Concurrent coevolution of intra-organismal cheaters and resisters.

The evolution of multicellularity is a major transition that is not yet fully understood. Specifically, we do not know whether there are any mechanisms by which multicellularity can be maintained without a single-cell bottleneck or other relatedness-enhancing mechanisms. Under low relatedness, cheaters can evolve that benefit from the altruistic behaviour of others without themselves sacrificing. If these are obligate cheaters, incapable of cooperating, their spread can lead to the demise of multicellularity. One possibility, however, is that cooperators can evolve resistance to cheaters. We tested this idea in a facultatively multicellular social amoeba, Dictyostelium discoideum. This amoeba usually exists as a single cell but, when stressed, thousands of cells aggregate to form a multicellular organism in which some of the cells sacrifice for the good of others. We used lineages that had undergone experimental evolution at very low relatedness, during which time obligate cheaters evolved. Unlike earlier experiments, which found resistance to cheaters that were prevented from evolving, we competed cheaters and noncheaters that evolved together, and cheaters with their ancestors. We found that noncheaters can evolve resistance to cheating before cheating sweeps through the population and multicellularity is lost. Our results provide insight into cheater-resister coevolutionary dynamics, in turn providing experimental evidence for the maintenance of at least a simple form of multicellularity by means other than high relatedness.

ABC transporters required for endocytosis and endosomal pH regulation in Dictyostelium.

In Dictyostelium, the RtoA protein links both initial cell-type choice and physiological state to cell-cycle phase. rtoA- cells (containing a disruption of the rtoA gene) generally do not develop past the mound stage, and have an abnormal ratio of prestalk and prespore cells. RtoA is also involved in fusion of endocytic/exocytic vesicles. Cells lacking RtoA, although having a normal endocytosis rate, have a decreased exocytosis rate and endosomes with abnormally low pHs. RtoA levels vary during the cell cycle, causing a cell-cycle-dependent modulation of parameters such as cytosolic pH (Brazill et al., 2000). To uncover other genes involved in the RtoA-mediated differentiation, we identified genetic suppressors of rtoA. One of these suppressors disrupted two genes, mdrA1 and mdrA2, a tandem duplication encoding two members of the ATP binding cassette (ABC) transporter superfamily. Disruption of mdrA1/mdrA2 results in release from the developmental block and suppression of the defect in initial cell type choice caused by loss of the rtoA gene. However, this is not accomplished by re-establishing the link between cell type choice and cell cycle phase. MdrA1 protein is localized to the endosome. mdrA1- /mdrA2- cells (containing a disruption of these genes) have an endocytosis rate roughly 70% that of wild-type or rtoA- cells, whereas mdrA1- /mdrA2- /rtoA- cells have an endocytosis rate roughly 20% that of wild-type. The exocytosis rates of mdrA1- /mdrA2- and mdrA1- /mdrA2- /rtoA- are roughly that of wild-type. mdrA1- /mdrA2- endosomes have an unusually high pH, whereas mdrA1- /mdrA2- /rtoA- endosomes have an almost normal pH. The ability of mdrA1/mdrA2 disruption to rescue the cell-type proportion, developmental defects, and endosomal pH defects caused by rtoA disruption, and the ability of rtoA disruption to exacerbate the endocytosis defects caused by mdrA1/mdrA2 disruption, suggest a genetic interaction between rtoA, mdrA1 and mdrA2.

A protein containing a serine-rich domain with vesicle fusing properties mediates cell cycle-dependent cytosolic pH regulation.

Initial differentiation in Dictyostelium involves both asymmetric cell division and a cell cycle-dependent mechanism. We previously identified a gene, rtoA, which when disrupted randomizes the cell cycle-dependent mechanism without affecting either the underlying cell cycle or asymmetric differentiation. We find that in wild-type cells, RtoA levels vary during the cell cycle. Cytosolic pH, which normally varies with the cell cycle, is randomized in rtoA cells. The middle 60% of the RtoA protein is 10 tandem repeats of an 11 peptide-long serine-rich motif, which we find has a random coil structure. This domain catalyzes the fusion of phospholipid vesicles in vitro. Conversely, rtoA cells have a defect in the fusion of endocytic vesicles. They also have a decreased exocytosis rate, a decreased pH of endocytic/exocytic vesicles, and an increased average cytosolic pH. Our data indicate that the serine-rich domain of RtoA can mediate membrane fusion and that RtoA can increase the rate of vesicle fusion during processing of endoctyic vesicles. We hypothesize that RtoA modulates initial cell type choice by linking vegetative cell physiology to the cell cycle.

