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.