Using force to probe single-molecule receptor-cytoskeletal anchoring beneath the surface of a living cell

The ligation of cell surface receptors often communicates a signal that initiates a cytoplasmic chemical cascade to implement an important cell function. Less well understood is how physical stress applied to a cell surface adhesive bond propagates throughout the cytostructure to catalyze or trigger important steps in these chemical processes. Probing the nanoscale impact of pulling on cell surface bonds, we discovered that receptors frequently detach prematurely from the interior cytostructure prior to failure of the exterior adhesive bond [Evans, E., Heinrich, V., Leung, A., and Kinoshita, K. (2005). Nano-to-micro scale dynamics of P-selectin detachment from leukocyte interfaces: I. Separation of PSGL-1 from the cell cytoskeleton. Biophys. J. 88, 2288-2298]. Retracting cells from receptor-surface attachments at many different speeds revealed that the kinetic rate for receptor-cytoskeletal unbinding increased exponentially with the level of force, suggesting disruption at a site of single-molecule interaction. Since many important enzymes and signaling molecules are closely associated with a membrane receptor-cytoskeletal linkage, pulling on a receptor could alter interactions among its constellation of associated proteins, perhaps switching some aspect of their function. Thus, if used in conjunction with cleverly engineered cell lines targeting receptor-cytoskeletal linkages, probing the kinetics of receptor-cytoskeletal unbinding with ultrasensitve force techniques can provide unique physical insight into the interactions involved in the chemical functions of a molecular adhesion complex. The aim of this chapter is to describe the nanomechanical methods needed to probe receptor-cytoskeletal anchoring beneath the surface of a living cell and to provide the analytical "thinking" needed to extract dissociation kinetics from the statistics of the various failure events observed in pulling. As demonstrations of the experimental approach and concepts, we will use examples taken from probing selectin and integrin receptors immobilized on glass microspheres and expressed on surfaces of leukocytes.