The Cheating Amoeba

Scanning electron micrograph of spore towers of the slime mold Dictyostelium discoideum.
David Scharf / Photo Researchers, Inc

What genes contribute to social interactions such as cheating or altruism? And what could cheating genes tell us about sociality, multicellularity, and cancer? A social soil amoeba could hold the answers.

By Gad Shaulsky

I was writing my thesis on the role of p53 in cancer in the early 1990s with a gas mask at my side. It was the first Gulf War, and the threat of Scud missiles that Iraq was launching at Israel made focusing on my thesis difficult. I had already decided to leave cancer research. I welcomed the distraction of reading about altruism, and about the strange little...

Those tumultuous days in Israel marked a major turning point in my career, but my interest in altruism had started years before. When I was an undergraduate at Tel Aviv University in the early 1980s, I'd spend hours discussing the evolution of multicellularity with my good friend Zeev Pancer, who now works at the Center of Marine Biotechnology at the University of Maryland Biotechnology Institute. I had concentrated my studies on genetics, biochemistry, and molecular biology while Zeev, who focused on zoology and animal behavior, took a course with Amotz Zahavi, a world-renowned scientist known for his controversial views on evolution. Zahavi ruffled the feathers of many evolutionary biologists when he suggested that altruism doesn't really exist - he said that there was no such thing as kindness for the sake of kindness.

His argument flew in the face of established experimental research and mathematical models1 that demonstrated how altruism could confer an evolutionary advantage at the species level - even at a cost to the individual. Leaders in evolutionary biology argued that an organism might give up personal advantage under conditions that would benefit its close relatives. I was an avid reader of Ayn Rand's novels and philosophy as a teenager, so I preferred Zahavi's point of view. So did Zeev.

The paradox of altruism was always in the back of my mind, even as I started my doctoral work on p53 and the role it played in B-cell differentiation at the Weizmann Institute of Science. I started to realize that testing altruism using the immune cells I had become familiar with would be impossibly cumbersome. Zeev had a better idea. He told me about an organism named Dictyostelium discoideum, often inaccurately called a slime mold, which seemed to exhibit altruism as it transitioned from a group of single cells into a multicellular organism.

D. discoideum (Dicty to its friends) lives as an individual single-celled organism as long as food is plentiful. But when threatened with starvation, tens of thousands of related and unrelated cells aggregate into a multicellular organism. The aggregate differentiates into a mass, or slug, with coordinated movement and senses. This slug then travels upwards in the soil towards light and heat and turns into a lollipop-shaped fruiting body. The fate of each cell is not equal: About 80% of the cells become viable spores that can survive drought and starvation; the remaining 20% die while forming a cellular stalk that lifts the spores high above the surface for dispersal. Was this an example of cellular altruism? Was there a gene that programmed the cells to give up their lives for the success of the group? I was captivated.

Was this an example of cellular altruism? Was there a gene that programmed the cells to give up their lives for the success of the group? I was captivated.

In the evolutionary jump from the independence of single-celled existence, where no sacrifices are required, a cell within a multicellular system makes a huge sacrifice: compromising the ability to survive and reproduce independently of the mass. Looking at Dicty's pattern of development always made me wonder about another multicellular organism: the human. What if the inclination to live as an independent entity still exists under the veil of suppression in every cell in a multicellular organism?

Back in the early 1990s, when I first became interested in it, Dicty wasn't amenable to the kind of experiments I wanted to do. While it was possible to make Dicty mutants with different traits using chemical mutagenesis, it was nearly impossible to clone the mutated genes. Nevertheless, I was intrigued by what could be done with Dicty, so I decided to interview for a postdoctoral position with Bill Loomis, a luminary in Dicty research.

During my visit to Bill's laboratory at the University of California San Diego (UCSD), I learned that a postdoc in the lab, Adam Kuspa, was developing new methods for genetically manipulating the organism. I could see that those methods might one day let me delve further into the genetic basis of altruism. Bill was very open-minded towards my unusual ideas, and I left that interview full of excitement, already with plans for future experiments.

