Tuesday, 18 February 2014 09:19

Will Aiptasia, a Sea Anemone with an Algal Symbiont, Become the Next C. elegans?

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Anemone-2Can Aiptasia model the symbiotic relationship
of corals and their internal algae (small golden
spots), revealing the cell biology mechanisms
driving destructive coral "bleaching?"
Photo by Jan DeNofrio
John Pringle has been going to different sorts of meetings this last decade. He is still a regular at the ASCB Annual Meeting and at smaller yeast biology gatherings. Indeed he was in New Orleans for the ASCB Annual Meeting in December to receive the E.B. Wilson Medal, the ASCB's highest scientific honor, for his pioneering work on cell polarization and cytokinesis. But Pringle also goes, when he can, to the International Coral Reef Symposium, the Society for Microbial Ecology, and the International Symbiosis Society. He still has a small yeast group in his lab although his other interests have represented the majority since 2007. He is becoming known at these marine biology and ecology meetings, but Pringle says that he wishes there were more cell biologists there. John Pringle aims to correct that.

Since 2005, his lab has been developing an in vivo laboratory system that can model intercellular relationships between coral animals, the multicellular reef builders that support a major share of all oceanic biodiversity but feed themselves by hosting endosymbiotic dinoflagellate algae, intimate converters by photosynthesis of CO2 into coral energy. Pringle's model is not a coral but a related sea anemone, Aiptasia pallida, a member of the same phylum, Cnidaria, and Class, Anthozoa. Aipatsia also hosts its own endosymbiont algae. A coral reef in a tropical sea is a dazzling feast for the eyes but no place for experimental genetic manipulations, Pringle explains. Besides he's a model systems guy.

When you ask Pringle how he got from yeast to coral symbionts, he takes you back to nature and to water. "As a kid I was led to natural history by my parents," Pringle explains, "particularly my dad who was an avid natural historian while working as a CPA and treasurer for a company in Chicago." Pringle grew up hiking, bird watching, and eventually collecting moths and butterflies. Oddly enough, he never took a biology course in high school and went through Harvard College as a math major. But halfway through, Pringle realized both that some other Harvard math majors seemed to have a gear that he didn't and also that he was actually more interested in biology. So he headed for graduate school intending to use his math in population biology.

But long before that, there was Pringle's immersion in competitive swimming, first in high school and then at Harvard. Indeed, he's never stopped. "I swam 2,000 yards this morning," he confesses. But outdoor swimming was another story and despite dips in murky Lake Michigan, Pringle didn't have his eyes opened until a snorkeling vacation in the Florida Keys shortly after college. "I still swim up and down pools, looking at the line on the bottom, but it's much better to swim over a coral reef." From his first view, the colors and the complex communities around corals astounded him. Pringle toyed with the idea of switching to marine biology. But his scientific life took a fateful turn when he joined, as a postdoc, Leland Hartwell's yeast lab at the University of Washington. Actually Pringle was Hartwell's first postdoc. Pringle became a vital part of the group that earned Hartwell the 2001 Nobel Prize in Physiology or Medicine. Pringle went on to his own labs and his own great successes in yeast, elucidating the principles of cell polarity such as the role of Cdc42, the Rho GTPase that shapes the cytoskeleton in both yeast and animal cells. He moved from the University of Michigan to the University of North Carolina, Chapel Hill, and finally to Stanford.

But corals continued to intrigue him. There was a graduate student with an unusual background in a lab across the hall from Pringle's in Chapel Hill. Kim Ritchie had done undergraduate marine biology research at the University of South Carolina on fungal diseases of Caribbean corals. She went to UNC for her PhD to learn the cell and molecular toolkit by working on yeast. Yeasts are fungi, so Pringle and Ritchie would trade perspectives on yeasts in petri dishes vs. fungal pathogens on coral reefs. Eventually, Ritchie told Pringle that the quadriennial International Coral Reefs Symposium was coming up in 2004 in Okinawa and that he should go.

Pringle had turned 60 the year before. He'd had been quietly wondering if this was his last chance to tackle a new problem, something related but outside yeast biology. Corals and their fungal problems might be that problem. "I knew that coral reefs around the world were in trouble," he recalls. He flew to Okinawa, Japan. "I dutifully went to all the sessions on coral diseases, of which there were many, but somehow I didn't find anything that resonated. But what struck me was that there was almost no genetics or cell biology. There was some population genetics but there was no genetic analysis of organismic function, which I'd been doing for most of my life. And there was essentially no cell biology." As for coral animals and their symbiotic algae, "No one knew much of anything about their cell and molecular biology."

All of Pringle's yeast work had been curiosity driven even if much of it turned out to be powerfully relevant to the biology of other cells, including human cells. But coral reefs are in trouble all over the world; victims of pollution, global climate change, and pandemic diseases. So here was a problem where he could see in advance that his cell science should make a practical difference.

