Tag, you’re it: The fluorescent proteins lighting up tumours

A luminous cloud of neon blue expelled by a deep-sea shrimp has rematerialised as a new form of paint to label tumours.

 
  A ‘brainbow’ depicting neural networks in the brain. Each individual neuron is labelled by one of many fluorescent proteins.   Jeff Lichtman/Flickr  (CC BY-NC-ND 2.0)

A ‘brainbow’ depicting neural networks in the brain. Each individual neuron is labelled by one of many fluorescent proteins. Jeff Lichtman/Flickr (CC BY-NC-ND 2.0)

 

Off the west coast of North America, the waters are awash with crystal jellies. These small fluorescent jellyfish sequin the ocean with millions of glowing green pinpricks. The glow comes from Green Fluorescent Protein (GFP), a protein that allows the jellyfish to absorb blue light and then emit green light.

In recent decades, GFP has become a stalwart of the genetics laboratory. Once isolated from the jellyfish, the gene encoding the protein can be introduced into the cells of other species, causing particular cellular components to glow green for easy identification. Now its reach has extended beyond the laboratory and toward the surgical table.

  Crystal jelly ( Aequorea victoria ) in its natural state (top) and exhibiting fluorescence (bottom).   Jim G/Flickr  (CC BY 2.0);  Phil Blackburn/Flickr  (CC BY-NC-ND 2.0)

Crystal jelly (Aequorea victoria) in its natural state (top) and exhibiting fluorescence (bottom). Jim G/Flickr (CC BY 2.0); Phil Blackburn/Flickr (CC BY-NC-ND 2.0)

In research published this year, US researchers attached GFP to NanoLuc, a subunit of luciferase, the chemical compound used by the deep-sea shrimp Oplophorus gracilirostris to produce glowing neon blue clouds in the water. The result of this combination is a light of great intensity that easily penetrates tissue, revealing the location of cancer cells, even those that have spread to new areas. Effectively, they’ve created a biological flashlight. It’s called LumiFluor, equal parts deep-sea shrimp chemical and jellyfish protein.

So just how does a glowing protein from a jellyfish or shrimp help scientists in the lab? It’s called biofluorescence, an organism's ability to produce its own light by absorbing natural light and re-emitting it as a different colour. It is an ancient, innate ability shared by hundreds of species, even evolutionarily distant ones like crustaceans and corals. And scientists are still stumbling upon more species that can glow, including recently discovered neon green marine eels, and a Hawksbill sea turtle sporting a psychedelic red and green mottle on its shell.

However, the act of plucking the genetic origins of colour from marine animals and placing them on scientists’ palettes began over 40 years ago with the North American crystal jelly, Aequorea victoria. When the 2008 Nobel Prize in Chemistry was awarded to the scientists instrumental in the discovery and development of GFP, it was in the wake of a renaissance of fluorescent protein research that began in the 1960s.

Since the first purification of GFP from jellyfish, its potential as a unique tool to observe biological mechanisms has been elevated to an art. GFP can be inserted into a limitless array of cellular and molecular components, causing them to gleam green.

The reason why GFP and other fluorescent proteins are so genetically versatile lies in their structure. The protein’s shape folds into a near perfect cylinder, creating a highly stable barrel wrapped around a colour molecule called a chromophore. This unique shape allows the protein to glow without any prompting from additional substances.

  The structure of GFP, showing its almost perfect cylindrical shape (left), and illumination seen in purified GFP (right).   Richard Wheeler/Wikimedia Commons  (CC BY-SA 3.0);  OpenCage/Wikimedia Commons  (CC BY-SA 2.5)

The structure of GFP, showing its almost perfect cylindrical shape (left), and illumination seen in purified GFP (right). Richard Wheeler/Wikimedia Commons (CC BY-SA 3.0); OpenCage/Wikimedia Commons (CC BY-SA 2.5)

Another advantage is that the genes that carry instructions to make fluorescent proteins are at home no matter where the gene is inserted, regardless of species and location. This combination of their resilience and easy-going genetic nature results in a harmless tag that is malleable enough to be attached to any region with minimal disruption to any natural processes. Scientists can now direct a flashlight onto the inner workings of any biological organism.

“GFP has this widespread applicability in every size scale,” said Assoc Prof Stuart Turville, a virologist at the Kirby Institute based at the University of New South Wales. “You can label whole organisms or bacteria, and you can even track single molecules over time.”

GFP transformed researchers into colour connoisseurs, and the entire natural world was suddenly their colouring book. Just as early painters found Tyrian or royal purple within the cream-coloured shells of the spiny murex sea snail, scientists have been mining nature for new colours.