M.J. Grimson and R.L. Blanton, Biological Sciences Electron Microscopy Lab / Texas Tech University

I moved to San Diego in May of 1991 and was delighted to work with Bill and get to know Adam, who soon became my good friend and colleague. Adam was mapping the Dicty genome, laying the groundwork for the genome-sequencing project that would follow a few years later. While teaching me how to work with Dicty, he also found time to debate altruism and development. It was clear he was a bigger fan of altruism than I.

A few years earlier, researchers discovered that Dicty cells express cell type-specific genes during their development into a fruiting body. They found the cotB gene, which encodes a spore-coat protein and is expressed in prespore cells about halfway into the developmental process. They also discovered the ecmA gene, which is induced exclusively in prestalk cells shortly after cotB induction in prespore cells. Bill and I hypothesized that cells would ?prefer' the prespore pathway, so we wanted to learn how some cells ended up in the "bottom 20%" as stalk cells of the terminal lollipop structure (see infographic).

To address this question, I designed a genetic ablation experiment. I put a gene for the toxin ricin-A under the promoters of the prespore gene (cotB) and the prestalk gene (ecmA) and expressed them in separate strains. These strains allowed us to track the behavior of each cell type as we selectively killed the other. When we killed prespore cells, the prestalk cells converted into prespore cells and then died as they began expressing the toxin from the cotB promoter. It was a fairly simple experiment, but it told us something very important: Every Dicty cell had the capacity and the tendency to take the coveted spore fate. So why did some cells end up as stalk cells?

When we did the converse experiment, killing only the prestalk cells, none of the prespore cells were "willing" to express the ecmA-prestalk gene, and the slugs formed neither stalks nor spores. It looked as though, once starvation set in, all Dicty cells were in a race to acquire the prespore fate. The ones that were able to do so first produced a signal that suppressed the remaining cells from becoming prespore, thus sending them down the deadly prestalk path. Since I did not favor the altruistic point of view, this experiment suggested to me that the prestalk cells may not be altruistic at all, but rather the losers in the race. It also showed that prestalk cells are essential for spore formation.

The genetic ablation experiment2 was very important to me. When I left the p53 field a year earlier, most of my mentors and friends thought that I was making the wrong career move. p53 was a popular molecule at the time and my publication record in the field was very strong. I was advised not to give up p53 for an organism with a name that most people can't even pronounce, but I preferred scientific interest to fame. I never had second thoughts about my decision, but after my first successful experiment and my first publication in Dictyostelium I must admit, I felt vindicated.

It was exciting to think that Zahavi's ideas, which were developed around animal behavior, might function at the scale of cellular differentiation. In my mind, there are two big problems with thinking that stalk cells are altruistic: How could altruism evolve in the first place, and how could it be maintained despite the possibility of the converse behavior - cheating. Cheaters are individuals that enjoy the benefits of being in a society without paying the price. In a human society, a cheater may avoid paying taxes but enjoy the emergency services, the paved roads, and other benefits provided by the society. Cheaters are known in other social organisms, and societies restrain them through various mechanisms, such as policing. I was aware of earlier experiments by Leo Buss that demonstrated the occurrence of cheaters in natural populations of a related species, D. mucoroides.3 Buss referred to them as somatic cell parasites; we refer to them as cheaters - cells that make more than their fair share of spores.

By 1992 Adam had developed restriction enzyme-mediated integration (REMI) - the method he had been working on when I applied to the Loomis lab that allowed us to mutate Dicty genes one at a time and clone the mutated genes. Adam and I started to characterize mutants that exhibited morphological defects during development and I began to learn how to do genetic screens. After Adam got a faculty position at Baylor College of Medicine, Bill and I adapted his method to perform saturation mutagenesis - generating a population of strains that contain mutations in nearly all of the genes in the genome, one mutation per strain.

