The discovery that allowed us to develop a formidable tool to study the living cell.
“A very intense light filled the interior of the hangar. It was so bright that it temporarily blinded me. Less than a minute later an explosion boomed and a strong pressure wave caused my ears to hurt. I noticed that the sky had suddenly become overcast. Everything “It was very mysterious. When I got to my grandmother’s house, it was covered in black ash. I took a bath and changed my clothes, although at the time I didn’t know that the ashes gave off dangerous radiation.”
Only on rare occasions has Osamu Shimomura, Nobel Prize winner in chemistry in 2008, described what it was like to witness the explosion of the Nagasaki atomic bomb in August 1945, just 15 kilometers from the epicenter, when he was a teenager and working in a workshop. aircraft repair. But those memories are a fundamental part of the story of a boy born in Kyoto who grew up in one of the most difficult eras in Japanese history and later identified a fluorescent substance in a jellyfish that, years later, would illuminate—literally— the field of biology.
The discovery of green fluorescent protein (GFP) was the first step in building a very powerful tool for investigating the living cell: fluorescent proteins that emit light of different colors. The technique, developed by Martin Chalfie and Roger Tsien (who shared the Nobel Prize with Shimomura) three decades after the discovery, allows us to see, for example, how cancer spreads or how a cell with Alzheimer’s disease deteriorates; also understand how neurons are organized within the brain and how proteins interact within the cell.
Today, the range of fluorescent proteins of all colors that can be injected into any tissue is an essential tool in molecular biology.
Bioluminescent glow
I met Osamu Shimomura by chance in 2007, during a summer course at the venerable Marine Biological Laboratory (LBM), in Woods Hole, Massachusetts, where he still maintains a laboratory despite having retired in 2001. At that time, before the Nobel , his life was calm and granting unexpected interviews had not become a permanent headache.
Tall, lean, and deceptively frail, Shimomura moves around his LBM lab as if carrying his own force field. His lean face with its benevolent expression has at the same time something monastic, academic, and iron; that is, something of a Jedi knight. He guides me through the small, cramped room, equipped with the latest in electron microscopy, and stops before a shelf with several glass containers.
Shimomura dims the lab lights. From a jar labeled “Cypridine 1944″ takes a handful of tiny stuffed crustaceans that look like sesame seeds, puts them in a porcelain mortar, adds a few drops of water and begins to grind them. Soon a soft sky blue luminescence emerges from the mortar that intensifies when the He applies pressure. “Ah, this is very good,” he murmurs. “I have done this demonstration many times and it always amazes me to see that the glow still appears after so many years of the small organisms being dissected.” His English is heavily infused with Japanese. and requires total concentration.
The first time Shimomura saw that bioluminescence he was 27 years old, had just graduated as a pharmacist and had just accepted a position as an assistant to Professor Yoshimasa Hirata at Nagoya University. Pharmacy was not of his interest, but the war had changed the rules of the game: he could not finish high school because most of his teachers had died and he was not accepted into universities because his academic records had been broken. been destroyed. His only way out was the College of Pharmaceutical Sciences, a makeshift university with inexperienced instructors, with which Shimomura ended up educating himself independently. Once graduated, he tried unsuccessfully to find employment at a major pharmaceutical company, until luck finally took him to Nagoya, where Hirata was dedicated to isolating and purifying natural compounds. “We don’t know anything about this,” the professor told his new assistant, showing her the light of the organism. Cypridina hilgendorfii. “Only it glows. Are you interested in finding out why?”
Shimomura was not the first to face this task. A group from Princeton University, in the United States, had been trying for years in vain to determine the nature of the chemical reaction that makes the crustacean glow. Cypridina hilgendorfii, which has no common name. During World War II, the Japanese military used the tiny animals ground up and soaked in saliva to read maps without using flashlights. As a result, hundreds of kilos of Cypridine, a resource that would later be extremely valuable for science. Bioluminescence has fascinated people for centuries. In 1887, Raphael Dubois, professor at the University of Lyon, France, had discovered that this “light of animal origin” appears thanks to two substances: one that acts as fuel and another that is the igniter. Dubois isolated these two components and named them luciferin (the fuel) and luciferase (the catalyst, that is, the substance that precipitates the chemical reaction), alluding to the name Lucifer, which means “bringer of light.”
