MIT researchers have found a way to glimpse
interactions between molecules on the surface of a cell.
By measuring the force generated by these cell surface interactions, the
MIT team was able to image and measure the rate at which individual
molecules join and separate from receptors on the cell surface. These
interactions are not visible with traditional light microscopy.
"We were able to measure regions of strong intermolecular binding on the
cell surfaces, which enabled us to map the locations of the receptors,"
said Sunyoung Lee, a graduate student in the Department of Materials
Science and Engineering and lead author of a paper on the work in the June
5 issue of the Proceedings of the National Academy of Sciences.
The technique, known as functionalized force imaging, could allow
researchers to better understand the strength and rates of interactions
between molecular ligands outside the cell and the molecular receptors on
the cell surface. These interactions play a critical role in cell growth,
proliferation and differentiation. It could also assist in the design and
testing of new drug molecules that bind strongly or quickly to the target
cell.
Receptors on the cell surface allow the cell to maintain constant
communication with its environment-they bind to molecules that convey
information about the environment and instructions telling them what
functions to carry out. In this study, the researchers looked at a
receptor called vascular endothelial growth factor receptor-2 (VEGFR2),
which is important for the proliferation, migration and differentiation of
the vascular endothelial cells that line blood vessels.
Researchers in the lab of Krystyn Van Vliet, senior author of the PNAS
study, are working to understand the kinetics of cell-molecule
interactions and how a cell responds to mechanical and chemical changes in
its environment. These changes in function can be evidenced by the number
and type of receptors displayed on their surfaces.
"You can ask specific questions about how the mechanical and chemical
stimuli outside the cell generate changes in cell surfaces and structures
within the cell," said Van Vliet, the Thomas Lord Assistant Professor of
Materials Science and Engineering.
With traditional light-based (optical) microscopy, you can see large cell
structures like the nucleus and cytoskeleton, but not tiny molecules such
as individual receptors on the living cell surface. To achieve the
nanometer-scale spatial resolution required to see these molecules on the
cell surface, the researchers used mechanical force, rather than light.
To pull a bound molecule from its target receptor, a very small force of
about 100 piconewtons is required. The researchers measured that force by
attaching anti-VEGFR2 antibody molecules to the end of a cantilevered
probe in a scanning probe microscope. The cantilever oscillates in a
regular pattern as it scans along the cell surface, and whenever the
pattern is disturbed, the researchers can infer that the antibody on the
probe has bound or "stuck" to its target receptor, VEGFR2.
By mapping those reversible interactions at every point on the cell
surface, the researchers can determine where the receptors are located
with respect to other cell structures. More importantly, said Van Vliet,
they can follow the molecular interactions on the cell over time, allowing
them to determine the binding kinetics, or the rate at which molecules
join and separate from the cell surface.
The force-based imaging also allows for visualization of the stiff
cytoskeleton underneath the cell surface, which provides internal
structure for the cell. By overlapping images of the cytoskeleton and the
VEGF receptors, the researchers found that most of the receptors were
located near the cytoskeleton. Such correlations support the current
hypothesis that VEGF receptor function is linked to that of other cell
surface proteins including integrins, which transmit mechanical forces
from the outside to the inside of the cell, Van Vliet said.
Other researchers have used this approach to measure binding forces in
isolated proteins, but the MIT team shows that these tiny forces can also
be used to visualize binding kinetics on chemically fixed (nonliving)
cells and living cells.
"It's challenging to do this on cells because their surfaces are made up
of many different kinds of molecules, which makes them topographically
rough, chemically diverse, and mechanically compliant," said Van Vliet.
Van Vliet and Lee outlined several possible applications of functionalized
force imaging on endothelial, cancer and stem cells, including
identification of target receptors for cell-specific drugs; comparison of
kinetics for individual and clustered receptors; and visualizing how the
mechanical stiffness of extracellular materials alters cell function and
cell surface receptor activity over time.
The research was funded by the National Science Foundation Nanoscale
Exploratory Research, the Center for the Integration of Medicine and
Innovative Technology, the Hugh Hampton Young Memorial Foundation and the
Beckman Foundation Young Investigators Program.
Anne Trafton
mit.edu