NANOSCALE MAPPING OF CELL SURFACE RECEPTORS WITH AN ATOMIC FORCE MICROSCOPE TIP HAVING A SINGLE LIGAND

MasterAtomic force spectroscopy (AFM) is a high resolution imaging tool, and it is suitable to investigate surface of biological samples. No necessity of labeling and the minimal pre-treatments are advantages. More recently, AFM is widely used to measure the interaction force between biomolecules at single molecule level. To record the force reproducibly and realize truly one-to-one interaction, it is important to control placement of the biomolecules on surface. It was demonstrated that a cone-shaped dendron that ensured homogeneous and sufficient spacing between the immobilized biomolecules was useful. As an extension, this lab set up a pathway leading to a single ligand tethering AFM tip through picking a DNA and ligating a DNA-ligand conjugate. Formyl peptide receptor (FPR) belongs to the seven-transmembrane domain G-protein coupled receptor family and it is expressed at high levels on neutrophils and monocytes. The ligand-FPR interaction triggers multiple changes in eukaryotic cells including rearrangement of the cytoskeleton which in turn facilitates cell migration and the synthesis of chemokines. Therefore, high resolution mapping of FPR with AFM will help to understand various physiological function of leukocyte. Because WKYMVm showed a high affinity to FPR, a single molecule of the peptide was tethered at the apex of an AFM tip through the above picking-ligation process. The force-based map of FPRs on a fixed cell surface was obtained by using the AFM tip. As a result, it was observed that there were 1~7 FPRs in 300ⅹ300 nm2. The number was roughly consistent with the one from the radio-labeled ligand assay. The interaction force of the ligand-FPR pair was 60 ~ 89 pN, and the value is similar to the one for the other ligand-receptor interaction force.As a summary, a single peptide tethering AFM tip was prepared through picking a DNA and subsequent ligation of a DNA-heptapeptide conjugate. Using the tip, individual FPRs were mapped and the distribution of FPRs on the fixed cell was observed. It is believed that the approach is applicable to understand the binding behavior of various ligands at the single receptor level