page 1241, that clustering does not precede binding but rather functions in adhesion strengthening following binding to multivalent ligands.To probe the question of whether clustering or affinity are primary factors in leukocyte integrin binding, Kim et al. devised a FRET system in which either the α or β subunits of the integrin LFA-1 (αLβ2) were labeled with nondimerizing forms of YFP and CFP. Under basal conditions, no micro-clustering was evident, as cells expressing αL-mCFP, αL-mYFP, and wild-type β2 showed little FRET. Nor did the researchers observe FRET when cells were stimulated to activate LFA-1 adhesiveness. Furthermore, activation of LFA-1 with cytoskeletal-disrupting agents did not induce microclusters or macroclusters, visible by confocal microscopy, in the absence of multivalent ligands.
However, addition of multivalent (but not monovalent) ICAM-1 ligand to stimulated cells induced FRET. Macro- and microclusters did form when cells expressing both the ligand and the integrin heterodimer were cultured together in a manner that stimulated aggregation.
Kim et al. conclude that clustering is involved in strengthening the adhesion force, after initial binding of a multivalent ligand. They hypothesize that conformational changes within the integrin heterodimer—which they were able to confirm with intramolecular FRET—is the key factor in activating integrin adhesiveness.