Single-Molecule Mechanics of the Talin-Integrin Bond

Abstract

Integrins are large heterodimeric proteins that play an important role in force transduction across the cell membrane. In order to bind the extracellular matrix integrins have to be in an activated open form. The activation of integrins requires the binding of the talin N-terminal F3 domain to the integrin b-tail and the association with the contractile actomyosin cytoskeleton. Despite its critical role in force transduction, the affinity between integrin b-tail and talin-F3 domain is surprisingly weak for all isoform pairs (K d >10 mM). This raises the question how such a weak bond resists the actomyosin pulling forces and ensures a stable connection across the cell membrane. To address this question we designed fusion constructs between the talin-F3 domain (but also entire talin head domain) and the b1-integrin cytoplasmic tail. Using a dual-beam optical tweezer setup we probed the mechanics of the talinintegrin tail bond at the single molecule level. Our results confirm the dynamic character of the talin-integrin tail bond, which shows an unbinding-rate of z 50/s. The binding energy of the talin-integrin tail bond is weak and ranges around 4-6 k B T, and the bond is mainly in an open state at forces higher than 5 pN. Interestingly, we discovered that the talin-integrin tail bond is stabilized by the presence of kindlin, which is also a FERM-domain protein that synergizes with talin to activate integrins. When measuring the talin-integrin tail interaction with kindlin in solution, the talin-integrin bound states become longer. Surprisingly, the length of the bound states is not influenced by force. This kindlin-induced stabilization effect might be of crucial importance for integrin activation, clustering and development of adhesion site assemblies.

DNA-based ForceChrono Probes for Deciphering Single-Molecule Force Dynamics in Living Cells

“…This suggests that the force on the integrin gradually increases and sustains for a period before abruptly terminating. This abrupt termination is likely due to either a break in the integrinligand bond outside the cell membrane or an internal protein-protein bond break, such as talin-integrin 33 . This contrasts with a slow relaxation scenario, where the two fluorescent molecules would extinguish sequentially due to the different refolding forces of the hairpins.…”