Many of the complex organic dyes, or labels, that biologists and biochemists use to track molecules inside cells were invented in Germany over 100 years ago. As a result, dye chemists such as Luke Lavis sometimes have to refer back to the original German-language manuscripts for guidance. Lavis doesn't speak German, so he looks to his wife and her Swiss parents for help. None are scientists, but aside from technical words he looks up in a specialized dictionary, "my mother-in-law is very good" at translating, he says.
However, Lavis points out that even translated, the papers represent chemistry a century old. One project Lavis is undertaking at Janelia Farm is to use modern methods to revamp some of the techniques. "There are more and more chemical reactions available," says Lavis. "Can we apply those new chemistries to synthesizing dyes?"
As with the other projects Lavis is working on, the ultimate goal in revamping dye chemistry is to improve the ability of biologists and biochemists to tackle complex questions. "I don't want to do interesting chemistry and simply stop there," he says. "The point is to make better tools and then learn something new about biology."
Lavis has been designing and fabricating dyes since he graduated with a degree in chemistry from Oregon State University in 2000. As an undergraduate, he worked in a natural products chemistry lab, recreating complex organic molecules from tree sap and red wine. Lavis loved it, but was also frustrated by the lack of applications. The natural products synthesis he was doing, he says, was to some extent an academic exercise. The mentality was, "I'm going to make this molecule because it's there." The usefulness of the molecule was secondary.
Uncertain about whether to pursue a research career or go to medical school, he took a break after graduation by going to work for Molecular Probes, a chemical maker based in Eugene, Oregon. Now a part of Life Technologies, Molecular Probes was, at that time, focused on making fluorescent dyes. While he was there, Lavis got a chance to make molecules with real-world uses. "It was very gratifying to make something, give it to a biochemist or biologist, and have them say `Wow; this really works well. We can now answer some biological questions.'"
Lavis spent less than a year at Molecular Probes before his supervisor moved to Molecular Devices, in Sunnyvale, California, and convinced Lavis to join him. At Molecular Devices, Lavis again worked on fluorescent dyes, creating drug discovery kits that scientists could use to measure such things as ion concentration in cells and voltage differences across cell membranes.
Lavis spent three years at Molecular Devices, enough time to decide that research, not medicine, was the path for him. He joined a chemistry/biochemistry group at the University of Wisconsin in Madison as a Ph.D. student.
There, Lavis worked in the lab of Ronald Raines, doing basic research on an enzyme called ribonuclease A. Variants of RNase A can selectively kill cancer cells, and Raines's group was trying to figure out how this process worked. "We had to develop new tools to look at the trafficking of the protein—how does it get into the cell and where does the RNase go," says Lavis. He developed a fluorescent dye that could be attached to ribonuclease A variants, but which remained dark outside the cell. When the protein-dye pair entered the cell, natural cellular enzymes called esterases automatically snipped a bit of the dye off and made the molecule fluoresce. "The protein outside the cell remained invisible and only the molecules that were internalized would light up," says Lavis. "This enabled some sophisticated imaging experiments to visualize only the protein inside the cell and accurately measure the rate of uptake."
Toward the end of his time at the University of Wisconsin, Lavis heard about Janelia Farm from another graduate student. He was attracted to the highly collaborative, focused atmosphere—similar to what he had encountered in industry and in the group in Madison. "I decided to go for it," says Lavis, because of the twin focuses of high-end imaging and neuroscience. "If I want to make useful molecules to answer biological questions, I want to be near biologists." But, he says, given his interest in imaging agents, he also wants to be near the microscope makers. At Janelia, Lavis gets both.
Along with his effort to modernize dye chemistry, at Janelia Lavis works with group leaders Eric Betzig and Harald Hess to develop new fluorescent tags for their PALM (photoactivated localization microscopy) systems. PALM obtains super-high-resolution images by shining low levels of a specific frequency of light at special proteins that fluoresce in response.
The photoactivated fluorescent proteins have only been developed in recent years, and according to Lavis, they have revolutionized imaging techniques. But, he says, the repertoire of such large, complex proteins remains limited, restricting multicolor experiments. Lavis thinks that more traditional "small-molecule" labels, such as the ones he attached to ribonuclease A during his Ph.D. research, still have a place as complements to the newer proteins. Lavis has developed new small-molecule tags for use with PALM and similar systems.
Lavis's third project focuses on improving the ability of small molecule fluorescent-ion indicators to operate in living tissue. Ions such as calcium have key roles in the cell, and visualizing their concentrations is important for cell biologists, but he notes that "most of the available fluorescent indicators were designed to work in cultured cells. … Scientists want to use them in live animals." Lavis is improving the properties of these molecules, developing better ways to deliver the compounds into cells, and also—again extending his Ph.D. work—see if he can devise ways to use cells' own enzymes to unmask his tags, in the same way he used them to unmask his dyes bound to ribonucleases.
Lavis collaborates extensively with many groups at Janelia to explore further refinements of this strategy. He wants to see if he can create fluorescent indicators that would be unmasked only in certain cells within the brain of a mouse. "We could look at changes in calcium concentration in just a defined population of cells, rather than the whole thing."
"To accomplish the audacious goals of Janelia, it will take collaborative research. Along with the fantastic biologists asking important biological questions, we require tool developers to develop state-of-the-art, next-generation tools to enable critical experiments and answer some of those questions."CONTACT
RESEARCH ABSTRACT SUMMARY:
Luke Lavis works at the interface of chemistry and biology, assembling small-molecule fluorescent dyes to facilitate biological studies.
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Photo: Paul Fetters