biology

Stanford Scientists
+
medicine

Discover a New Method

to Monitor Proteins in vivo
by Adrienne Sussman

B
ioluminescent imaging is one of the most important
tools available to scientists trying to understand how

Photo Credit: www.biochem.arizona.edu/dept/mouse10min.jpg

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 Technology

Molecular biologists currently utilize β-galactosidase (β-
gal) or luciferase enzymes as important visualization
tools. β-gal is widely used as a reporter gene, as different
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 octopuses 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. Both of these imaging methods
are relatively recent discoveries, but today they are some
of the most important tools for researchers in molecular
biology and medicine.
A mouse expressing luciferase in a tumor in its leg. This visualization
technique is especially important for cancer research, but can only
show proteins that are within the cell.
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 β-
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

Wehrman and von Degenfeld set out to find a new technique that would combine
the benefits of β-gal and luciferase. After a year of fruitless pursuit, the pair finally
hit upon a solution: a compound called Lugal.
30 stanford scientific

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
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 β-gal and luciferase and
allow them to visualize secreted proteins in living animals.
The camera can capture the light of glowing luciferase only when it is inside cells.
β-gal can be tracked even if a protein is secreted or transported through the
bloodstream, but only within a tissue sample.
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 β-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
Photo Credit: http://www.stanford.edu/group/blau/research-technology.html
The image on the left shows a mouse injected with luciferase only in its
left leg and Lugal in the right leg. While the left leg has almost no lu-
minescence, the right leg glows strongly. On the right, a mouse shows
luminescence in both legs
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 β-gal, the side-group is cleaved off, forming
a compound somewhat similar in structure to luciferin.
layout design: Mac Sriyanong
biology
When this processed version of Lugal encounters +
luciferase, the characteristic glow that is captured medicine
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 β-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 β-gal imaging, a technique that
many researchers already utilize, scientists will be able to
conduct research with Lugal right away on the available β-
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. S
ADRIENNE SUSSMAN is a senior majoring in Biology, with a minor in
Spanish Literature.
To Learn More:
Visit the Blau Lab website at http://www.stanford.edu/
group/blau/index.html or read about the discovery in Na-
ture Methods, vol. 3, No. 4, April 2006 (Pg. 295-301).
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