Shining Light on Molecular Darkness

Things that glow are cool. I'm still delighted every time I see the fireflies come out on a warm summer evening. It turns out that bioluminescent organisms do much more than provide idle joy. For scientists, they are biological lamps that can be used to illuminate the darkest corners of molecular machinations.

Ultimately, this piece is about how bioluminescence is a useful tool for investigating events on the cellular and molecular levels. But I would be remiss if I didn't share some of the amazing facts and stories I learned along the way, so let's start there.

For the Biologist, Historian, and General Seeker of Knowledge

Bioluminescence has evolved independently at least 40 times, in organisms ranging from bacteria to glowworms to fish in the deep ocean (1). In fact, I discovered that over 50 percent of deep-sea inhabitants luminesce.

Humanity has been aware of bioluminescence for a long time—some of the earliest recorded mentions of “glowing wood” come from Aristotle and Pliny the Elder. Pliny also wrote about a clam that would produce a stream of phosphorescent liquid when frightened. These writings led to a fashion trend of glow-in-the-dark banquets after Roman party-people discovered that eating the clams made one’s mouth luminescent for a while.

It isn’t until about the 17th century that we begin to find writings from people trying to understand how different organisms produce light. Robert Boyle, an Irish chemist, physicist and natural philosopher, observed what he called “shining wood”, a piece of decaying wood that glowed. He wondered whether oxygen was required to sustain the glow. After placing a sample of wood into a sealed compartment and pumping out the air, he “could not perceive any Light at all to proceed from the Wood.” (2) His conclusion that oxygen is required for luminescence was the first documented report on the nature of bioluminescence. We now know the luminescence likely came from bacteria or fungus growing on decaying wood, but Boyle’s observation that oxygen is needed to produce bioluminescence still holds up, over 350 years later.

panellusstipticusaug12-2009 — *Panellus stipticus* is a species of luminescent fungus found in Asia, Australia, Europe and North America. Photo credit: Ylem [Public domain], from Wikimedia Commons

hawaiian-bobtail-squid04 — The Hawaiian bobtail squid forms a symbiotic relationship with the bioluminescent bacterium *Alliivibrio fischeri*. Photo credit: William Omerod, courtesy of Margaret McFall-Ngai

In the 1940’s, William McElroy, a curious young biochemistry professor at Johns Hopkins University, began to question the molecular basis for bioluminescence. At the time, a German biochemist working at Cornell University named Fritz Lipmann had just discovered that adenosine triphosphate (ATP) is the basic energy carrier for living cells. McElroy knew that it takes energy to generate light, and after seeing Lipmann’s work, it seemed reasonable that ATP might be a source of that energy. Sure enough, by adding ATP into firefly extracts, he could produce light. McElroy continued researching the mechanics of firefly luminescence for decades, at one point even offering local children 25¢ per 100 fireflies they brought to his lab.

The pioneering work of McElroy and his wife, fellow biochemist Marlene Anderegg DeLuca, inspired a younger generation of scientists. Keith Wood was one of them.

"I thought the coolest thing to do would be to engineer new molecules using the building blocks of life."

Enter the Luciferase

Keith is a man with a confidence born of 40 years working on the same thing: luciferases, the enzymes organisms use to produce light. When I asked him why he first started studying bioluminescence, he told me: “Ever since I was a teenager, I thought the coolest thing to do would be to engineer new molecules using the building blocks of life.”

Keith wanted to understand how nature’s Legos work. By the time he got to graduate school, Keith had learned what some of the building blocks of life are—proteins, DNA, RNA, etc. A new technique for making targeted changes to protein-coding genes, site-directed mutagenesis, had just been invented, and he wanted to be able to use that tool to show how changing protein structure changed function. This is what led him to join DeLuca’s lab to study luciferases for his PhD work. Luciferase was a magnificent tool for studying how enzymes work because its output is so easy to measure: just put your sample into a luminometer and you’ll get a reading of how much light is being produced (Relative Light Units).

Keith’s expertise in luciferase is what brought him to Promega in the late eighties. At that time, radioactive isotopes were common tools for molecular biologists. Radioactive labeling was an extremely useful technique, but came with the obvious drawback of having to deal with radioactivity and radioactive waste. Bill Linton, Promega’s founder and CEO, recognized the need for technologies that would replace radioactive isotopes for labeling. Though he didn’t yet know what those technologies would be, he kept his eyes open for new detection techniques. One day, an image of a glowing tobacco leaf in a Science paper—the first report of a transgenic plant expressing luciferase—caught his eye. Keith co-authored that paper, so Bill invited him to give a talk at Promega. Keith later joined Promega where he spent over 30 years developing new and exciting tools for researchers in the biomedical sciences.

transgenic-luciferase-plant — The first luminescent, transgenic tobacco plant, carrying the firefly luciferase gene. Reproduced with permission from Ow, D.W. *et al.* (1986), *Science* **234**, 856–9.

“In a world filled with fluorescence, why should we even care?”

For the Biomedically Curious

Is luciferase really all that different from other reporters, like green fluorescent protein (GFP)? To answer that question, we have to dig deeper into how these different luminescent processes work. Let’s get technical.

