Monday, 18 August 2014 00:00

Proteins and Prejudice—The Mr. Darcy of Protists, Giardia, Defies the Canonical Mitotic Checkpoint Proteins

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Giadia11This scanning electron micrograph (SEM)
reveals why Giardia is classed as a flagellate.
SEM by the Cande lab,
University of California, Berkeley
It is a truth all but universally acknowledged that a eukaryotic cell entering mitosis must be in want of the canonical proteins for mitotic checkpoints. And then there is Giardia intestinalis. A notorious flagellate pathogen, this binucleate protist belongs to one of the major eukaryotic lineages now called the "Excavates." Like all other Excavates, Giardia is weird, says Zacheus Cande of the University of California, Berkeley, but weird in a good way because of its ancient evolutionary divergence from the better-known branch of eukaryotes where everything from humans to yeast hang out. Just how weird, Cande and Juan-Jesus Vicente, now at the University of Washington School of Medicine, reveal in a paper recently accepted by Molecular Biology of the Cell (MBoC).1 The researchers demonstrate that Giardia manages its mitotic operations without any of the canonical Anaphase Promoting Complex/Cyclosome (APC/C) proteins but using components associated with the Mitotic Checkpoint Complex (MCC) in a strictly non-canonical way to control chromosome segregation, spindle morphology, and nuclei formation and positioning.

Giardia pushes on through the cell cycle by deploying three MCC proteins, Bub3, Mad2, and Mps1, to perform what would be in other eukaryotes double duty, according to Cande and Vicente. The researchers first identified these MCC proteins by searching through the Giardia protein sequence database, looking for MCC proteins that had homologs in humans and yeast, which have eight MCC proteins in common. Giardia has only three homologs in common—Bub3, Mad2, and Mps1. When the researchers knocked down these MCC proteins using morpholinos, they created havoc during mitosis for Giardia, resulting in abnormal numbers of or severely misplaced nuclei.

Working in Giardia is both exciting and puzzling, according to Cande. In canonical eukaryotes, the MCC halts cycle progression until all the duplicated elements—sister kinetochores and sister chromatids—are in place, firmly attached to microtubules, and at proper tension. Then the MCC switches off, releasing the canonical proteins of the APC/C complex to ubiquitinate cell cycle regulators and allowing the dividing chromatids to irrevocably separate. Yet in Giardia, the MCC proteins themselves stand in for the missing APC/C and move the cycle forward without canonical ubiquitination.

Giardia's divergent weirdness comes as no surprise to Cande. In earlier work, he and colleagues explored how Giardia, the first eukaryote shown to lack all the canonical actin-binding proteins, successfully managed its actin cytoskeleton in cell morphogenesis, membrane trafficking, and cytokinesis.2 The researchers also noticed that the Giardia genome, which was the first pathogenic flagellate genome to be sequenced, lacked the canonical proteins for anaphase regulation through ubiquitination. That launched this follow-up, says Cande.

He recalls, "When we first said that ubiquitination is not involved in the cell cycle in Giardia, everyone and his brother said, 'These are very difficult assays and you missed it.' We had to do it over and over, but we showed that there is no APC and no ubquitination by any of the known cell cycle components. If there's no APC, how can you have a checkpoint?"

Cande is cautious. He acknowledges that this is far from the complete mitotic story in Giardia. "One of the reasons that people don't work in Giardia is that there are not that many molecular tools. It's a small community. Tools that work in other organisms like CRISPR and TALENs don't work well in Giardia." With morpholinos, the researchers were able to target the three MCC proteins, but Cande estimates that, "We've found only a few of the checkpoint proteins so this is very incomplete." But from what is known, it is clear that there is so much more to learn.

"Do we really have a checkpoint? We're still agnostic on that. If it's a checkpoint, it's really different. But what we found when we knocked them (the MCC proteins) down, they had direct effects on cell cycle progress." Cande proposes three possible solutions to the mitotic checkpoint question: "One, it has it but we can't recognize it. Two, it doesn't need it, or three, it's evolved something else that takes its place." All three possible answers are intriguing to Cande. "Either way, it's interesting."

MAD2-KDWhen the researchers knocked down the
MCC protein, Mad2, it created mitotic havoc
for Giardia with misplaced microtubules, actin,
and chromatin.
Imaging by Juan-Jesus Vicente
This MBoC paper was edited by Kerry Bloom of the University of North Carolina, Chapel Hill, who works in yeast on chromosome division and placement. A revelation from editing this paper for Bloom was just how different these organisms are from more familiar eukaryotes and how differently they operate. "These proteins that we've been told have these canonical functions can function in other aspects of the process in other organisms," says Bloom.

"In my view, this is like the microbiology of the biome," Bloom says. "We're just starting to realize how these parasites have this wacky biology. The APC is conserved from yeast to man yet it's completely lacking here. I'm not shocked. I think it's the tip of the iceberg of how many different ways that biology can work."

Giardia is more than a theoretical challenge for cell biologists. It is also a potent and active disease agent. In the United States, roughly 20,000 cases of Giardia are reported each year but the figure could be five times higher, says Cande, as well-nourished American adults can shrug off an infection, thinking it's flu. But in developing countries where a third of the population has had giardiasis, Giardia is a familiar player in the cycle of malnutrition, unsafe water, inadequate sanitation, and re-infection that holds generation after generation in poverty. Like so many diseases in developing countries, giardiasis doesn't attract enough research funding or drug development interest, says Cande, even though resistance is developing to the most commonly prescribed drugs. Cande isn't sure that Giardia's weird mitotic proteins could become targets for a new effective (and profitable) giardiasis drug, but he says that this work could help both those who study Giardia infection and those who study eukaryotic mitosis to see its biology in broader dimensions.

There is the take-home lesson for cell biologists, says Cande. "It's necessary to look at organisms that are much more evolutionarily diverse to understand the cell cycle. By studying just yeast and man, we're missing out on something." It's the extreme evolutionary divergence of Giardia and the other Excavates (which include pathogens such as Trichomonas, Trypanosoma, and Naegleria) that make them so valuable, says Cande. "We've only studied the medically relevant Excavates. We have no idea of what the other Excavates are like."

Says Cande, "It's time for an evolutionary cell biology that's more comparative and is looking at highly divergent organisms."

The final version of the paper is scheduled for publication in the September 15, 2014, issue of MBoC.

 

1Vicente JJ, & Cande WZ (2014). Mad2, Bub3 and Mps1 regulate chromosome segregation and mitotic synchrony in Giardia intestinalis, a binucleate protist lacking an Anaphase Promoting Complex. Molecular biology of the cell PMID: 25057014

2Paredez AR, Assaf ZJ, Sept D, Timofejeva L, Dawson SC, Wang CJ, & Cande WZ (2011). An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins. Proceedings of the National Academy of Sciences of the United States of America, 108 (15), 6151-6 PMID: 21444821

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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