Single-molecule measurements of the effect of force on Thy-1/αvβ3-integrin interaction using nonpurified proteins

Thy-1 and αvβ3 integrin mediate bidirectional cell-to-cell communication between neurons and astrocytes. Thy-1/αvβ3 interactions stimulate astrocyte migration and the retraction of neuronal prolongations, both processes in which internal forces are generated affecting the bimolecular interactions that maintain cell–cell adhesion. Nonetheless, how the Thy-1/αvβ3 interactions respond to mechanical cues is an unresolved issue. In this study, optical tweezers were used as a single-molecule force transducer, and the Dudko-Hummer-Szabo model was applied to calculate the kinetic parameters of Thy-1/αvβ3 dissociation. A novel experimental strategy was implemented to analyze the interaction of Thy-1-Fc with nonpurified αvβ3-Fc integrin, whereby nonspecific rupture events were corrected by using a new mathematical approach. This methodology permitted accurately estimating specific rupture forces for Thy-1-Fc/αvβ3-Fc dissociation and calculating the kinetic and transition state parameters. Force exponentially accelerated Thy-1/αvβ3 dissociation, indicating slip bond behavior. Importantly, nonspecific interactions were detected even for purified proteins, highlighting the importance of correcting for such interactions. In conclusion, we describe a new strategy to characterize the response of bimolecular interactions to forces even in the presence of nonspecific binding events. By defining how force regulates Thy-1/αvβ3 integrin binding, we provide an initial step towards understanding how the neuron–astrocyte pair senses and responds to mechanical cues.