Single Molecule Force Spectroscopy Maps to Study Receptors Clustering

An atomic force microscope (AFM) allows molecular resolution imaging of hydrated specimens. However, it is often limited in providing identity of the imaged structures, especially in a complex system such as a cellular membrane. Cell surface macromolecules such as ion channels and receptors serve as the interface between the cytoplasm and the extracellular region and toward which many regulatory signals are directed. Their density, distribution and clustering are key spatial features influencing effective and proper physiological responses. We used a method that uses AFM “force-volume maps” to identify and map regional distribution as well as ligand-, or antibody-induced real-time clustering of receptors on the cell surface. This technique also allows simultaneous imaging of the resultant changes in cellular micromechanical properties, such as elasticity and cytoskeletal reorganization of the cell. As an appropriate physiological sample, we have examined spatial distribution and real-time clustering of VEGFR, the receptor for vascular endothelial growth factor which is an important angiogenic factor in human and animal tissues. We have used AFM probes conjugated with anti-VEGFR-antibody (anti-Flk-1 antibody) to examine binding (or unbinding) forces between VEGF-R2 (Flk-1) in both in vitro as well as in live endothelial cells. A quantal set of binding and unbinding forces was measured between the antibody conjugated to the AFM tip and purified VEGFRs adsorbed on to a mica surface (Fig 1). The unbinding force varied between 60 and 240 pN and was a multiple of discrete quantized strength of approximately 60 pN (Figure 1B).