Coral animals are marine invertebrates of the phylum Cnidaria that live in closely knit communities of multicellular polyps. Each polyp nurses one species of dinoflagellate algae inside, feeding them on CO2 and trace nitrogen. The algae, in turn, convert this by photosynthesis into glucose and other nutrients for the coral. To Pringle's mind, this relationship presented an array of fundamental cell biology issues. How did the symbiotic partners recognize each other such that the animal did not view the alga as a pathogen or a piece of food? How did they coordinate their reproduction? In stressed coral reefs where the corals are succumbing to "bleaching," which partner makes the call that ends the symbiosis?

To Pringle, these were all classic cell biology questions, touching on cell cycle control, intra and intercellular signaling, and immune recognition. Studying the cell biology of symbiosis on an ocean reef was out of the question, but when Pringle questioned the marine biologists who kept corals in aquaria, he heard frightening tales of the animals' delicacy, requiring keepers to live with multiple alarm beepers that watched 24/7 over temperature, gas concentrations, and contaminants in the reef tanks. Pringle was looking to build a laboratory model system, not Sea World. "I was looking for a weed, something that grows fast, and is hard to kill," Pringle says. In short, he was looking for a Cnidarian version of baker's yeast.

He found it in Aiptasia, a sea anemone in the Cnidaria phylum that lives in symbiosis with dinoflagellate algae closely related to those in corals. Aiptasia sets up on mangrove roots, bridge abutments, docks, rocks, and in aquaria where it is considered a particular pest. Aiptasia had a small cell biology literature going back to the 1970s. It had a strong supporter for the idea of using it for a model system in Virginia Weis, a zoologist at Oregon State University whom Pringle had met at the meeting in Okinawa. Pringle says his explicit models for his Aiptasia program were Sydney Brenner with the roundworm Caenorhabditis elegans and Elliot Meyerowitz and Chris Somerville with the flowering cress Arabidopsis thaliana, not to mention his own and others' efforts on Saccharomyces cerevisiae. Pringle would build a model around his sea anemone. "I thought I knew how to make a model organism work and how to build a community around a model organism."

Anemone-3Eating out, an Aiptasia pallida snags a passing
brine shrimp but its main nutrition comes
internally from its symbiotic dinoflagellate algae.
Photo by Natalya and Cody Gallo
He also knew that he would be outside the usual boxes for funding. Pringle admits he "bootlegged" the project to life with undergrad volunteers and his own time until he was able to horse trade service as a graduate school dean at Stanford for a small research grant in lieu of a salary increment. With that, he had his first fulltime postdoc working on Aiptasia.

Through Stanford's flexible system of graduate student research grants, he built a team. He also formed a collaborative partnership with Stephen Palumbi at Stanford's Hopkins Marine Station and Arthur Grossman in the Department of Plant Biology of the Stanford branch of the Carnegie Institution for Science. Three years ago, the three of them were able to convince the Gordon and Betty Moore Foundation (GBMF) that countering the environmental threats that were imperiling coral reefs required a better understand of symbiont physiology. The GBMF grant included funding for Pringle's experimental model system, Aiptasia. (Check out the Pringle lab video on the Aiptasia project here.) It's also available through GBMF here

Building the Aiptasia system has not been easy, says Pringle. Beyond the funding difficulties, Aiptasia genetics have proven difficult, although the lab now has a way to steer the usually asexual anemone into sexual reproduction. RNAi to create loss-of-function phenotypes is still a work in progress. Yet collaborating with Grossman, Palumbi, and other colleagues, Pringle was able to uncover a critical clue about the mechanisms behind coral bleaching during heat stress. The standard theory, supported by many experiments, is that the pathway that leads to algal loss is set in motion by light-dependent generation of reactive oxygen species (ROS) by heat-damaged chloroplasts.

Prompted by postdoctoral fellows Dimitri Tolleter and François Seneca, the researchers wondered if bleaching could occur in the dark. Using Aiptasia and a few captive coral species, Pringle says that they were able to demonstrate that with heat stress, "We could show bleaching as rapid in the dark as in the light." This does not necessarily contradict the importance of a light-dependent bleaching process, says Pringle, but there must at least be another cellular pathway that leads to the same result.

The existence of redundant pathways is old news in cellular physiology, but Pringle thinks that cell biologists could learn a lot by working in less familiar organisms. Yeast geneticists, he says, including himself, have been working with blinders on. We have been too focused on animal and fungal biology, he says. "One of the lessons is that there are other whole worlds of interesting biology out there, waiting to be explored." Marine bacteria, viruses, algae, and protozoa perform cellular functions like those in animals and fungi, but the mechanisms they employ are mostly unknown. Pringle's favorite is the formation of cleavage furrows that pinch off dividing cells from each other and have been thought to depend on an actomyosin "contractile ring." Pringle says that brown and green algae, among others, also form furrows for the same purpose, but because the algae split off from the evolutionary line that leads to the animals and fungi so long ago, they do not have the type of myosin found in contractile rings. So how do these algae produce their cleavage furrows? No one knows, says John Pringle, a thought which delights him. "The ocean is full of stuff that no one knows anything about," he says.

John Fleischman

John is ASCB Senior Science Writer and the author among other things of two nonfiction books for older children, "Phineas Gage: A Gruesome But True Story About Brain Science" and "Black & White Airmen," both from Houghton-Mifflin-Harcourt, Boston.

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