Imaging Protein Transport in Live Cells
by Adrienne Sussman
Bioluminescent imaging is one of the most important tools available to scientists trying to understand how cells and proteins function. This technique has been revolutionized following a new discovery by Dr. Thomas Wehrman and Dr. Georges von Degenfeld, two postdoctoral fellows working with Dr. Helen Blau, Stanford professor in the department of Microbiology and Immunology. Their work will allow researchers to monitor protein transport in living cells in a new way. Until now, those studying how cells work have been frustrated by the shortcomings of the available technology. By combining older methods into a single new technique, Wehrman and von Degenfeld have created a powerful tool for future research in molecular biology.
The limits of current visualization technology
Molecular biologists currently utilize B-galactosidase (B-gal) or luciferase enzymes as important visualization tools. B-gal is widely used as a reporter gene, as different colored compounds can be produced from its reaction with appropriate substrates. In reactions involving luciferase, light is produced by the oxidation of a pigment called luciferin. The reaction between luciferin and oxygen is extremely slow until it is catalyzed by luciferase. This is the same reaction that produces the glow of a firefly.
Using each of these techniques as a visualization tool, scientists engineer cells to produce one of these two proteins in conjunction with another protein of interest. The scientists can then assay for the color or light produced, and by extension, determine the expression and movement of the protein of interest. However, both of these methods have serious drawbacks. Scientists must look at thin slices of tissue when analyzing B-gal experiments, making it impossible to study the transport of the protein in vivo (in living cells). Luciferase, on the other hand, produces light which can be detected by a sensitive camera designed at Stanford by Christopher Contag, Faculty Director of the Stanford Center for Innovation in In vivo Imaging. The camera can capture the light of glowing luciferase while it is still inside a living animal. However, luciferase can only be identified inside the cell. If the protein is secreted or transported through the bloodstream, it cannot be detected.
A difficult search for a new visualization technique
Frustrated by the limits of these two systems, Wehrman and von Degenfeld set out to find a new technique that would combine the benefits of B-gal and luciferase and allow them to visualize secreted proteins in living animals. The search for a useful substrate was complicated by faulty protein shipments and slow orders; however, according to lab director Helen Blau, both researchers were “determined to find a way to make it possible to image B-gal — they were as persistent as Sherlock Holmes.”
After a year of fruitless pursuit, the pair finally hit upon a solution. Their inspiration came from a twenty-year-old paper that mentioned a compound called “Lugal”, a “caged galactoside-luciferin” that had previously been used to detect low levels of β-gal. No one had ever thought to use this substrate for live cell imaging. Looking at the structure of the compound, Wehrman and von Degenfeld realized that Lugal might be just what they were searching for.
Lugal is similar to luciferin but is “caged” by a bulky side group. This “cage” allows it to pass from cell to cell – an important advantage over luciferase. When Lugal encounters B-gal, the side-group is cleaved off, forming a compound somewhat similar in structure to luciferin. When this processed version of Lugal encounters luciferase, the characteristic glow that is captured by Contag’s camera can still be seen, even when the luciferase compound moves out of the cell. In this way, Lugal takes advantage of the best of B-gal and luciferase technology. Only one question remained: would it work? The pair wasted no time in ordering a custom version of Lugal and testing its imaging capacity in mice. To their delight, when the mice were placed under the camera, they glowed. Using Lugal, the scientists could target specific tissues and track proteins of interest inside and outside of live cells.
The future of Lugal research
According to Dr. Sanjiv Sam Gambhir, the Director of the Molecular Imaging Program at Stanford, the best aspect of this new technique is its convenience. Since the new technology uses B-gal imaging, a technique that many researchers already utilize, scientists will be able to conduct research with Lugal right away on the available B-gal-expressing lab animals. In a paper published in Nature Methods last April, Wehrman and von Degenfeld speculate that this discovery will be useful to biologists “determining the distribution of circulating factors, detecting extracellular antigens, or labeling endogenous cells.” Blau elaborated on that claim, explaining that the new technique would “shorten the time to drug discovery enormously,” allowing pharmaceutical researchers to determine rapidly if a new drug is binding to its expected targets.
Since this discovery last spring, the Blau lab has continued its study with protein imaging and is currently testing whether Lugal can be used to visualize entire signaling pathways. The new technique has already been helpful in Blau’s lab, where Lugal has been employed to examine stem cell function. Both Wehrman and von Degenfeld have left Stanford and are now working for pharmaceutical companies. Wehrman is currently creating kits that will make the Lugal technology widely available to scientists, widening its impact on imaging research.
Sidebar: The term “bioluminescence” refers to the conversion of chemical energy into light in living organisms. Organisms as diverse as fire flies, worms, deep sea fish, and types of mushrooms, clams, and octopi all have the ability to create their own light. In bioluminescent imaging, a gene that encodes for glowing proteins is inserted into a laboratory animal. The bioluminescent protein is attached to another protein of interest, so by following the light, scientists can also track the tagged protein.
This technique is related to fluorescent imaging methods such as GFP. The difference is that in fluorescent imaging, the inserted protein only glows when an external light is shined on it. Fluorescent proteins convert external light to a different wavelength so that they appear to glow a particular color, while bioluminescent proteins actually produce their own light.