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

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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?