The cheater cells abounded. Many clones contributed more than their fair proportion to the prespore region at the expense of their counterparts; some contributed as little as 10% or 5% to the prestalk, rather than the requisite 20%.

We had a lot of fun with our new tool and used it to discover a signaling mechanism that coordinates differentiation and morphogenesis during development. In 1997 I got a faculty position at the Department of Molecular and Human Genetics at Baylor College of Medicine and revived my collaboration with Adam, who was already at the Department of Biochemistry. I started to develop genomic tools in anticipation of the genome sequence of Dictyostelium that Adam was working on, and to explore the molecular mechanisms of intercellular communication. Altruism, cheating, and other evolutionary questions had to wait.

In 2000, my friend Rich Kessin and his colleague Herb Ennis at Columbia University were the first to use REMI to look for the cheaters that Buss had described - strains that contributed a larger percentage of their cells to the spores in chimera with wild type cells. The mutated gene they found in that screen was named cheater A (chtA), which was later renamed fbxA. This was the first demonstration of a cheater mutation in Dictyostelium and a proof that social behavior was amenable to genetic exploration. The test also made it clear that cheating could be costly. Obligatory cheaters, like the one Rich and Herb found, were completely dependent on others for spore production. My experiments suggested to me that altruism could not explain Dicty's submission to the stalk fate, and Rich and Herb's work showed that cheating was not a sustainable trait either.

It was around that time that I came across two evolutionary biologists who had recently started using Dicty as a model for their studies on the evolution of social behavior. Joan Strassmann and Dave Queller from Rice University had been using insects in their research, but they realized that Dicty was a better model for genetic studies. They tested whether cheating was possible in wild Dicty populations. Unlike Rich's carefully selected mutant clones, Dicty in the wild would form slugs not only with cells from the same clone, but also with genetically heterogeneous neighbors. Instead of using mutants, Joan and Dave mixed clones from the wild at known proportions and tested whether these proportions were maintained in the prespore and prestalk compartments during development.

The cheater cells abounded. Many clones contributed more than their fair proportion to the prespore region at the expense of their counterparts; some contributed as little as 10% or 5% to the prestalk, rather than the requisite 20%. Moreover, the cheaters did not seem developmentally compromised, indicating that they were perfectly capable of cooperation under the right conditions. Clearly these were facultative cheaters that have not been recognized before in Dictyostelium.4

Joan and Dave were leaders in the study of evolution of sociality. Adam and I had honed the molecular biology techniques in this little organism. The first time we met to talk about our ideas, the sparks flew. We fell in love with each other instantly - in a purely collaborative sense. We knew that facultative cheaters were abundant in nature, and now cheating was amenable to genetic study. The time was right and the tools were ready for a genome-scale exploration that could tell us how many genes participate in social behavior and what functions they might perform.

We decided to work together on the project, but we didn't want to look for obligatory cheaters, because we already knew that these mutants were self-limiting and couldn't be abundant in nature. We wanted to find strains that were facultative cheaters, more like the "natural" strains Dave and Joan had found.

Lorenzo Santorelli, who was a graduate student in Joan and Dave's lab, came to my lab to work on the problem. Together with other lab members he generated 20,000 mutant strains and performed a selection similar to the one that Kessin and his colleagues had done. We were surprised at the number of Dicty clones we found with cheating behaviors. When we analyzed the strains further we found well over 100 genes that could confer cheating behavior when mutated. The chtA/fbxA gene was among them, but it wasn't the only one - not by far. These ?social' genes turned out to be involved in many different cellular activities and many were novel genes. More than finding various genetic programs that could confer cheating, we also discovered that our mutants behaved ?badly' only when they formed a chimera with another clone. Surprisingly, when they were starved as genetically homogenous populations, they appeared to submit the full 20% to the stalk fate. It seemed somehow that Dicty knew when to cheat and when to cooperate.

