P2X receptors are membrane ion channels gated by extracellular ATP1,2 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 μM). Site-directed mutagenesis revealed essential conservation of structure-function relations with P2X receptors of higher organisms. The receptor was insensitive to the usual P2X antagonists3 but was blocked by nanomolar concentrations of Cu 2+ 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.The D. discoideum genome contains five sequences predicted to encode proteins homologous to vertebrate P2X receptors. The protein most closely related to the seven human receptors is DDB0168616 (Supplementary Fig. 1). HEK cells expressing a humanized version of this complementary DNA responded to ATP with robust inward currents (Fig. 1). ATP evoked unitary currents (conductance 8.2 pS at -100 mV) in outsideout patches (Fig. 1a, b), establishing that this gene (D. discoideum p2xA) encodes an ATPgated ion channel (DdP2X). Whole-cell currents evoked by ATP were concentrationdependent (10-300 μM) with kinetic properties most similar to those observed for human P2X 2 or P2X 4 receptors1 (Fig. 1d). With sodium as the only cation, the current-voltage relation reversed at zero and showed moderate inward rectification (Fig. 1c). By measuring the current reversal potentials in different extracellular ions, we found that the channels were freely permeable to Ca 2+ ions but less so to larger cations (the relative permeability P X /P Na was 1.5 ± 0.11 (n = 8 cells), 0.54 ± 0.01 (5), 0.48 ± 0.21 (5), 0.38 ± 0.16 (5) and 0.21 ± 0.05 (8), where X represents Ca 2+ , choline, Tris, tetraethylammonium and N-methyl-D-glucamine, respectively; Fig. 1c). These values are in the range of vertebrate P2X receptors1. Western blotting of the expressed receptor showed two bands at 48 and 55 kDa (Fig. 1e). The
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.
Cheaters disrupt cooperation by reaping the benefits without paying their fair share of associated costs. Cheater impact can be diminished if cooperators display a tag (‘greenbeard') and recognise and preferentially direct cooperation towards other tag carriers. Despite its popular appeal, the feasibility of such greenbeards has been questioned because the complex patterns of partner-specific cooperative behaviours seen in nature require greenbeards to come in different colours. Here we show that a locus (‘Tgr') of a social amoeba represents a polychromatic greenbeard. Patterns of natural Tgr locus sequence polymorphisms predict partner-specific patterns of cooperation by underlying variation in partner-specific protein–protein binding strength and recognition specificity. Finally, Tgr locus polymorphisms increase fitness because they help avoid potential costs of cooperating with incompatible partners. These results suggest that a polychromatic greenbeard can provide a key mechanism for the evolutionary maintenance of cooperation.
SummaryCooperation is ubiquitous across the tree of life, from simple microbes to the complex social systems of animals [1]. Individuals cooperate by engaging in costly behaviors that can be exploited by other individuals who benefit by avoiding these associated costs. Thus, if successful exploitation of social partners during cooperative interactions increases relative fitness, then we expect selection to lead to the emergence of a single optimal winning strategy in which individuals maximize their gain from cooperation while minimizing their associated costs [2]. Such social “cheating” appears to be widespread in nature [3], including in several microbial systems [4–11], but despite the fitness advantages favoring social cheating, populations tend to harbor significant variation in social success rather than a single optimal winning strategy. Using the social amoeba Dictyostelium discoideum, we provide a possible explanation for the coexistence of such variation. We find that genotypes typically designated as “cheaters” [12] because they produce a disproportionate number of spores in chimeric fruiting bodies do not actually gain higher fitness as a result of this apparent advantage because they produce smaller, less viable spores than putative “losers.” As a consequence of this trade-off between spore number and viability, genotypes with different spore production strategies, which give the appearance of differential social success, ultimately have similar realized fitness. These findings highlight the limitations of using single fitness proxies in evolutionary studies and suggest that interpreting social trait variation in terms of strategies like cheating or cooperating may be misleading unless these behaviors are considered in the context of the true multidimensional nature of fitness.
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.
SummaryCell-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.
Rab GTPases play key roles in the delivery, docking and fusion of intracellular vesicles. However, the mechanism by which spatial and temporal regulation of Rab GTPase activity is controlled is poorly understood. Here we describe a mechanism by which localized calcium release through a vesicular ion channel controls Rab GTPase activity. We show that activation of P2XA, an intracellular ion channel localized to the Dictyostelium discoideum contractile vacuole system, results in calcium efflux required for downregulation of Rab11a activity and efficient vacuole fusion. Vacuole fusion and Rab11a downregulation require the activity of CnrF, an EF hand containing Rab GAP found in a complex with Rab11a and P2XA. CnrF Rab GAP activity to Rab11a is enhanced by the presence of calcium and the EF-hand domain. These findings suggest that P2XA activation results in vacuolar calcium release, which triggers activation of CnrF Rab GAP activity and subsequent downregulation of Rab11a to allow vacuole fusion.
Background: Dictyostelium discoideum P2X receptors are found on the contractile vacuole.Results: Four of the five receptors (P2XA, P2XB, P2XD, and P2XE) form ATP-activated channels but differ in their optimal ionic conditions.Conclusion: Properties of five P2X receptors correlate with their rescue of an osmoregulatory phenotype in P2XA-deficient Dictyostelium cells.Significance: A P2X receptor is required for normal contractile vacuole operation.
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