ABOVE: Epithelial cells in the upper intestine © iStock.com, Dr_Microbe

Every day, billions of our cells die and new, healthy ones take their place. In a healthy gut lining, as in most tissues, a type of cell death called apoptosis is thought to mediate this process almost entirely on its own. But researchers from RIKEN in Kobe, Japan, suspect they have discovered a new kind of cell death in the gut of a fruit fly. The new process, which they call erebosis or “deep darkness,” may be present in other tissues, the team reports April 25 in PLOS Biology and if found in humans, it could affect how we understand diseases of the gastrointestinal tract. 

María Domínguez Castellano, a neuroscientist at the Institute of Neurosciences in Alicante, Spain who didn’t work on the study, tells The Scientist she found the paper “very intriguing,” and hopes to see more from the researchers about the new putative form of cell death. “They’ve clearly done so much work but . . . [Erebosis] is clearly there and it’s so intriguing that you want more.”

Accidentally uncovering an alternative to apoptosis

“The dogmatic idea is apoptosis is involved in cell turnover in the gut,” study coauthor Sa Kan Yoo, a biologist at RIKEN, tells The Scientist. He says that this dogma is so strong that he was initially resistant to the idea that he was looking at a new form of cell death.

The project began several years ago, when Yoo and RIKEN PhD student Hannah Ciesielski were searching for a factor that regulates the systemic effects of cancer. A protein called Ance—the fruit fly form of the human angiotensin-converting enzyme, which is involved in the regulation of blood pressure and electrolyte balance in humans—was a top candidate. The researchers began to map the locations of Ance in tissues throughout the body of the fruit fly Drosophila melanogaster using an antibody that labels the protein with Green Fluorescent Protein (GFP) and that brought them to the gut.

Their imaging experiments revealed that some gut cells are full of Ance, while others produce very little. Intrigued, they decided to find out why. 

Yoo and his team thought they had stumbled on a new cell type that strongly expressed Ance, which they decided to call Ance cells. These Ance cells “were very weird,” says Yoo. When the team watched Ance cells, they saw that many started to lose proteins, organelles, and important molecules they need to survive. ATP production slowed. Their nuclei swelled, then flattened, and eventually disappeared. The cells also started losing their GFP, glowing less and less brightly. The fact that the cells appeared to lose fluorescence over time signaled to the researchers that the Ance cells were “very dynamic,” and must be going through a period of change. They soon came to think that the cells might be nearing the end of their lives.

A million (or at least three) ways to die in the gut

All cells have a limited lifespan, and their death can come about in several ways. As they age and accumulate mutations, internal or external signals trigger apoptosis, which can be thought of as an organized auto-destruct. The cell shrinks and dissolves into discrete packages called apoptotic bodies, which are later consumed by cell-eating immune cells called phagocytes. Less commonly, damaged, oxygen-starved, or cancerous cells can undergo necrosis, swelling and eventually bursting open to spill their contents into the body. Cells can also die via autophagy, a process akin to consuming themselves, which is thought to be brought about by a lack of food. In autophagy, cells dissolve their internal contents through autophagosomes, large vesicles that break down the cell’s contents. 

Infographic showing different modes of cell death including erebosis.
DEEP DARK DEATH: The researchers of the new study discovered a new type of cell death in Drosophila melanogaster gut cells, which differs from other types of cell death in a few key ways: in apoptosis, the most common cell death process, internal and external signals trigger the cell body, organelles, and nucleus to shrink as the cytoskeleton and DNA break down. The cell breaks down into discrete packages containing parts of the cell called apoptotic bodies, which are later consumed by phagocytes (not shown). Necrosis occurs when tissue is damaged or deprived of oxygen. The cell body and organelles expand, causing the cell to become porous. The organelles break down. Eventually, the cell lyses and dumps its contents into the extracellular sera. During autophagy, a cell forms autophagosomes, vesicles that fuse with lysosomes to create autophagolysosomes, which break down the cell’s contents. The cytoskeleton stays intact. Throughout erebosis or "deep darkness," the gut cell loses organelles. Cells also lose important cytoskeletal proteins, and the microvili on the gut cell’s apical surface shrink. The cell’s nucleus goes through a period of expansion, then flattens and shrinks, eventually fragmenting.
Erin Lemieux

