Transcript Slide 1
Chemomechanical
mapping of ligand-receptor
biding kinetics on cells
Sunyoung Lee, Jelena Mandic, and Krystyn Van Vliet
Aditya Kohli, 20.309, 11/20/08
Roadmap
Summary
Background
Results
Analysis / Future Work
Roadmap
Summary
Background
Results
Analysis / Future Work
Summary
Experiments
Authors mapped individual
VEGF receptors and
determined whole cell ligand
binding kinetics by means of:
Scanning probe microscopy
Molecular force spectroscopy
Results
Developed a novel approach
to understanding both
individual receptor location and
binding kinetics on a single
molecule level
Gained a spacio-temporal
visualization of cell surface
dynamics that regulate
receptor mediated behavior
Roadmap
Summary
Background
Results
Analysis / Future Work
Limitations exist in current imaging
techniques
Need for simultaneous access to spacial, temporal, and intermolecular
force dynamics at a single cell and molecule level
Spatial distribution and quantity of receptors are needed to understand
how ligand binding may depend on environment
Method: Flow cytometry, immunocytochemical staining, FRET, and FRAP
reveal binding affinity and kinetics of receptor ligand interactions via time
course monitoring of labeled ligand levels
Limitation: Spatial distribution of active receptors is not resolved
Method: AFM can resolve both spatial distribution and binding kinetics
Limitation: Slow process with low resolution (500nm)
Goal of this paper is to overcome these shortcomings by
developing a novel imaging process
Chemochemical imaging
Cell surface scanned with a magnetically driven oscillating,
cantilevered probe to which monoclonal anti-VEGFR2 antibodies
are tethered (1 Ab/probe)
Retardation of full oscillations indicative of pico-newton level force
between antibody probe and cell receptor lead to image contrast
Dark regions appear on image as recognition sites and are
indicative of receptor site position
Receptor mediated behavior regulate
critical cell behaviors
Vascular endothelial growth factor
receptor (transmembrane
tyrosine kinase) expressed by
vascular endothelial cells
Involved in focal adhesion
turnover, actin cytoskeleton
remodeling, and angiogenesis
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Roadmap
Summary
Background
Results
Analysis / Future Work
Determination of receptor location and
binding specificity
Chemochemical imaging used to visualize and measure the binding kinetics
of VEGF2 receptors in fixed and living human umbilical vein endothelial
cells (HUVECs)
Binding kinetics analysis
Koff 1.05104 s 1
K on 5.83104 s 1M 1
K
K D off
1.80 10 9 M
K on
Visualization of receptors on living cell
surfaces
In live cell imaging, the position and number of receptors varies over
time due to diffusion and recycling
Receptors have D .02 m2 s
Receptor immobility is evidence of cytoskeletal confinement
Roadmap
Summary
Background
Results
Analysis / Future Work
Chemomechanical mapping allows for
individual cell and receptor analysis
Demonstrated a general and versatile approach for simultaneously
measuring receptor position and resolving binding kinetics
Measured binding kinetics on a single cell basis, demonstrated
specificity of binding events using competitive binding of soluble
antibodies
Showed that VGEFR2 position is correlated with cytoskeletal
structure - supports the hypothesis that VEGFR2 function is related
to transmembrane integrin complexes that convey force from the
ECM to the actin cytoskeleton
Future work
Full analysis of binding kinetics by light fixation of receptors
Are binding kinetics altered in mechanically stiff regions of cell
surfaces?
How do ligand binding properties and receptor position change in
the presence of a particular drug agonist/antagonist or between
tumor and differentiating cells?
Questions?