Bioluminescence is a relatively rare characteristic in terrestrial animals, but very common among marine creatures (see As you see? No. 41). In the abyssal depths of the ocean live bacteria, protozoa, fungi, jellyfish, squid, worms, crustaceans, mollusks and sharks that have this gift of producing light in a chemical reaction so effective that it barely releases heat; a cold light that can serve as an alarm signal, an escape artist’s disguise, an object of seduction and a bluff to find food.
The most notable bioluminescent organisms in the ocean are dinoflagellates, whose single cell can sometimes be seen with the naked eye. These organisms are responsible for the beautiful blue-green trails that transform beaches at night into surreal landscapes. The dinoflagellates make the foam shine and illuminate the passage of the fish as if there were arrows of light in the water.
“I knew that to determine the chemical structure of luciferin that causes the night glow Cypridine “I was going to need the compound in a completely pure state to then crystallize it,” says Shimomura, his face bathed in blue. “But I had no idea what type of molecule it was. A sugar? A protein? An amino acid? Something unknown?”
The task was titanic because it would have to extract only the luciferin molecules from among thousands of different molecules that make up the crustacean. As if that were not enough, luciferin is extremely unstable and degrades immediately when in the presence of oxygen. Shimomura decided to do all of his experiments in a hydrogen chamber, which was very dangerous due to the explosive nature of that gas.
Creating crystals of a substance requires producing an increasingly pure extract (see As you see? No. 162). In Shimomura’s case, each purification attempt required seven days and seven nights of continuous work. For almost a year the young chemist persevered without paying much attention to the danger of hydrogen. But after 10 months of increasingly pure preparations, he had not managed to produce a single luciferin crystal.
One day, frustrated with another failed experiment, he accidentally left open a container with a luciferin solution in contact with an acidic medium. The next day he saw with amazement that small red crystals had formed on the surface of the solution. They were pure luciferin crystals, and the treatment that made them grow was acid. The luminosity of the crystals turned out to be 37,000 times greater than the dry powder of the crustacean.
The serendipitous discovery gave Shimomura the tools to establish the exact nature of luciferin. Cypridine. News of his feat reached Princeton University, where Frank Johnson had tried in vain to do the same. Frustrated with his own attempts, Johnson invited the young Japanese to move to the United States to work on what would be his great contribution to science. His former professor Hirata’s parting gift was to give him a doctorate in chemistry, because he knew that would double his salary: from $300 to $600 a month.
The massive collection of Cypridine that took place in Japan during the war was what gave Shimomura the advantage over the Princeton scientists. The crustacean is abundant in the shallow waters of the Sea of Japan and scarce in the rest of the world, and Shimomura had unlimited amounts of material to work with. Thus, ironically, a byproduct of the war that nearly killed him was what launched his scientific career.
The mystery of the jellyfish
As soon as he got off the train in New Jersey after a long sea crossing from Japan—the first time he had left his country—the researcher was greeted by Johnson, who took him to a laboratory located in one of the Gothic-style buildings of the Princeton University. And history repeated itself: Johnson turned off the lights, gave him a jar with a whitish powder and mixed it with water. But, unlike the previous experience with the Cypridine, this time there was no light reaction. “This is bioluminescent jellyfish powder Aequorea victory. Its effect only lasts a few hours while it is fresh and cannot be reactivated. Would you be interested in studying this jellyfish?” Johnson asked the new collaborator. Of all the bioluminescent animals observed at Princeton, this was the only one for which the experiment recipe failed. Johnson wanted to know why.
The Aequorea —sometimes called a glass jellyfish—has a pretty two-inch transparent umbrella shape; It has long, hair-like tentacles and about 100 pinhead-sized bioluminescent spots on the outer edges of the dome. In those days the Aequorea They were abundant in the cold waters of Friday Harbor, in the San Juan Islands, Washington state (shortly after they almost disappeared), and Shimomura and Johnson went there one summer.
The same afternoon of their arrival they began collecting jellyfish on the pier of the University of Washington laboratories using nets to clean pools. They filled several buckets and then sat down with a pair of scissors to trim the edge of each jellyfish’s dome, which contained the light organs. The next step was to put the strips of light organs on a cotton cloth and squeeze out the juice, which continued to glow for several hours. During the summer of 1961 they collected and squeezed more than 9,000 jellyfish.
The focus of the study was to stop the chemical reaction that…