Across all major classes of taxa, the ability to bioluminesce has remained largely untraceable throughout evolution as it does not appear to follow any specific phylogenetic path. This fascinating natural phenomenon allows for the emission of light in living creatures through chemical reactions in the body, and for centuries, people have often pondered the potential applications of bioluminescence in the human world. This vision, however, was only made a reality upon the isolation of the green fluorescent protein (GFP) from the jellyfish species Aequorea victoria by scientist Osamu Shimomura, which would go on to completely revolutionize cell tracking and imaging as we know it today.
Bioluminescence Throughout Evolution
Convergent evolution of bioluminescence in a diverse variety of phyla suggests the importance of the trait to each organism as well as the ease at which bioluminescence is able to evolve. At the molecular level, the oxidation of luciferin allows for the emission of light with the reaction rate controlled by a catalyzing enzyme (typically luciferase or a photoprotein). While the evolution of luciferins has remained largely conserved, the array of luciferases and photoproteins is rather diverse and unique . When a photoprotein binds to another ion (e.g. Ca2+ or Mg2+), it prompts a structural change in the protein, and light is emitted . The claim that the evolution of bioluminescence is relatively simple is further supported by the fact that predators must only develop luciferase in order to prompt light emission from a bioluminescent prey. Bioluminescence is especially remarkable in its vast distribution across the ocean, acting as the primary visual stimulant for many organisms as sunlight grows increasingly scarce .
GFP Discovery and Isolation
An especially groundbreaking discovery in modern science is that of the green fluorescent protein, responsible for bioluminescence in the marine cnidarian hydrozoan Aequorea victoria. Isolated by Nobel laureate Osamu Shimomura, green fluorescent protein (GFP) plays a vital role in the transduction of blue chemiluminescent light into a green fluorescent light. The protein was first harvested as a by-product of a luminous photoprotein aequorin from the jellyfish Aequorea victoria . Isolation of GFP was made possible through a performance of polymerase chain reaction on the DNA fragment coding for GFP taken from Aequorea victoria DNA . Upon stimulation by Ca2+ ions, Aequorea victoria’s catalyzing photoprotein aequorin releases blue light, which is then absorbed by GFP and re-emitted as green light . In 1992, the full green fluorescent protein gene was cloned by microbiologist Douglas Prasher, though Prasher himself was unable to see through the expression of GFP in microbes due to lack of funding. His work was continued by biologist Martin Chalfie, who successfully achieved GFP expression in E. coli and C. elegans and thus extinguished doubt that jellyfish-specific cellular conditions are necessary for fluorescence .
Specific to neuroscience, GFP is especially useful in its ability to illuminate and allow for the visualization of neural networks. In a broader sense, GFP is most often fused with other proteins in order to observe the distinct protein in action. The protein of interest now expresses green fluorescent properties without losing its original function. Another application of GFP lies in bacterial pathogenesis, specifically in the study of the host-pathogen interaction wherein GFP is used as a biological marker. Insertion into the pathogen prior to replication enables a thorough visualization of the colonization and spread of bacteria in vivo. GFP is successful in this role due to its low toxicity and ability to continuously replicate .
Roger Tsien, an American cell biologist, first utilized the fundamental principle of mutation to reap the full benefits of GFP. By altering wild GFP (with two excitation maxima) to have only a single excitation maximum at 484 nm, a single point mutant (S65T) of GFP was introduced, bearing a higher intensity and stability than naturally occurring GFP. S65T came to be known as enhanced GFP or EGFP . Many colored mutants of GFP have been designed by scientists shifting the emission spectra, allowing different proteins in a system or process to fluoresce in different colors and thus making large-scale visualization plausible. Tsien and colleagues were able to create multiple fluorescent variants upon further research, such as T203Y or YFP (yellow), CFP (cyan) and BFP (blue) .
Fluorescence in Research
The institution of GFP in the bioscientific world paved the way for the discoveries of a plethora of light-emitting molecules that would go on to stake their own claims in bioluminescence imaging. One such discovery was made in 2003 by Russian biochemist Sergey Lukyanov, who stumbled upon red fluorescence in Discosoma (a coral) and was able to trace this property to a red fluorescing protein he dubbed DsRed . Furthermore, FLuc (firefly luciferase), cloned from the North American firefly Photinus pyralis, has become a standard reporter gene for in vivo cell observation due to its prolonged light emission. A codon-optimized variant has been modified specifically for cell tracking in mammalian cells. RLuc, isolated from the sea pansy Renilla reniformis, calls upon the catalysis of coelenterate luciferin oxidation to exhibit bluish-green bioluminescence. Although RLuc is unable to sustain luminescence for extended periods of time, dual imaging in the same subject using both FLuc and RLuc is applicable in tracing two molecular events or two cell populations simultaneously .
The discovery, isolation, and application of green fluorescent protein have had a revolutionary impact on the field of biomedical and neuroscientific research. Luminescence has given way to the most stable and clear cellular and biochemical imaging achievable thus far. In a field developing at such a rapid pace, the utilization of fluorescent proteins and luciferases in visualizing neural networks is vital to understanding the connections between individual neurons. Ultimately, the most fascinating verity in this study is that the tools for progressing the global database of scientific knowledge often lie within nature itself.
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