Biophysical methods to probe claudin-mediated adhesion at the cellular and molecular level

Claudins are a family of tetraspan membrane proteins that localize at tight junctions in an epithelial monolayer forming a selective barrier to diffusion of solutes via the intercellular spaces. It is widely accepted that the interaction between the extracellular loops of claudin molecules from adjacent cells is critical for this function. Though previous experiments utilizing traditional biological, biochemical, morphological, and electrophysiological approaches have provided significant insights into the role of claudins in regulating ion permeability, the interaction kinetics between these molecules has not been characterized. In this chapter, we describe two experimental procedures to study the adhesion forces imparted by claudins: (a) dual micropipette assay to quantify the adhesion forces at the cellular level and (b) single molecule force spectroscopy using atomic force microscopy to characterize the interaction kinetics at the molecular level. Though the experimental procedures are described for claudins, they can be easily modified for studying the interaction properties of a wide variety of other proteins.