"Find me" and "eat me" are not phrases one would expect to find in scientific literature, but they are common in Dr. Kodi Ravichandran's University lab. "Millions of cells are born and millions die in our bodies everyday," said Michael Elliott, a research associate in the Ravichandran lab. A large number of these cells die through a process of programmed cell death called apoptosis. "Removing these dead cells is critical to maintain normal function in most tissues," Elliott explained.
In this process, specialized cells called phagocytes find and engulf, or eat, the dying cells. Impaired removal of dying cells has been linked to the development of a variety of diseases, including heart disease, cancer and diabetes.
Currently, Ravichandran and his colleagues are focused on how phagocytes locate these dying cells. "Conceptually it made sense that the phagocytes must be able to detect and move toward these dying ... cells from some distance in a tissue environment," Elliott said. "However, the means by which a dying cell could 'talk' to the phagocyte to encourage [it] to come and eat [the cell] was not known."
Elliott and his coworkers set out to investigate this process and published their findings in the journal Nature in 2009. By using a variety of techniques in cultured cells and in laboratory mice, the researchers determined that dying cells release small molecules called nucleotides. These nucleotides diffuse outward into the cellular environment, much like a spray of perfume in a room. Phagocytes then detect, or "smell," these nucleotides and follow them to their source.
The researchers tested this by taking liquid surrounding the dying cells, as it contained a variety of substances excreted by the cells. They then placed this mixture near phagocytes in specialized dishes and measured the movement of the phagocytes toward it.
"At the beginning, the molecular identity of the [find-me] cues was a real mystery," Elliott said. "After months of unsuccessful attempts, I finally found that depletion of nucleotides [from the mixture] almost completely blocked the 'find-me' response. This was the watershed moment for this project."
These findings inspired further investigation by Faraaz Chekeni, a medical student first in the master's and doctorate program before working in the lab as a graduate student. "I thought it was very interesting that we could harvest factors released by apoptotic cells and use them to attract [phagocytes]," Chekeni said. "When Rusty [Elliott] demonstrated that the find-me signals were in fact nucleotides, I wanted to know how these factors were released."
The subsequent experiments were published in a paper in the journal Nature last year, outlining a series of experiments wherein they identified the role of a protein on the surface of cells called pannexin 1. This protein creates a hole, or channel, in the cell membrane which can be opened or closed, much like a door, to allow nucleotides to leak out of the cell.
"One of the most surprising findings," Chekeni said, "was that cleavage of the channel by caspases resulted in the activation of the channel." Caspases are enzymes known to be turned on during apoptosis to chop up a variety of molecules in the cell. In this case, caspases cut off one end of the protein, thereby causing the channel to remain open, akin to removing the door from its hinges.
Further evidence showing that cleavage of the pannexin 1 protein leads to nucleotide release came from experiments in which Chekeni put into cells a mutated form of the protein without the cleaved end, much like putting in an extra door jam without a door. These cells then constantly leaked nucleotides, even when they were not undergoing apoptosis.
"I think the 'coolest' data is that expression of the truncated version of pannexin is enough to open the channel," said Dr. Allison Armstrong, co-author and postdoctoral fellow in the Ravichandran lab. "This is a critical piece of data that has advanced the field's understanding of how pannexin channels function."
Collectively, these studies have opened a variety of new avenues to the Ravichandran and other labs. One of the main focuses now is to determine what role nucleotide release plays in disease by studying its function in laboratory animals. "We hope to eventually use this understanding to develop more effective prevention and treatment strategies for diseases such as cancer and autoimmunity," Elliott said.
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