to oscillate between several distinct states (Kaern et al. 2005). Since this cell-cell variability does not derive from physical differences in the genetic code it is termed non-genetic heterogeneity. Non-genetic heterogeneity endows cell populations with useful capabilities they could never achieve if each cell were the same as its neighbors (Raj & van Oudenaarden 2008; Eldar & Elowitz 2010). One such example is seen during multicellular development and "salt and pepper" cell type differentiation. In this review, we will first examine the importance of non-genetic heterogeneity in initiating "salt and pepper" pattern formation during Dictyostelium discoideum development. Second, we will discuss the various ways in which non-genetic heterogeneity might be generated, as well as recent advances in understanding the molecular basis of heterogeneity in this system.

Dictyostelium discoideum: An Alternative Nonanimal Model for Developmental Toxicity Testing.

A critical aspect of toxicity evaluation is developmental and reproductive toxicity (DART) testing. Traditionally, DART testing has been conducted in vivo in mammalian model systems. New legislation aimed at reducing animal use and the prohibitive costs associated with DART testing, together with a need to understand the genetic pathways underlying developmental toxicity means there is a growing demand for alternative model systems for toxicity evaluation. Here we explore the potential of the eukaryotic social amoeba Dictyostelium discoideum, which is already widely used as a simple model system for cell and developmental biology, as a potential nonanimal model for DART testing. We developed assays for high-throughput screening of toxicity during D. discoideum growth and development. This allowed the toxicity of a broad range of test compounds to be characterized, which revealed that D. discoideum can broadly predict mammalian toxicity. In addition, we show that this system can be used to perform functional genomic screens to compare the molecular modes of action of different compounds. For example, genome-wide screens for mutations that affect lithium and valproic acid toxicity allowed common and unique biological targets and molecular processes mediating their toxicity to be identified. These studies illustrate that D. discoideum could represent a predictive nonanimal model for DART testing due to its amenability to high-throughput approaches and molecular genetic tractability.

What can microbial genetics teach sociobiology?

Progress in our understanding of sociobiology has occurred with little knowledge of the genetic mechanisms that underlie social traits. However, several recent studies have described microbial genes that affect social traits, thereby bringing genetics to sociobiology. A key finding is that simple genetic changes can have marked social consequences, and mutations that affect cheating and recognition behaviors have been discovered. The study of these mutants confirms a central theoretical prediction of social evolution: that genetic relatedness promotes cooperation. Microbial genetics also provides an important new perspective: that the genome-to-phenome mapping of social organisms might be organized to constrain the evolution of social cheaters. This constraint can occur both through pleiotropic genes that link cheating to a personal cost and through the existence of phoenix genes, which rescue cooperative systems from selfish and destructive strategies. These new insights show the power of studying microorganisms to improve our understanding of the evolution of cooperation.

Conditional expression explains molecular evolution of social genes in a microbe.

Conflict is thought to play a critical role in the evolution of social interactions by promoting diversity or driving accelerated evolution. However, despite our sophisticated understanding of how conflict shapes social traits, we have limited knowledge of how it impacts molecular evolution across the underlying social genes. Here we address this problem by analyzing the genome-wide impact of social interactions using genome sequences from 67 Dictyostelium discoideum strains. We find that social genes tend to exhibit enhanced polymorphism and accelerated evolution. However, these patterns are not consistent with conflict driven processes, but instead reflect relaxed purifying selection. This pattern is most likely explained by the conditional nature of social interactions, whereby selection on genes expressed only in social interactions is diluted by generations of inactivity. This dilution of selection by inactivity enhances the role of drift, leading to increased polymorphism and accelerated evolution, which we call the Red King process.

Curcumin and derivatives function through protein phosphatase 2A and presenilin orthologues in Dictyostelium discoideum.

Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using in silico docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds.

Cell Cycle Heterogeneity Can Generate Robust Cell Type Proportioning.

Cell-cell heterogeneity can facilitate lineage choice during embryonic development because it primes cells to respond to differentiation cues. However, remarkably little is known about the origin of heterogeneity or whether intrinsic and extrinsic variation can be controlled to generate reproducible cell type proportioning seen in vivo. Here, we use experimentation and modeling in D. discoideum to demonstrate that population-level cell cycle heterogeneity can be optimized to generate robust cell fate proportioning. First, cell cycle position is quantitatively linked to responsiveness to differentiation-inducing signals. Second, intrinsic variation in cell cycle length ensures cells are randomly distributed throughout the cell cycle at the onset of multicellular development. Finally, extrinsic perturbation of optimal cell cycle heterogeneity is buffered by compensatory changes in global signal responsiveness. These studies thus illustrate key regulatory principles underlying cell-cell heterogeneity optimization and the generation of robust and reproducible fate choice in development.