Old compound finds new life in molecular imaging studies
Scientists' yearlong hunt turns up new way of watching proteins in real time in living animals
BY AMY ADAMS
Until recently two of the popular tools molecular biologists had at their disposal for watching molecules within cells both fell short of ideal. This situation was irking postdoctoral fellows Thomas Wehrman, PhD, and Georges von Degenfeld, PhD, who hoped to harness the best of both techniques.
With some molecular ingenuity and a touch of luck, the pair managed to hook the approaches together and open up a new world of possible molecular biology experiments. This technique was published in the April issue of Nature Methods.
According to Helen Blau, PhD, the Donald E. and Delia B. Baxter Professor of Pharmacology, who led the work, the breakthrough was a long time coming. "Many people were frustrated by the situation and had been looking for ways to make the two methods work together," she said.
Here's what they were up against. One method of watching molecules involves a protein called ß-galactosidase. Researchers can engineer cells that make ß-gal either alone or hooked up to their protein of interest. In thin slices of tissue—but not in live animals—it's then possible to see where that protein was located inside or outside of the cell. This approach is handy for studying proteins that attach themselves to the outside of cells or that travel in either the spaces between cells or the bloodstream.
The other method is similar in many regards, but instead uses a protein called luciferase. The advantage here is that a sensitive camera developed by Christopher Contag, MD, associate professor of pediatrics and of microbiology and immunology, and his colleagues can detect light produced by luciferase in a living animal. This approach allows researchers to watch a cell that makes luciferase as it travels throughout the body, or to detect which tissues are making the protein. The glitch is that the luciferase has to stay within the cell in order to be seen and isn't visible in tissue sections, unlike ß-gal.
You want to watch a protein that gets secreted from the cell to see where it travels in real time? No luck. Only ß-gal is visible outside the cell and it can't be detected in a live animal. The answer, obviously, is to hook the two together. What wasn't so obvious is how.
Wehrman and von Degenfeld spent a year searching for a substrate that would somehow detect the presence of ß-gal in living animals. After testing all the known options and striking out, Wehrman and von Degenfelf came across the perfect solution in a paper—a compound called Lugal that had been around since 1986 without anyone realizing its potential to detect ß-gal in living animals. In both living cells and in tissue slices, ß-gal lops a molecular arm off of Lugal, which then produces a luminescent glow that can be detected in live animals by Contag's camera.
Throughout the pair's search, Lugal had been available as part of a kit in a Promega catalog—a catalog that's as common in labs as a phone book—but sold for a different purpose. To avoid buying the entire molecular kit, the group had the company manufacture a custom version of Lugal, which worked like a charm.
"Now researchers can have the advantages of both enzymes, since their activity is linked," Blau said.
The group successfully tested their technique in mice. Sure enough, they could detect ß-gal hooked up to a protein they wanted to study as its levels rose and fell in real time. Most importantly, they could detect where those proteins were located both inside and outside of cells.
Blau said she's now talking with Sanjiv Gambhir, MD, PhD, professor of radiology and director of the molecular imaging program at Stanford, about how the technique might be extended for other imaging applications.
Graduate student Peter Krutzik and Garry Nolan, PhD, associate professor of microbiology and immunology, contributed to the work.