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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 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 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) 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?