Chapter 14: Examining cell-cell interactions in the kidney using AFM single-cell force spectroscopy.

The ability of individual cells to synchronize activity is a basic feature of efficient and appropriate tissue function, depending on the physicochemical transduction of signaling through multiprotein complexes that functionally mediate adhesion. The direct connection of physical properties and intercellular signaling is of great importance to certain pathologies including diabetes. Atomic force microscopy (AFM) single-cell force spectroscopy (SCFS) is a high-resolution technique that provides a statistically reliable measurement of the minute forces involved in cell tethering and membrane dynamics. Detection of altered nanoscale forces underlying the loss of adhesion in early tubular injury is pivotal for the development of novel therapeutic strategies for diabetic nephropathy. Here we describe the step-by-step use of an integrated AFM-SCFS system designed to measure functional force-displacement in separating renal tubular epithelial cells. Parameters such as unbinding forces, detachment energy and distance to complete separation, can be obtained from force-displacement (F-d) curves and are critical in assessing how physical changes of cellular adhesion contribute to cell contact, coupling and communication in the diabetic kidney