Self-Recognition in Social Amoebae Is Mediated by Allelic Pairs of Tiger Genes

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Science  22 Jul 2011:
Vol. 333, Issue 6041, pp. 467-470
DOI: 10.1126/science.1203903


Free-living cells of the social amoebae Dictyostelium discoideum can aggregate and develop into multicellular fruiting bodies in which many die altruistically as they become stalk cells that support the surviving spores. Dictyostelium cells exhibit kin discrimination—a potential defense against cheaters, which sporulate without contributing to the stalk. Kin discrimination depends on strain relatedness, and the polymorphic genes tgrB1 and tgrC1 are potential components of that mechanism. Here, we demonstrate a direct role for these genes in kin discrimination. We show that a matching pair of tgrB1 and tgrC1 alleles is necessary and sufficient for attractive self-recognition, which is mediated by differential cell-cell adhesion. We propose that TgrB1 and TgrC1 proteins mediate this adhesion through direct binding. This system is a genetically tractable ancient model of eukaryotic self-recognition.

The ability to distinguish self from nonself is observed in many organisms and is a central component of social evolution (1). During Dictyostelium development, individual cells aggregate to form multicellular fruiting bodies where 20 to 30% die while constructing a cellular stalk, and 70 to 80% survive as spores (2). This altruistic behavior is enigmatic because fruiting bodies can contain cells with different genotypes, which facilitates social exploitation (3). We have identified a self-recognition system that allows Dictyostelium to cooperate preferentially with relatives, which provides potential defense from exploitation (4). We have also identified two polymorphic genes, tgrB1 and tgrC1 (previously lagB1 and lagC1, respectively), that are necessary for self-recognition and for development (5).

We hypothesize that tgrB1 and tgrC1 encode matching transmembrane proteins (TgrB1 and TgrC1, respectively) that mediate self-recognition by heterotypic interactions between the extracellular protein domains on adjacent cells. If the hypothesis were correct, we would expect that deleting the resident alleles would render the cells unable to recognize their own kind as self. Furthermore, replacing the resident alleles with a matching pair of alleles from a different strain would restore self-recognition but abolish cooperation with the parental strain.

We deleted the tgrB1-tgrC1 locus (fig. S1) and mixed the mutants with the parental AX4 cells (6). The strains started to segregate within the streams of cells that form at the onset of aggregation and eventually formed segregated multicellular structures (Fig. 1A). In pure populations, the tgrB1tgrC1 cells were arrested at the aggregate stage (Fig. 1F) and exhibited compromised spore production (Fig. 1J). These results suggest that an intact pair of tgrB1-tgrC1 alleles is necessary for self-recognition, consistent with our previous findings that tgrB1 and tgrC1 are individually necessary for self-recognition (5). We then reintroduced a pair of tgrB1-tgrC1 alleles from AX4 into the deletion strain, which restored the locus to its original structure with minor changes (fig. S1). Both alleles were fully functional because they complemented all the tgrB1tgrC1 phenotypes. This double gene–replacement strain (tgrB1AX4tgrC1AX4) mixed with the parental AX4 strain as seen in the even distribution of red- and green-labeled cells (Fig. 1B). The strain also produced fruiting bodies (Fig. 1G) and spores (Fig. 1J) in pure populations.

Fig. 1

Cells (all in the AX4 background) labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP) were grown separately, mixed in equal proportions, and allowed to develop together (A to E). Multicellular structures were photographed with epifluorescence microscopy during streaming aggregation (8 to 12 hours, main images) and at the finger stage (14 to 18 hours, insets). Scale bar, 0.5 mm. The cartoons are models of interactions between cells. Cells are illustrated as elongated amoebae. TgrB1 and TgrC1 are illustrated as membrane proteins; the shading indicates their origins. Interactions between matching pairs are highlighted. (A) The parental strain (AX4, red) mixed with a tgrB1-null tgrC1-null strain (green). (B) AX4 (red) mixed with a strain in which tgrB1-tgrC1 were replaced with matching AX4 alleles. (C and D) Strains (green) in which tgrB1-tgrC1 were replaced with alleles from wild strains QS4 (C) or QS38 (D), mixed with AX4 (red). (E) Double gene–replacement cells carrying tgrB1-tgrC1 from QS4 (red) mixed with double gene–replacement cells carrying tgrB1-tgrC1 from QS38 (green). Pure populations were also developed into fruiting bodies (48 hours). (F) tgrB1-null tgrC1-null cells. (G to I) Double gene–replacement cells carrying tgrB1-tgrC1 alleles from AX4 (G), QS4 (H), or QS38 (I). Scale bars: (F) 1 mm, (G) to (I), 0.5 mm. (J) Sporulation efficiencies of these and two additional double gene–replacement strains harboring tgrB1-tgrC1 from strains QS31 or QS45, as indicated under the x axis. The graph shows the percentage of cells that became spores. The means and standard errors of five or six replicates are given.