  The  Entacmaea quadricolor    sea anemone.   Jan Messersmith/Flickr  (CC BY-NC-ND 2.0)

The Entacmaea quadricolor sea anemone. Jan Messersmith/Flickr (CC BY-NC-ND 2.0)

One story begins almost like the precursor to a joke. Walking into a pet shop, a Russian scientist was so charmed by the brilliant red shade of the Entacmaea quadricolour sea anemone that he just had to have it, undeterred by the fact it was already sold. The result was a red fluorescent protein 10 times brighter than any existing red. Now there are red, blue, yellow and orange fluorescent proteins, existing alongside GFP in a catalogue of shades.

A menagerie of glowing green animals has since become synonymous with the GFP technique, unfortunately eclipsing the true scope of protein's impact. The ‘Brainbow’ is a far more evocative example, mapping the complex connections between individual neurons in the brain. Each individual interwoven neuron is flagged with a different fluorescent protein, the brain's intricate neural architecture revealed as a multicoloured mosaic.

Colour, in its many iterations, informs our perception and interpretation of the physical world. This is no different for scientists studying the biological world. Fluorescent proteins are now multicoloured workhorses in research, and fluorescence bioimaging is an indispensable feature of the laboratory, one with special resonance in the field of virology.

  Neurons illuminated with GFP showing the intricate connections in the somatosensory cortex of a mouse brain.   Robert Cudmore/Wikimedia Commons  (CC BY-SA 2.0)

Neurons illuminated with GFP showing the intricate connections in the somatosensory cortex of a mouse brain. Robert Cudmore/Wikimedia Commons (CC BY-SA 2.0)

A persistent limiting factor in virus research was the incredibly small size of viruses, which makes them difficult to study. Information was gleaned from black and white images that did not capture the dynamism with which viruses infect and spread throughout their hosts. Today, the remarkable progress in the virology field has matched pace by pace with the development of GFP and other fluorescent proteins. GFP opened up a new dimension by allowing us to study virus movement and behaviour during infection.

“By inserting GFP in certain parts of the virus genome, you can see when the GFP-tagged gene is scheduled to be switched on during virus life cycles,” said Turville.

Multiple fluorescent proteins used in tandem can help understand even finer details. “You can construct a virus to express different colours, for example in HIV [Human Immunodeficiency Virus], where you can have it transition from GFP to mCherry [red] during virus entry into a cell.”

Indeed, scientists have observed the activity of viruses by watching green virus particles glissade down structural filaments in cells, like infection highways. It is now also possible to witness the single-minded efficiency of immune cell movement in lymph nodes, and to see them converge around an infectious intruder.

  The deep-sea shrimp  Oplophorus gracilirostris , which was crucial in the development of LumiFluor.   SEFSC Pascagoula Laboratory/Flickr  (CC BY 2.0)

The deep-sea shrimp Oplophorus gracilirostris, which was crucial in the development of LumiFluor. SEFSC Pascagoula Laboratory/Flickr (CC BY 2.0)

The epoch of fluorescent proteins is yet to wane, with new tools making use of GFP’s genetic dependability. And so enters LumiFluor, the part-shrimp, part-jellyfish biological flashlight that may help surgeons remove tumours with more precision than ever before.

LumiFluor has been proposed to stain target cells, determining tumour margins so that surgeons can excise a tumour without leaving behind any residual cancer cells. However, generating specificity to recognise human tumours would be the biggest obstacle.

"Dyes would bypass the need for genetic specificity," said Turville, citing previous  research that successfully used dyes rather than a genetic tag to enable colour coded surgery. It’s work helmed by Roger Tsien, one of the recipients of the Nobel Prize for his work on GFP.

However, he adds, the intensity of LumiFluor is 40 times brighter than any existing labelling product and would have a distinct impact on imaging methods in animal models.  He sees LumiFluor as a tool that could be made stronger if combined with another cancer detecting strategy – synthetic viruses that deliver death signals to tumours.

“If LumiFluor specificity to human tumours could be genetically engineered, it could be used together with synthetic viruses to label residual tumours," said Turville. "It could possibly be the tool to bring it all together.”

From deep-sea to surgical table, the role of fluorescent proteins has been chameleonic, but their impact resonates across the entire research field. By pulling the scales off researchers' eyes, fluorescent proteins like GFP have cemented their place in the modern laboratory. They will continue to move forward through scientific history, fulfilling their evolved purpose – to illuminate.