Filamin's Force Sensing Mechanism Revealed by Optical Tweezers

Abstract

Cell adhesion, spreading and migration, as it occurs during embryogenesis or wound healing and hemostasis, involve a complex interplay between the extracellular matrix, transmembrane adhesion receptors and the cytoskeleton. A prominent role plays human filamin A, since it is a widely expressed actin cross-linker which additionally binds a multitude of transmembrane receptors such as beta integrins or the von Willebrand Factor receptor GPIbα.Although it has been shown that beta integrins or GPIbα bind to the Ig-like domain 21 of filamin A, the molecular-level mechanism for filamin binding, especially its regulation, is still unclear. Filamin can auto-inhibit the interaction by hiding the binding site through intra-molecular interactions and it has been suggested that the binding site can be exposed by a force-induced conformational change.To address the question whether filamin's binding site in domain 21 was activated by force, we adapted a highly sensitive optical tweezers setup to pull at single domain pairs of human filamin A containing the receptor binding site and the auto-inhibiting region. We were able to apply and detect low forces in the physiological relevant regime around 3 pN and monitor the conformational change by measuring the length increase of the protein on a sub-millisecond timescale. This allowed us to distinguish in real-time between the inhibited closed conformation and the active open one. Additionally, we were able to detect the binding of different ligands added in solution and how the applied force increased the binding rate. Therefore, filamin A can be regarded as a force sensor.