The difference between what was previously seen on the cell surface (left image) is different from what Jeri Timlin, Jesse Aaron, and Bryan Carson are now able to image (right). Orange areas correspond to the bacterial lipopolysaccharide (LPS), derived from E. coli, and the green areas correspond to the cell’s TLR4 receptors. Image: Jeri Timlin, Jesse Aaron, and Bryan Carson
Researchers at Sandia
National Laboratories have developed a super-resolution microscopy technique
that is answering long-held questions about exactly how and why a cell’s
defenses fail against some invaders, such as plague, while successfully fending
off others like E.coli. The approach
is revealing never-before-seen detail of the cell membrane, which could open
doors to new diagnostic, prevention, and treatment techniques.
“We’re trying to do
molecular biology with a microscope, but in order to do that, we must be able
to look at things on a molecular scale,” says Jesse Aaron, postdoctoral
appointee at Sandia Labs.
The cell membrane, while
providing structure and housing the cell’s interior, regulates movement of
materials in and out of the cell, controls adhesion to other objects, and
coordinates the cell’s communications and subsequent actions through signaling.
Receptor proteins on the surface of immune cells, known as toll-like receptors
(TLRs), are tasked with recognizing intruders, or antigens. The TLR4 member of
this receptor family responds to certain types of bacteria by detecting
lipopolysaccharides (LPS) present on their surface. TLR4 proteins then alert
the cell and activate an immune response.
techniques they developed, Sandia researchers Aaron, Jeri Timlin, and Bryan
Carson discovered that TLR4 proteins cluster in the membrane when confronted
with LPS derived from E.coli, which
increases cell signaling and response. Interestingly, LPS derived from the
bacteria that cause plague, Yersinia
pestis, do not cause the same effects. This could explain why some
pathogens are able to thwart the human immune system.
The plague studies
marked the first time such small events have been imaged and compared, the
Sandia researchers said. Previously, even the most sophisticated optical
microscopes could not image the cell surface with enough spatial resolution to
see the earliest binding events, due to the diffraction barrier, which limits
what can be resolved using visible light.
Jesse Aaron, left, Jeri Timlin, and Bryan Carson in their laboratory working with new imaging techniques to view cell-level activity with unprecedented detail. Photo: Randy Montoya
traditional visualization methods, you can’t see the level of detail you need.
It’s important to look at not only what’s present, but also when and where it’s
present in the cell,” Timlin said.
The technique used by
Timlin and Aaron builds on super-resolution capabilities developed in recent
years, but goes another step by adding dual-color capabilities to the
relatively new stochastic optical reconstruction microscopy, or STORM. The
combination enables the Sandia team to get a more complete picture by
simultaneously imaging LPS and TLR4 receptors on the membrane.
microscopy capabilities are akin to looking out the window of an airplane and
seeing the irrigation circles. You know that plants are there, but you can’t
tell what kinds of plants they are or what shape the leaves are,” said Carson,
a Sandia immunologist who was an integral part of the project. “But with this
technology, it’s like zooming in and seeing the leaves and the structure of the
plants. That buys you a lot in terms of understanding what’s happening within a
cell and specifically how the proteins involved interact.”
In 2009, the National
Institutes of Health awarded Timlin a five-year, $300,000-a-year innovation
grant. Next on the team’s agenda is developing the capability to image live
cells in real time using spectral Stimulated Emission Depletion, or STED. “We’re working toward using a version of superresolution that’s much more
live-cell friendly, and extending that in terms of what colors are available to
do multiple colors, while maintaining the live-cell friendliness. I see this as
a beginning of a long development in this type of imaging technology,” Timlin
applications likely will expand as the technology reveals previously
unattainable details of cell signaling. Eventually, the Sandia team would like
to be able to visualize protein/protein interactions.
process that goes on in your body is somehow controlled by proteins forming
complexes with other proteins or complexes in the membrane, so this would give
you this ability to look, with high spatial resolution and multiplexed color
capabilities, at four or more things in a living cell, which can’t be done very
easily right now. It can be done in pieces, but we want to see the whole
biological process,” Timlin said.
The technology has
exciting potential in immunology and drug discovery. Improved imaging could
show the mechanisms viruses use to invade cells, which might lead to drugs that
would block entry. “We’re hoping to do something like label the viral particles
and watch them in real time, or as close as we can to real time, in the
internalization process,” Carson
said. “With the superresolution technique, we can actually watch them move
through the membrane and see if there are other structures being recruited by
the virus to the site of internalization.”
developed the technology in support of its biological national security
programs, but the team wants to expand the technology into other areas such as
biofuels to better understand where and when different pigments are located on
the membrane of oil-producing algae. This would provide valuable insight into
their photosynthesis functions, which could lead to more efficient biofuel
“A lot of this work
is in its early stages, but we’re encouraged by what we’re seeing and excited
about its future potential,” Aaron said.