The tgrB1 and tgrC1 sequences are polymorphic (5). We cloned them from four wild strains that are different from one another and from AX4 (fig. S2) and recombined them into the resident locus of the tgrB1tgrC1 cells. We then mixed the cells in equal proportions and found that the double gene–replacement strains carrying alleles from QS4 (tgrB1QS4tgrC1QS4) or QS38 (tgrB1QS38tgrC1QS38) segregated from AX4 (Fig. 1, C and D) and eventually formed separate fruiting bodies (fig. S3). Segregation was observed at other proportions as well (fig. S4). The double gene–replacement cells developed and produced spores in pure populations (Fig. G to I), as did strains carrying either tgrB1QS31tgrC1QS31 or tgrB1QS45tgrC1QS45 (Fig. 1J). We also tested a mixture between the tgrB1QS4tgrC1QS4 and the tgrB1QS38tgrC1QS38 cells and observed that the two strains segregated (Fig. 1E).

In the models (Fig. 1), we propose that cells lacking both tgrB1 and tgrC1 segregate from AX4 because they are not recognized as self. They also fail to develop in pure populations because they do not recognize one another as self. Reintroducing a matching pair of alleles from AX4 restores self-recognition and allows the cells to cooperate with AX4. Introducing a pair of polymorphic alleles from any wild strain restores self-recognition, as reflected in the cells’ ability to develop in pure populations, but these double gene–replacement strains do not cooperate with strains with nonmatching tgrB1-tgrC1 alleles, including the parental AX4. We also conclude that self- or nonself-recognition is not a result of compromised development—the double gene–replacement cells develop well as pure populations, but segregate from nonself cells in mixes.

The only differences between the double gene–replacement strains are the sequences of tgrB1-tgrC1. Therefore, matching pairs of these alleles are both necessary and sufficient for self-recognition in the AX4 background, which suggests direct involvement in recognition.

The donor strains of tgrB1 and tgrC1 are genetically distinct from AX4 and from each other, and segregation between them is directly correlated with their genetic distances (4). Moreover, segregation and tgrB1-tgrC1 polymorphism are correlated (5), which suggests that tgrB1 and tgrC1 are major determinants in self-recognition. To test that possibility directly, we examined recognition between the double gene–replacement strains and the donor strains (Fig. 2). In control experiments, the tgrB1AX4tgrC1AX4 cells segregated from the wild strains (Fig. 2, A and D). Segregation was evident at the streaming stage and later in development when the cells formed segregated fingers. Mixing QS4 with the tgrB1QS4tgrC1QS4 strain resulted in an even distribution of cells during early aggregation, and the two strains remained in the same multicellular structures, although sorting was observed within the slugs (Fig. 2B). We observed similar patterns when we mixed QS31 and tgrB1QS31tgrC1QS31 cells (with less homogeneous mixing during early aggregation, but with both strains present in the same slug) (Fig. 2E). The reciprocal nonmatching mixes resulted in segregation—QS4 segregated from tgrB1QS31tgrC1QS31 (Fig. 2C), and QS31 segregated from tgrB1QS4tgrC1QS4 (Fig. 2F). In the models (Fig. 2), two cells recognize each other via matching tgrB1 and tgrC1 alleles, despite other genetic differences between them. These observations suggest that tgrB1 and tgrC1 are major determinants in self-recognition. The segregation observed at the finger stage may reflect physiological differences, because the wild strains are different from AX4 at various genetic loci (4).

Fig. 2

We labeled the wild strains QS4 (A to C) and QS31 (D to F) with GFP (green text) and the double gene–replacement strains (in AX4 background) with RFP (red text): tgrB1-tgrC1 were from AX4 (A and D), QS4 (B and F), or QS31 (C and E). We grew the cells separately, mixed them in equal proportions as indicated, and allowed them to develop together. We photographed the structures as in Fig. 1. Scale bar, 0.5 mm. Cartoons are as in Fig. 1. The shadings indicate genetic differences between cells and the respective origins of the alleles.

TgrC1 is a heterotypic cell-cell adhesion protein (7), and TgrB1 is its potential binding partner (5); together these findings suggest that self-recognition is mediated by differential binding of matching TgrB1 and TgrC1. However, tgrC1 also affects adenosine 3′,5′-monophosphate signaling and gene regulation (8, 9), so self-recognition could be mediated by differential signaling. To test the role of differential cell-cell adhesion directly, we developed AX4 and double gene–replacement strains separately until they aggregated, disaggregated the structures into single-cell suspensions, mixed the cells, and tested cell-cell adhesion in suspension (10). Control experiments showed even mixing between AX4 and the tgrB1AX4tgrC1AX4 cells (Fig. 3A). However, AX4 and tgrB1QS4tgrC1QS4 cells adhered into distinct aggregates (Fig. 3B), whereas differentially labeled tgrB1QS4tgrC1QS4 cells formed mixed aggregates (Fig. 3C). We also tested single gene–replacement strains in pure populations and in mixes and found that tgrB1 and tgrC1 interact “in trans,” such that TgrB1 on one cell interacts with a matching TgrC1 on the other cell (fig. S5). In the models (Fig. 3), we propose that TgrB1 on one cell binds the matching TgrC1 on a neighboring cell, such that cells with matching tgrB1-tgrC1 alleles adhere to one another.