At that point, Yoo and his team were still trying to explain Ance cell activity within the context other forms of cell death, especially apoptosis, as it is thought to be the most common driver of the gut’s quick (once every four-day to three-week) tissue turnover. They began searching for evidence that Ance cells were producing markers of necrosis and autophagy, the other, less-common forms of cell death. But they failed to find evidence that any of the three were taking place. Furthermore, inactivating caspases (which are molecules typically found in cells undergoing apoptosis that signal cells to start breaking down) with microRNAs failed to stop the cells from losing organelles, proteins, or ATP. 

To figure out what was going on, the researchers used a general cell death marker called TUNEL, which labels fragmented DNA. TUNEL labeled some Ance cells but not others. The cells that were labeled had lower GFP signals and squatter nuclei, which strongly indicated that these cells were indeed approaching the end of their lives. 

The researchers also looked at whether this newly-described, Ance-related pathway to death still occurred in Drosophila mutants that lacked important apoptosis, necrosis, and autophagy-related proteins. In all cases, erebosis persisted. In all, their findings pointed to one conclusion: Ance was a marker for a cell’s eventual fate—a kind of cell death no one had described before, which they decided to call erebosis.

Erebosis: more questions than answers

Yoo acknowledges that, technically, the team didn’t prove that cells are dying through erebosis, nor have they worked out a lot of the details. Though they’ve documented that these cells are undergoing a process that seems difficult to bounce back from, they haven’t shown them disappearing in real-time. They could still be alive, Yoo supposes, existing indefinitely in a new, low-metabolic state. Also, exactly how erebotic cells begin to lose organelles or break down cytoplasmic proteins is still unknown. “It’s really hard to prove that a cell is dying,” says Yoo, “It’s almost . . . a philosophical question.” But without organelles or a nucleus, says Yoo, it only makes sense that death is on the horizon for these cells. 

“It’s still unclear how [erebosis] fits into homeostasis . . . and I want to know more about where else erebosis is happening,” says Dominguez.

If erebosis is a death pathway, it could help explain confusing results from other studies, says Andreas Bergmann, a biologist the University of Massachusetts Medical School not involved in the study who wrote a perspective on the paper that was published April 26 in PLOS Biology. “I was really excited when I saw this work,” Bergmann says, as for years, his lab has had trouble showing apoptosis “with standard apoptotic markers.” And in some previous studies, inhibiting apoptosis in gut cells has slowed cell turnover, while in others, it didn’t. This indicates that some other mechanism may be involved.

The findings could also have clinical implications. Defective cell turnover, Yoo says, is related to several gastrointestinal diseases, including ulcerative colitis and gastroenteritis. If erebosis occurs in the human gut, it could go wrong and play a role in certain diseases. Now, Yoo is working on understanding which genes and proteins are involved in erebosis, while his collaborators are checking if this process exists in mammals and in other Drosophila tissues.

And in a strange twist, the researchers have already found that Ance isn’t actually required. The process of molecule- and organelle-dumping and nuclei flattening continued unabated when Ance was knocked out using miRNAs. So, although gut cells tend to take up Ance during erebosisthe researchers don’t yet know why. 

The importance or lack thereof of Ance is not the only erebosis mystery remaining to be solved. Both Bergmann and Dominguez Castellano say that there’s a lot more investigating to do. For instance, they’re eager to learn more about what genes and proteins control erebosis and what other tissues it might occur in. “This [story] isn’t just one paper,” Bergmann says, “It’s ten papers.”

Correction (May 19): This article has been edited to reflect that the new research was published in PLOS Biology, not PLOS ONE. The Scientist regrets the error.