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Luciferin and GFP: The Fluorescent Chemicals Used by Insects, Sea Creatures and Humans!

Experiments aimed at finding drugs to kill cancer cells make use of luciferin, one of the molecules responsible for the glow of a firefly.

Eons ago in a remote village in China, a promising young lad was studying for the almighty civil service registry exam. His parents were too poor to afford an oil-burning lamp so that he could read at night. One evening, the lad noticed some bugs that glowed in the darkness as they danced in and out of the tall grass. He had an idea! He captured several dozen, sealed them in a thin white silky bag, and hung the bag on a small tree. Thanks to the luminescent bugs, the boy was able to hit the books well into the night. He passed the exams with flying colors.

Or at least that’s how the story goes. No one can confirm whether the story is fact or fable, but true or not, it is an ancient record of bioluminescence, light produced by living creatures such as fireflies.

How do fireflies create their telltale glow? It differs slightly depending on species—there are more than 2000 species of fireflies found across the world, including many that do not glow—but the one we know the most about is the North American Firefly (Photinus pyralis). It uses a molecule named luciferin and its enzyme buddy luciferase. Luciferase reacts with luciferin, causing it to break down into two compounds and release CO2 One of those two compounds has a bit of excess energy that it releases as light!

The production of this light has three requirements, other than luciferin and luciferase: magnesium, oxygen and ATP. That ATP requirement is a big part of why the luciferin assay has become an important tool for biochemical research. Adenosine-5'-triphosphate (ATP) is the universal "energy molecule" of all forms of life. So, luciferase and luciferin can be used to test if something like a cell is alive and still producing ATP.

At Memorial Sloan Kettering Cancer Center, for example, experiments aimed at finding drugs to kill cancer cells make use of luciferin chemistry. Various types of cancer cells are exposed to candidate drugs in different concentrations for different lengths of time. Add in luciferin and luciferase, and if a cell is alive, it will produce ATP and a glow is observed. The less glow, the more cancer cells that were killed by the drug, and that is music to everyone’s ear.

Bugs that emit light are not the only game in town when it comes to natural glows. The ocean is richly endowed with green-glowing creatures. In fact, the deeper the ocean, the darker it becomes, and the greater the variety of glowing deep-sea creatures. According to NASA, 90% of deep-sea creatures glow! The glow serves a utilitarian purpose for these creatures. Depending on the situation, the light produced can help entice mates or deter a predator.

Some varieties of jellyfish can also glow. They produce a green fluorescent protein (GFP) that is also quite handy for cancer research. Assays involving GFP are more complex than the quick luciferase test, but by incorporating the glowing protein into cancer cells, scientists can easily trace their locations. These shiny green trackers are more stable and have a longer shelf life than luciferin both in vitro (cells grown in a Petri dish) and in vivo (in a whole living organism like laboratory mice).

One of the authors of this article had a personal “eureka encounter” with this technology. During her graduate school days she had an interview with a leading expert in liver cancer at the Cold Spring Harbor Laboratory, on the Gold Coast of Long Island. After a brief polite chitchat, the scientist switched to a discussion of science. With a visible glow in his eyes, he said “I am going to show you our new data, and it is absolutely brilliant! Are you ready?” What appeared on the super-sized computer monitor was a laboratory mouse with glowing green spots in selective parts of its body, from head to tail. These were liver cancer cells that had spread from the original organ via the lymph nodes. Thanks to GFP, no cancer cells were able to hide!

In 2008, Dr. Osamu Shimomura, Dr. Martin Chalfie, and Dr. Roger Y. Tsien were awarded the Nobel Prize for their contribution to fluorescent technologies. Dr. Shimomura tells of having captured more than a million jellyfish along the coast of Washington, squeezing each through a rayon gauze to obtain enough green glowing substance for protein extraction. A reminder of Thomas Edison’s dictum of discoveries being based on 1% inspiration and 99% perspiration!

Today, bioluminescent research extends past the green part of the spectrum. Thanks to engineered mutations created by Dr. Roger Y. Tien, there is red fluorescent protein (RFP) with hues that can be tomato, cherry or strawberry. There is also yellow fluorescent protein (YFP), orange fluorescent protein (OFP), and blue fluorescent protein (BFP).

Clearly, humans can use luciferin and luciferase for a variety of scientific purposes. But what do fireflies use it for? Most species of fireflies use their flashing lights to help them find mates. In many species, the females stay stationary while the male flies buzz around and blink their lights. In response, the females will change their own glowing pattern and frequency to guide the males to them.

One group of fireflies, however, use their glowing abdomens to hunt. Females of the genus Photuris engage in aggressive mimicry by imitating the flashing patterns of other species' females to lure and eat the males who seek mates.

Unfortunately, due to habitat loss and climate change, firefly numbers are declining across much of the world. The lack of appropriate green spaces for fireflies to live and mate is compounded by the sedentary nature of many firefly species. The larvae of the common European glow-worm are reported to move only about 5 meters (16.4 feet) per hour. Light pollution as well may be impacting fireflies' ability to thrive. In one study, light pollution reduced the flashing of Photuris versicolor by almost 70%.

So, on a warm summer night, when the darkness is interrupted by tiny pinpricks of flashing lights, know that firefly season has come once again. However, for many scientists, it never ended.

Ada McVean, BSc & Nancy Liu-Sullivan, Ph.D

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