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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
Authors mapped individual VEGF receptors and determined whole cell ligand binding kinetics by means of: Scanning probe microscopy Molecular force spectroscopy
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
ExperimentsExperiments ResultsResults
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
www.sigmaaldrich.com
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)
Receptors non-uniformly distributed around cytoskeletal elements
Measured ~1.47 E5 VEGFR2/cell, data matches with radio-labeled ligand measurements
Receptors uniformly distributed along length of cytoskeleton
Binding kinetics analysis
141005.1 sK off1141083.5 MsK on
MKK
Kon
offD
91080.1
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 Receptor immobility is evidence of cytoskeletal confinement
smD 202.
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?