A cell-counting factor regulating structure size in Dictyostelium.

Developing Dictyostelium cells form large aggregation streams that break up into groups of 0.2 x 10(5) to 1 x 10(5) cells. Each group then becomes a fruiting body. smlA cells oversecrete an unknown factor that causes aggregation streams to break up into groups of approximately 5 x 10(3) cells and thus form very small fruiting bodies. We have purified the counting factor and find that it behaves as a complex of polypeptides with an effective molecular mass of 450 kD. One of the polypeptides is a 40-kD hydrophilic protein we have named counting. In transformants with a disrupted counting gene, there is no detectable secretion of counting factor, and the aggregation streams do not break up, resulting in huge (up to 2 x 10(5) cell) fruiting bodies.

RtoA links initial cell type choice to the cell cycle in Dictyostelium.

In Dictyostelium, initial cell type choice is correlated with the cell-cycle phase of the cell at the time of starvation. We have isolated a mutant, ratioA (rtoA), with a defect in this mechanism that results in an abnormally high percentage of prestalk cells. The rtoA gene has been cloned and sequenced and codes for a novel protein. The cell cycle is normal in rtoA. In the wild type, prestalk cells differentiate from those cells in S or early G2 phase at starvation and prespore cells from cells in late G2 or M phase at starvation. In rtoA mutants, both prestalk and prespore cells originate randomly from cells in any phase of the cell cycle at starvation.

A Dictystelium mutant with defective aggregate size determination.

Starved Dictyostelium cells aggregate into groups of roughly 10(5) cells. We have identified a gene which, when repressed by antisense transformation or homologous recombination, causes starved cells to form large numbers of small aggregates. We call the gene smlA for small aggregates. A roughly 1.0 kb smlA mRNA is expressed in vegetative and early developing cells, and the mRNA level then decreases at about 10 hours of development. The sequence of the cDNA and the derived amino acid sequence of the SmlA protein show no significant similarity to any known sequence. There are no obvious motifs in the protein or large regions of hydrophobicity or charge. Immunofluorescence and staining of Western blots of cell fractions indicates that SmlA is a 35x10(3) Mr cytosolic protein present in all vegetative and developing cells and is absent from smlA cells. The absence of SmlA does not affect the growth rate, cell cycle, motility, differentiation, or developmental speed of cells. Synergy experiments indicate that mixing 5% smlA cells with wild-type cells will cause the wild-type cells to form smaller fruiting bodies and aggregates. Although there is no detectable SmlA protein secreted from cells, starvation medium conditioned by smlA cells will cause wild-type cells to form large numbers of small aggregates. The component in the smlA-conditioned media that affects aggregate size is a molecule with a molecular mass greater than 100x10(3) Mr that is not conditioned media factor, phosphodiesterase or the phosphodiesterase inhibitor. The data thus suggest that the cytosolic protein SmlA regulates the secretion or processing of a secreted factor that regulates aggregate size.

Mutagenesis and gene identification in Dictyostelium by shotgun antisense.

We have developed a mutagenesis technique that uses antisense cDNA to identify genes required for development in Dictyostelium discoideum. We transformed Dictyostelium cells with a cDNA library made from the mRNA of vegetative and developing cells. The cDNA was cloned in an antisense orientation immediately downstream of a vegetative promoter, so that in transformed cells the promoter will drive the synthesis of an antisense RNA transcript. We find that individual transformants typically contain one or occasionally two antisense cDNAs. Using this mutagenesis technique, we have generated mutants that fail to aggregate, aggregate but fail to form fruiting bodies, or aggregate but form abnormal fruiting bodies. The individual cDNA molecules from the mutants were identified and cloned using PCR. Initial sequence analysis of the PCR products from 35 mutants has identified six novel Dictyostelium genes, each from a transformant with one antisense cDNA. When the PCR-isolated antisense cDNAs were ligated into the antisense vector and the resulting constructs transformed into cells, the phenotypes of the transformed cells matched those of the original mutants from which each cDNA was obtained. We made homologous recombinant gene disruption transformants for three of the novel genes, in each case generating mutants with phenotypes indistinguishable from those of the original antisense transformants. Shotgun antisense thus is a rapid way to identify genes in Dictyostelium and possibly other organisms.