We published our results in Nature in February 2008,5 but like any good science, the results only led to even more fascinating questions. How did cells know when they were in a pure population and when exploitable neighbors were nearby, how did these cells talk, and how did they listen? Were they capable of recognizing each other and altering their behavior? Even before we finished our work on the screen, we had started to work on these questions.

Dictyostelium cells may not garner as much attention as the venerable p53, but they give us a glimpse into a system that grappled with the problems of cellular sociality long before complex multicellular organisms arose. It is pretty clear to me that Dictyostelium cells compete among themselves for the prize of becoming spores, but I don't think the competition we see on the cellular level with Dicty is so unique. One of my graduate students, Anupama Khare, and I have argued that the inclination to compete is conserved, even between the genetically identical cells that comprise multicellular organisms such as humans.6 During early development, competition between cells to remain a proliferative stem cell rather than die as a terminally differentiated skin cell could be more significant than we've imagined so far. This intercellular competition takes on yet another meaning, when we think about it in terms of malignant proliferative processes such as cancer.

Our experiments certainly haven't closed the book on altruism; we've simply added another chapter.

If any of the social genes we found in Dicty can be mutated to generate a cheater, one that takes advantage of surrounding cells for its own benefit, then perhaps there is an inclination toward unfair advantage in all cells - an inclination that was suppressed during the transition to multicellularity, but never eliminated. If we think of Dicty as a model for multicellularity, then it would appear that what happened during that transition was not a loss of competitive tendencies, but rather only suppression. Because we found so many genes that can confer cheating when mutated, we think that the social genes, which restrain cheating, have probably evolved many times and under various selective pressures. It suggests that the social system might be rather vulnerable to cheaters. When I imagine a correlate to cheating genes in human cells, the genes I would choose would be tumor suppressors. These genes are abundant and cover a wide variety of pathways, similar to Dicty's cheater genes. When mutated, tumor suppressors release a cell from its requirement to cooperate within the multicellular system. This single cell, unfettered by its social requirements, can reproduce at will, even though its unilateral action eventually kills the organism.

The terms we use - cheating, competition, altruism, etc. - imply reason and emotion. These terms are borrowed from our human experience, but they should not be misinterpreted. The genes and pathways we find in Dictyostelium are components of intercellular interactions that have been shaped by selective pressures that are quite different from the evolutionary pathways shaping human behavior. The Dicty social genes might have common functions with human genes that regulate cellular and tissue behavior, but they are not likely involved in regulating human behavior the way they regulate Dicty behavior. The deeper concepts, however, may be common. All organisms compete with one another for survival and proliferation; the mechanistic details are different, but the motivation and the consequences are similar.

Our experiments certainly haven't closed the book on altruism; we've simply added another chapter. I'm sure the debate will continue, within the walls of my laboratory and beyond. Just the same, given Dicty's long-standing image as an altruist, I imagine Zahavi would appreciate what a little cheater Dicty has turned out to be.

Gad Shaulsky is a Professor and Director of Graduate Studies of Molecular and Human Genetics at Baylor College of Medicine. The work has been supported with a Frontiers in Integrative Biological Research grant from the National Science Foundation.

1. W.D. Hamilton, "The genetical evolution of social behaviour I and II," J Theor Biol, 7:1-52, 1964.
2. G. Shaulsky, W.F. Loomis, "Cell type regulation in response to expression of ricin A in Dictyostelium," Dev Biol, 160:85-98, 1993.
3. L.W. Buss, "Somatic cell parasitism and the evolution of somatic tissue compatibility," Proc Natl Acad Sci, 79:5337-41, 1982.
4. J.E. Strassmann et al., "Altruism and social cheating in the social amoeba Dictyostelium discoideum," Nature, 408:965-7, 2000.
5. L.A. Santorelli et al., "Facultative cheater mutants reveal the genetic complexity of cooperation in social amoebae," Nature, 451:1107-10, 2008.
6. A. Khare, G. Shaulsky, "First among equals: competition between genetically identical cells," Nat Rev Genet, 7:577-83, 2006.

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