Watching worms die may not sound particularly exciting, but what if when they kicked the bucket they started glowing blue? That’s what a team of biologists has observed in the roundworm Caenorhabditis elegans. A blue “death wave” ripples down the worms’ bodies for up to six hours as the life drains out of them—a phenomenon that is yielding insights into how death spreads throughout an organism.
“Death actually propagates,” says David Gems, a biogerontologist at University College London (U.C.L.) and co-author of the study. “The presence of a dead cell triggers destruction in a neighboring cell.” Gems and his colleagues describe their findings in the July 23 edition of PLoS Biology.
The cascade of cell death in C. elegans rides a wave of calcium ions that travels through the worm from front to back. The influx of calcium into a cell triggers processes that dismantle cellular structures. Lysosomes—the waste-processing centers of cells—burst and “that’s when all hell breaks loose,” as Gems puts it. The lysosome eruptions cause the cell to digest itself. The calcium ions then jump to a neighboring cell and the death wave continues.
The blue fluorescence, seen only when the worms are under ultraviolet light, comes from anthranilic acid, an organic compound produced inside little granules that line the worm’s intestine. As the death wave propagates, the granules burst, releasing the acid into a lower pH environment. The drop in pH amplifies the anthranilic acid’s natural fluorescence, generating a blue flash that follows the calcium wave. The flash “is a marker of death,” Gems says.
By knocking out proteins called innexins that are essential to transferring calcium from one cell to the next, the researchers were able to stop the spread of death in the worm—but only in the case of injury. “Blocking necrosis doesn't expand life span,” Gems says. In the case of aging “there are other things happening in parallel that are contributing to death.”
So does this mean that targeted drugs can stop or slow injury-related death in humans? Not yet. “We're so much more complicated than that,” says Cassandra Coburn, also of U.C.L. and lead author on the paper. “I don’t think you can make that comparison.” But the finding could lead to a better understanding of tissue damage. The propagation of cell death in worms is a model for understanding death in higher order animals “during injury from stroke, infection or mechanical injury like a bone-break or a stab,” Gems explains. “Essentially, that’s universal. What we found is that the pathway involved in this chain of death [in C. elegans] is pretty much the same as in mammals. It's an ancient biology of spreading of death in organisms.”
“People are very excited” about this new insight, says Malene Hansen, a microbiologist who researches aging at the Sanford-Burnham Medical Research Institute but is not involved in the work. “The implication of this systemic collapse as a real process to the event of organismal death is something we had just not thought about before,” she says. For a long time biologists have viewed aging as something that just builds up over time: a wear-and-tear process. This study, however, shows aging and death as a triggered, controlled event. “The emerging picture,” Hansen explains, “is that there’s all these cellular processes that talk to each other…and these processes have been linked to aspects of disease that hopefully we can pin together in a cool way.”
The discovery of the worm’s glowing death wave owes a lot to luck. Coburn was studying blue fluorescence in C. elegans for her PhD project. Other researchers had reported an increase in fluorescence in worm populations over time, leading many biologists to assume it was a marker of aging. Coburn, however, still wanted to figure out what caused it. When she started watching individual worms—as opposed to an entire population—she got a surprise. Rather than slowly brightening as they aged, the worms flashed blue at death. “I stayed in the lab until 2 A.M. to see what was happening because I couldn't bear to not know,” Coburn recalls. “It was such a bizarre phenomenon to watch...it really took my breath away.”
Once Coburn and Gems discovered the death fluorescence, they wanted to know what it was—“what was the blue stuff, how does it form and what’s its relationship to death?” she says. This initial investigation led them to the discovery that the anthranilic acid fluoresces and the calcium wave triggers the event.
Many mysteries remain. For one, what role do innexins—the proteins that shuffle calcium from one cell to the next—play? To identify the calcium pathway, Coburn worked with worms that had a gene mutation that switched off innexin production. No innexins meant no calcium propagation and no death wave. But did the absence of innexins since birth introduce any side effects? “I’m not so sure whether or not having these genes during development could be a problem,” Hansen says. The ideal experiment would leave the innexins intact as the worms mature and then switch them off later in life.
Remarkably, biologists can do this. By feeding the worms a special blend of their favorite food—genetically modified bacteria—researchers can introduce a genetic “off-switch” anytime they like. The technique won Andrew Fire and Craig Mello the Nobel Prize in Physiology or Medicine in 2006.
Researchers would also like to know what starts the calcium wave. “That's the big question,” Gems says. He wonders whether it begins in the same way as rigor mortis, the stiffening of muscles after death. In rigor mortis the inhibition of ATP, the basic unit of energy within a cell, triggers a release of calcium into the muscles. That is, without ATP the cell can’t keep out the calcium ions. Hansen also speculates about what role other tissues play: “It would be cool to ask if it starts in the neurons or muscle,” which could reveal insights into human death.
Both Gems and Coburn are at a loss to explain why the death wave only propagates from front to back. Coburn suggests it might have something to do with a bundle of nerve cells sitting next to the beginning of the intestine. The researchers also think there must be some undiscovered organization in the intestine that differentiates front from back. The intestine is the worm’s only major organ and so has to simultaneously function as a liver and stomach as well. “It’s just a long tube of cells,” Hansen explains, “but there may actually be different compartments, just like our intestinal tube…, that is compelling to us.”
For the moment they have no clear answers but they remain enthralled with the phenomenon. “People are fascinated by seeing this spectral glow of death in an organism,” Gems remarks. “I think that’s important. You don’t get much wonder in C. elegans.”