Luminescence is the process of photon (light) emission. When an electron transitions from an excited state to a ground state, if the conditions are right, a photon is emitted. Luminescent processes are categorized based on how the electron becomes excited.

There are two main kinds of luminescence: photoluminescence and chemiluminescence. In photoluminescent processes, like fluorescence, the electron gets excited by absorbing light. On the other hand, in chemiluminescent processes, such as bioluminescence, a chemical reaction provides the energy to excite the electron. This distinction makes a huge difference in terms of how photon emission is measured—and that’s the important part for experimental scientists, because only what is measurable matters.

Because fluorescence requires putting light in to get light out, there must be a way to distinguish between input light and output light. So, we use filters to keep some wavelengths of light out, and only let through the wavelengths we’re interested in measuring. Unfortunately, no filter is perfect, and some amount of the excitation light will make it through the filter, adding background noise.

Chemiluminescent processes do not require any external light. All the light you see is generated from the luminescent process. This means no background light. As a result, there is a strong linear correlation between the light produced and light measured, over a much greater range than is possible with fluorescence: 7–8 orders of magnitude, versus only 3–4 orders of magnitude for fluorescent processes.

Luminescence is also highly sensitive. “You can see very tiny amounts of light if you let your eyes adapt. If I get down to the tiniest amount of light I can see, luminometers can measure 10,000-100,000 times less than that, and the tiniest amount of light I can see is almost too much for my instruments to measure.”

Our aim as scientists is always to perturb the biological systems we study as little as possible, in order to get as close as we can to “natural” conditions. With the high sensitivity of bioluminescence, we can measure biological processes happening in live cells accurately and precisely—and without the need to make cells over-produce proteins just so we can see them. This sensitivity also means that we can acquire useful data from smaller samples, so screening tens or hundreds of thousands of samples becomes much easier. This is where the strength of luciferases lies: high-speed, quantitative data collection.

bioluminescent-dinoflagellates — Bioluminescent dinoflagellates producing light in breaking waves off the coast of New Jersey. Photo credit: catalano82, from Wikimedia Commons

"We're borrowing the biology from nature and using it to understand the natural biology of the cell."

For the Human

oplophorus — The deep-sea shrimp *Oplophorus gracilirostris* uses its bioluminescent spit as a burglar alarm to fend off predators.

When we first started investigating bioluminescence, it was to understand the process itself—the biology of luciferase. The light emitting reaction has subsequently been configured to measure a wide variety of cellular biology, from cell health to enzyme activity to the specific event of turning on a gene. Now, with the advent of new techniques for genetic manipulation, an enhanced understanding of bioluminescence and the discovery (or generation) of better luciferases, we are using bioluminescence as a tool to explore different biology in other organisms.

To understand some of the science that has been done using bioluminescence technologies, I spoke with Amy Landreman, a Senior Product Manager who oversees a number of our bioluminescent reporter-based products. She told me that the first studies to use luciferase as a reporter used luciferase from fireflies or Renilla reniformis, the sea pansy. As our knowledge of bioluminescence in nature expanded, we found luciferases that were brighter (making them easier to see) and smaller (making them less disruptive to biological systems). Directed evolution of the luciferase from the deep-sea shrimp Oplophorus gracilirostris led the creation of NanoLuc® luciferase in 2012, which has since been used for a variety of research, ranging from clinical drug development to studies of neurodegenerative diseases.

For example, researchers at the University of Wisconsin-Madison used NanoLuc® luciferase to create a luminescent influenza virus, enabling them to track the progress of influenza infection in live mice (3). Just this year, scientists employed bioluminescence to find novel drugs targeting natural killer cells, key players in the immune system that have important roles in conditions from chronic infection to cancer (4). Others have used luciferase to study protein:protein interactions that are involved in amyotrophic lateral sclerosis (ALS) (5).

The brightness and small size of NanoLuc® expand the possibilities to use bioluminescence in ways that minimize impact on normal cell biology. Cells can be engineered to express NanoLuc® as if it was part of the normal genome, and NanoLuc® can be monitored in live cells so that changing biology can be tracked over time. “We’re borrowing the biology from nature and using it to understand the natural biology of the cell,” Amy explained. “Where we [Promega] fit in the story is in that in-between space, with the directed evolution, etc.”: creating new tools to shine a light on molecular darkness.

bioluminscence-feature-article-images-r1-8b

References

Haddock, S.H., et al. (2010) Bioluminescence in the sea. Ann. Rev. Mar. Sci. 2, 443–93. [PubMed]
Boyle, R. (1666) New Experiments Concerning the Relation between Light and Air (in Shining Wood and Fish). Philos. Trans. 2, 581–600.
Tran, V. et al. (2013) Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread. J. Virol. 87, 13321–9. [PubMed]
Hayek, S. et al. (2019) Identification of primary natural killer cell modulators by chemical library screening with a luciferase-based functional assay. SLAS Discov. 24, 25–37. [PubMed]
Oh-Hashi, K., et al. (2016) SOD1 dimerization monitoring using a novel split NanoLuc, NanoBit. Cell Biochem. Funct. 34, 497–504. [PubMed]

Shining Light on Molecular Darkness

by Julia Nepper