As a key element in the cytoskeleton, actin filaments are highly dynamic structures that constantly sustain forces. However, the fundamental question of how force regulates actin dynamics is unclear. Using atomic force microscopy force-clamp experiments, we show that tensile force regulates G-actin/G-actin and G-actin/F-actin dissociation kinetics by prolonging bond lifetimes (catch bonds) at a low force range and by shortening bond lifetimes (slip bonds) beyond a threshold. Steered molecular dynamics simulations reveal force-induced formation of new interactions that include a lysine 113(K113):glutamic acid 195 (E195) salt bridge between actin subunits, thus suggesting a molecular basis for actin catch-slip bonds. This structural mechanism is supported by the suppression of the catch bonds by the single-residue replacements K113 to serine (K113S) and E195 to serine (E195S) on yeast actin. These results demonstrate and provide a structural explanation for actin catchslip bonds, which may provide a mechanoregulatory mechanism to control cell functions by regulating the depolymerization kinetics of force-bearing actin filaments throughout the cytoskeleton.single-molecule force spectroscopy | mechanotransduction | mechanosensing | nemaline myopathy T he actin cytoskeleton, primarily a force-bearing structure, controls the morphology, motility, and adhesion of the cell (1-4). Its core filamentous component, assembled from actin monomers via noncovalent interactions (5), undergoes rapid and controlled polymerization and depolymerization, allowing the dynamic reorganization of the actin cytoskeleton (1, 2).In cells, this dynamic process can be modulated by forces, and this is crucial to mechanosensitivity, mechanotransduction, and cellular adaptations to mechanical stresses (3, 6-8). For example, the assembly, stabilization, and reorganization of the actin stress fiber and the focal adhesion, where actin filaments constantly sustain tension, are induced by externally applied forces (9-12) dependent on myosin-generated contractility (4,8,13,14) and sensitive to substrate rigidity (3, 15, 16). These observations led us to investigate the molecular mechanism by which actin dynamics are regulated by force.The force-regulated kinetics of several molecular interactions important to adhesion and force-bearing functions of cells are governed by catch-slip bonds, in which the interaction is stabilized by tensile force in a low range and destabilized when force exceeds a threshold (17)(18)(19)(20)(21)(22). Various mechanisms, such as the allosteric model based on intramolecular conformational change under forces (23,24) and the sliding-rebinding model based on force-induced formation of new interactions due to intermolecular interface sliding (18,25), have been proposed to provide structural explanations for catch-slip bonds in different molecular interactions.Here we use atomic force microscopy (AFM) force-clamp experiments to determine how force regulates the off-rate of actin depolymerization and to elucidate the structural ...

BackgroundIntegrin αLβ2 (lymphocyte function-associated antigen, LFA-1) bears force upon binding to its ligand intercellular adhesion molecule 1 (ICAM-1) when a leukocyte adheres to vascular endothelium or an antigen presenting cell (APC) during immune responses. The ligand binding propensity of LFA-1 is related to its conformations, which can be regulated by force. Three conformations of the LFA-1 αA domain, determined by the position of its α7-helix, have been suggested to correspond to three different affinity states for ligand binding.Methodology/Principal FindingsThe kinetics of the force-driven transitions between these conformations has not been defined and dynamically coupled to the force-dependent dissociation from ligand. Here we show, by steered molecular dynamics (SMD) simulations, that the αA domain was successively transitioned through three distinct conformations upon pulling the C-terminus of its α7-helix. Based on these sequential transitions, we have constructed a mathematical model to describe the coupling between the αA domain conformational changes of LFA-1 and its dissociation from ICAM-1 under force. Using this model to analyze the published data on the force-induced dissociation of single LFA-1/ICAM-1 bonds, we estimated the force-dependent kinetic rates of interstate transition from the short-lived to intermediate-lived and from intermediate-lived to long-lived states. Interestingly, force increased these transition rates; hence activation of LFA-1 was accelerated by pulling it via an engaged ICAM-1.Conclusions/SignificanceOur study defines the structural basis for mechanical regulation of the kinetics of LFA-1 αA domain conformational changes and relates these simulation results to experimental data of force-induced dissociation of single LFA-1/ICAM-1 bonds by a new mathematical model, thus provided detailed structural and kinetic characterizations for force-stabilization of LFA-1/ICAM-1 interaction.