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
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Summary
Background
Results
Analysis / Future Work
Roadmap
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Summary
Background
Results
Analysis / Future Work
Summary
Experiments
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Authors mapped individual
VEGF receptors and
determined whole cell ligand
binding kinetics by means of:
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Scanning probe microscopy
Molecular force spectroscopy
Results
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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
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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
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Cell surface scanned with a magnetically driven oscillating,
cantilevered probe to which monoclonal anti-VEGFR2 antibodies
are tethered (1 Ab/probe)
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Retardation of full oscillations indicative of pico-newton level force
between antibody probe and cell receptor lead to image contrast
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Dark regions appear on image as recognition sites and are
indicative of receptor site position
Receptor mediated behavior regulate
critical cell behaviors
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Vascular endothelial growth factor
receptor (transmembrane
tyrosine kinase) expressed by
vascular endothelial cells
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Involved in focal adhesion
turnover, actin cytoskeleton
remodeling, and angiogenesis
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Roadmap
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Summary
Background
Results
Analysis / Future Work
Determination of receptor location and
binding specificity
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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  1.05104 s 1
K on  5.83104 s 1M 1
K
K D  off
 1.80  10 9 M
K on
Visualization of receptors on living cell
surfaces
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In live cell imaging, the position and number of receptors varies over
time due to diffusion and recycling
Receptors have D  .02 m2 s
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Receptor immobility is evidence of cytoskeletal confinement
Roadmap
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Summary
Background
Results
Analysis / Future Work
Chemomechanical mapping allows for
individual cell and receptor analysis
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Demonstrated a general and versatile approach for simultaneously
measuring receptor position and resolving binding kinetics
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Measured binding kinetics on a single cell basis, demonstrated
specificity of binding events using competitive binding of soluble
antibodies
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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
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Full analysis of binding kinetics by light fixation of receptors
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Are binding kinetics altered in mechanically stiff regions of cell
surfaces?
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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?