Analysis of Two-Dimensional Dissociation Constant of Laterally Mobile Cell Adhesion Molecules

Abstract

We formulate a general analysis to determine the two-dimensional dissociation constant (2D K d), and use this method to study the interaction of CD2-expressing T cells with glass-supported planar bilayers containing fluorescently labeled CD58, a CD2 counter-receptor. Both CD2 and CD58 are laterally mobile in their respective membranes. Adhesion is indicated by accumulation of CD2 and CD58 in the cell-bilayer contact area; adhesion molecule density and contact area size attain equilibrium within 40 min. The standard (Scatchard) analysis of solution-phase binding is not applicable to the case of laterally mobile adhesion molecules due to the dynamic nature of the interaction. We derive a new binding equation, B/ F = [( N t × f)/( K d × S cell)] - [( B × p)/ K d], where B and F are bound and free CD58 density in the contact area, respectively; N t is CD2 molecule number per cell; f is CD2 fractional mobility; S cell is cell surface area; and p is the ratio of contact area at equilibrium to S cell. We use this analysis to determine that the 2D K d for CD2-CD58 is 5.4–7.6 molecules/ μm 2. 2D K d analysis provides a general and quantitative measure of the mechanisms regulating cell-cell adhesion.