Science advances in tandem with a scientist's ability to see things. When Antonie van Leeuwenhoek built the first light microscope in the 1600s, the invention opened a whole world of previously unknown single-celled organisms to study. It also became clear that human tissue is composed of cells. Similarly, tools created by HHMI investigator Roger Tsien have enabled scientists to look into those cells in ways that were previously impossible.
In 2008, the Nobel Prize in Chemistry, which Tsien shared with Osamu Shimomura and Martin Chalfie, recognized Tsien's work on a genetic marker called green fluorescent protein (GFP). Tsien manipulated the GFP gene, which was originally isolated from the the jellyfish Aequorea victoria, to create a set of genetic markers that can be attached to any of the tens of thousands of proteins at work in the human body, permitting scientists to observe what they do. GFP markers, which are used by scientists around the world today, work in any species. GFP has been used to create bacteria that can identify explosives such as TNT and others that glow in the presence of arsenic, a significant problem in well water in Southeast Asia.
Tsien's work on GFP is part of a long career focused on visualization. In the mid-eighties, during his graduate studies at the University of Cambridge, he built a series of fluorescent dyes to label important messenger molecules: calcium, cyclic AMP, and nitric acid. Those molecules act as intracellular messengers, aiding in functions as varied as neurotransmitter release from neurons, muscle contraction, fertilization, hormone response, and glucose release. Tsien's markers let researchers study those processes in living cells. Even today, a 1985 paper on the calcium markers remains the most-cited of Tsien's reports.
Those dyes had limits, however, which Tsien understood: they had to be injected into individual cells, and each molecule he wanted to study required a new dye. Rather than build ever more individual dyes, Tsien wanted a genetic marker. This would let scientists create transgenic organisms with a fluorescent marker on any protein. Green fluorescent protein had been discovered by Osamu Shimomura in 1962. In 1985, a scientist at Woods Hole Oceanographic Institute, Douglas Prasher, had identified and isolated the gene that created the protein. Prasher provided a copy of the gene to Tsien, who quickly recognized both its promise and its limitations. Most of the protein it produced was in a nonuseful form, and the part that did glow produced a big peak in the UV spectrum, which is invisible. The visible green peak was much smaller. So Tsien used biochemistry techniques to improve the gene, first making a version that glowed a brighter green. Then he created modified versions in different colors, including yellow and cyan.
This variety of colors let scientists label multiple proteins simultaneously and observe their interactions. When two different fluorescent proteins get extremely close to each other, one steals excitation energy from the other, changing the color that is emitted in a process called fluorescence resonance energy transfer, or FRET. By measuring these color changes, scientists can determine how close the proteins are to each other.
Since that initial work, Tsien has further expanded the palette of fluorescent proteins available to researchers. In 2007, after two Russian scientists purified a red fluorescent protein from coral, Tsien's lab modified it to make a useful genetic marker, reducing the protein to a quarter of its original size and creating variants in colors that he gave names like mPlum and mCherry. In announcing the Nobel Prize, the Royal Swedish Academy of Sciences called the palette of fluorescent indicators Tsien has developed “a universal ‘tool box’ for studies of dynamic processes in living systems.”
Today, Tsien is expanding the tool box. With Xiaokun Shu, a postdoc in his lab, he's working on an infrared fluorescent protein, so researchers can observe an animal's internal organs without having to perform surgery. (Tissue and blood obscure the current palette of fluorescent proteins.)
He's also focusing on new projects: watching memories form and lighting up cancerous tumors for surgeons. When the brain lays down new memories, new neural synapses are created. By marking proteins that are crucial to synapse creation, he hopes to eventually identify new synapses as they are being created. And he has found a way to infect a cancer tumor with fluorescent proteins, so it glows on the operating table, making the edge of the tumor visible to the surgeon. In a small study of transgenic mice with an especially aggressive breast cancer, the mice whose tumors glowed during surgery had a 3-fold increase in tumor-free survival.
To tackle these problems, Tsien is developing new tools. To watch memory formation, he's developed a technique to measure when proteins form. To make tumors glow, he's developed a sophisticated delivery system that's especially attractive to cancer cells. When he makes those tools available, it's likely that, just as with his calcium dyes and GFP, other researchers will adopt them to study different questions as well.
Photo: Paul Fetters