Wednesday, 02 July 2014 09:23

Old Lab Animal Model Systems Gain New Respect—Paramecium Redux

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parameciumParamecium.
Illustration by Johnny Chang, ASCB
When the fledgling ASCB held its big meeting in a down-at-the-heels hotel on the Chicago lakefront in 1961, it was something of a carnival of animals, lab animals. Peter Satir, who is now at the Albert Einstein College of Medicine in the Bronx, NY, was present in Chicago. Fifty three years later when asked about the first scientific program, Satir couldn't help pointing out how many different organisms or parts thereof were being studied. There were papers using the rectal gland of the sucker shark, the salt gland of the herring gull, and the gas secreting epithelium of the Portuguese man-of-war. Many of today's common laboratory animal model systems were represented—Drosophila, Dictyostelium, Escherichia coli, and Saccharomyces. But there was only one HeLa presentation and Caenorhabditis elegans had yet to catch the eye of Sydney Brenner. In his first ASCB talk, Satir spoke on fine structure and motion in mussel gill cilia.

Mussel was the perfect organism for his work at the time on the microtubule bundles inside the central flagellar stalk, says Satir. He had two reasons to favor the mussel. "Keith Porter had studied it a little bit and he was my mentor (at what was then the Rockefeller Institute) and he'd also gotten some really nice EMs of clam gills. The other reason was that it (mussel) was a classic material in the ciliary field going back to the 1920s."

Mussels were also cheap and easy to buy fresh through New York City fish markets. More importantly, their cilia-lined gills exhibited metachronal waves, that is, rhythmic beating in which a pulse rippled through long rows of cilia like wind through wheat. With a blast of OsO4 fixative, Satir froze a metachronal wave in its tracks, capturing cilia in every position for closer study under EM. From frozen mussel gills, Satir worked out that the microtubules bundled in the axoneme moved not by extension but by sliding past each other, driven by a one-way motor protein now called dynein. (For this and other ciliary discoveries, Satir will receive the ASCB's E.B. Wilson Medal along with John Heuser of Washington University in St. Louis and Bill Brinkelye of the University of Texas, Southwest, at the 2014 ASCB/IFCB Annual Meeting in Philadelphia).

But Satir and most ciliary studies soon moved on to more portable and genetically malleable ciliates, particularly Tetrahymena and then Chlamydomonas. It was in this last humble protist that the big discoveries of recent years were made that eventually linked defects in primary (non-motile) cilia to a common and still fatal human disorder, polycystic kidney disease (PKD). This sparked off the "ciliopathy" land rush.

Satir has gone on to look at ciliary function in mammals, using mice in recent collaborations with Søren Christensen of the University of Copenhagen. While older model systems may go out of fashion, Satir says, these organisms often have capabilities experimenters can use to address fundamental questions too difficult in more popular model systems. And then sometimes the big questions change and suddenly an old organism is back in the spotlight. Such as Paramecium, suggested Satir.

While many non-scientists remember Paramecium from junior high school biology, the unicellular protozoans were critical in studying ion channels in ciliary motion. "Paramecium were one of the first organisms where it was realized that there were specific ion channels in the cell and one of them was in the cilium." Satir explains. "It's a voltage sensitive calcium channel and it was one of the first described channels. It goes back a long ways." To hear where Paramecium are going today, Satir recommended I speak with Judith Van Houten at the University of Vermont.

Paramecium are having a renaissance as a research model system, at least in the US, says Van Houten, thanks to the sequencing of the genome by a European consortium. "It was eclipsed for a while, more in the U.S. than in Europe where people stayed the course with Paramecium. But as you know, until an organism's genome is annotated and modern techniques for gene manipulation can be applied, it's just hard to use that organism. It's becomes less useful than Drosophila or yeast." The 2006 publication of the Paramecium tetraurelia genome revealed 40,000 genes, the result of three whole genome duplications, and opened new avenues of utility for the tiny ciliate.

"Once the genome was annotated by this consortium in Europe, things broke open again," Van Houten recalls. "I struggled for a long time with the biochemistry of membrane proteins involved in chemical sensing, the receptors and the signal transduction." The complete genome plus new tools of genetic manipulation like transfection through RNAi feeding freed the Van Houten lab to tackle new problems with Paramecium, especially as the link between primary cilia and human PKD that has put ciliopathies on the research map.

In a recent paper, Van Houten demonstrated what post-genome Paramecium can do. Her lab was able to get at the protein meckelin (MKS3), one of two genes linked to Meckel-Gruber Syndrome, a rare but catastrophic developmental disorder that is now considered a ciliopathy. Using RNAi to suppress MKS3 expression created a phenotype of wildly disordered and misaligned cilia in Paramecium, which Van Houten believes is a defect, not in the cilia themselves, but in the guidance and attachment of their ciliary roots to basal bodies.

Their highly patterned surface and its highly patterned cilia make Paramecium a great model on which to see small genetic alterations turn into highly visible phenotype. "If there's even a slight effect, it becomes magnified because of pattern disruption," says Van Houten. "Most of the cilia that are studied are one to a cell. With Paramecium, a slight change [in gene expression] give us a big change in swimming behavior because channels don't go where they should or because the pattern has been disrupted. That's what allowed us to find things about Mecklin [MKS3] that I don't think you would in tissue culture cells or other kinds of model systems."

Using different organisms to get at different questions is why Paramecium are even more useful in the genomic age, Van Houten believes. "We're not a mouse or C. elegans. But we have the ability to use a different protein or a different reaction [to see mechanisms] that aren't uncovered easily in other organisms. We hope we've shown that Mecklin [MKS3] must have various other cytoskeletal interactions and that people should have a look at that."

The long history of Paramecium study is itself an advantage in the genomic age, she says. In the 1970s, Paramecium were used for studies of non-nuclear inheritance by people like Satir and Ching Kung at the University of Wisconsin, Madison. "It was Kung who tried to put together a readout of Paramecium physiology by watching its behavior and putting that together with a genetic dissection of behavior," she says. "This was at a time when bacterial chemotaxis was coming forward and using applied genetic techniques to look for mutants that didn't behave properly." Kung made a name for himself, says Van Houten, creating a new place for Paramecium in research today which "rides on the shoulders of the all the electrophysiology work that had gone on before in genetic dissection."

So does Van Houten see new interest from other labs in her venerable lab model system? "I think it is increasing somewhat," she reports. "When you go to meetings like ASCB, you find a lot of kindred spirits who are interested in cilia. There are always more in Tetrahymena than Paramecium but with Paramecium we have this great history of electrophysiology that we use to support what we're doing and that's very helpful to us. Among that group, Paramecium is a respected organism that can continue to make contributions. And then Peter [Satir] and others have shown how important ciliates can be to understanding," says Van Houten.

Which was Satir's point back at Albert Einstein where he was throwing out other examples of old organisms that are finding new uses in research. "I think that we have not exploited these organisms thoroughly enough."

NEXT: Planaria Reveal a New Side in Their Modeling Careers

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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