Fig. 3

We developed pure populations of RFP- and GFP-labeled cells, all in the AX4 background, disaggregated and mixed them in equal proportions, allowed them to adhere in suspension, and photographed them with confocal fluorescence microscopy. Scale bar, 0.1 mm. (A) AX4 cells (red) mixed with a strain in which tgrB1-tgrC1 were replaced with matching alleles from AX4 (green). (B) AX4 (red) mixed with a strain in which tgrB1-tgrC1 were replaced with matching alleles from QS4 (green). (C) Strains (both red and green) in which tgrB1-tgrC1 were replaced with matching alleles from QS4. Cartoons are as in Fig. 1.

Self-recognition can be mediated by attraction of self or by rejection of nonself. To test which mechanism is utilized, we tested the effect of an extra tgrB1-tgrC1 pair. AX4 cells are haploid, so introducing extra alleles would make them merodiploid for these genes. If self-recognition were mediated by attraction of self, then introducing an extra pair of foreign alleles would allow the merodiploids to aggregate with two different strains, each sharing one pair of tgrB1-tgrC1 alleles. In the case of nonself rejection, an extra pair of foreign tgrB1-tgrC1 alleles would lead to self-rejection and result in aberrant aggregation. We constructed three merodiploid strains and found that they aggregated well and produced normal fruiting bodies and spores in pure populations. Mixing the tgrB1+/QS4tgrC1+/QS4 merodiploid strain with either AX4 (Fig. 4A) or with the tgrB1QStgrC1QS4 double gene–replacement strain (Fig. 4B) resulted in even cell mixing, which suggests that self-recognition is mediated by attraction. Mixing the tgrB1+/QS4tgrC1+/QS4 merodiploid with the tgrB1QS31tgrC1QS31 double gene–replacement strain (Fig. 4C) resulted in segregation, which suggests that the merodiploid genotype did not result in recognition promiscuity. The tgrB1+/QS31tgrC1+/QS31 merodiploid behaved in the same way (Fig. 4D to F). Finally, the tgrB1+/AX4tgrC1+/AX4 merodiploid coaggregated with AX4 but segregated from the two other double gene–replacement strains (Fig. 4, G to I), which suggests that doubling the tgrB1-tgrC1 dosage is not a confounding factor. In the models (Fig. 4), we propose that any matching pair of tgrB1-tgrC1 alleles in one cell is sufficient for recognition of a respective pair in another cell. In addition, the presence of a nonmatching pair of tgrB1-tgrC1 alleles does not interfere with this recognition. These data suggest that tgrB1 and tgrC1 mediate self-recognition through an inclusive attraction mechanism.

Fig. 4

We made merodiploid strains by introducing extra pairs of matching alleles into AX4: tgrB1-tgrC1 from QS4 (A to C), QS31 (D to F), and AX4 (G to I). We labeled these cells with RFP (red) and mixed them with GFP-labeled haploid cells: AX4 (A), (D), and (G); or double gene–replacement strains with tgrB1-tgrC1 from QS4 (B), (E), and (H); or QS31 (C), (F), and (I). Cell growth, mixing, photography, and cartoons are as in Fig. 1. Scale bar, 0.5 mm.

Our data implicate tgrB1 and tgrC1 as central components in Dictyostelium self-recognition because a matching pair of tgrB1-tgrC1 alleles is both necessary and sufficient for self-recognition in the AX4 genetic background and because the double gene–replacement strains cooperate with the genetically different donor strains. TgrC1 is a known heterotypic cell-cell adhesion protein (7), so we propose that TgrB1 is its binding partner and that TgrB1 on the surface of one cell binds a matching TgrC1 on the surface of another cell.

D. discoideum has other cell-cell adhesion genes, mainly cadA and csaA (11), that have no direct effect on self-recognition (5), but that may explain the adhesion of tgrB1tgrC1 cells to each other. tgrB1 and tgrC1 could mediate repulsion between cells with nonmatching alleles by modifying other adhesion complexes (11), but the inclusive attraction of merodiploid cells to one another, to the parental strains, and to the double gene–replacement strains, suggests that tgrB1 and tgrC1 mediate attraction rather than repulsion.

Other self-recognition systems rely on polymorphic membrane proteins with extracellular immunoglobulin-like folds (1), including the mammalian major histocompatibility complex (12), Botryllus fusibility/histocompatibility (13), Drosophila dendrite self-avoidance (14), and Hydractinia allorecognition (15, 16). These proteins are not related to tgrB1-tgrC1 or to each other, which suggests convergent evolution in which similar properties arose, despite the lack of extensive sequence similarity. The important properties are sequence polymorphism, which provides unique identities, and extracellular domains, which mediate cell-cell interactions. The immunoglobulin fold is common in these systems, probably because it is tolerant of sequence variation (17).

Supporting Online Material

Materials and Methods

Figs. S1 to S5

Tables S1 to S2


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We thank Y. Wang and W. J. Cordill for useful discussions. This work was supported by grant no. R01 GM084992 from the NIH. R.B. was supported by National Research Service Award grant F31 GM086131 from the NIH and by an NSF fellowship 2006042535.
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