Cyclic Mechanical Reinforcement of Actin Interactions

Abstract

The fundamental mechanism underlying force regulation in actin dynamics is not understood. To investigate actin depolymerization under dynamic force environments, single molecule experiments were conducted using atomic force microscopy (AFM).A custom-made AFM and force-clamped experimental procedures were used. To measure G-actin/G-actin (GG) interaction, G-actin was immobilized on the AFM cantilever tip and the polystyrene petri-dish was functionalized with G-actin. For G-actin/F-actin (GF) interaction, F-actin was prepared and immobilized on the petri-dish. To mimic dynamic force, once binding was detected during the tip retraction, force was applied to it via one of the programming paths; (1) loading the bond to 10, 15, 20 and 25 pN forces, and then reducing the force to 5 pN; (2) loading 1.5-, 2.5- or 3.5-cycle with a 10 pN peak force and holding at 10 pN to measure bond lifetime.GG and GF dissociations were qualitatively similar to each other. When loaded by a linear ramp, actin dissociations exhibited a biphasic transition from a catch bond to a slip bond. In the catch bond region, bond lifetimes increased as force increased to a maximum at 12 pN in GG and at 20 pN in GF interactions. Interestingly, after applying cyclic forces, the post-priming bond lifetimes of GG and GF were significantly prolonged at low force range (5-10 pN), instead of reverting instantly to a low affinity state. This shows that cyclic force is more effective in strengthening actin-actin bonds than a linear ramp.Our study demonstrated that kinetics of actin depolymerization is force dependent and the mechanical priming process by cyclic force application significantly enhanced bond lifetime of actin/actin. We hypothesize that the mechanical reinforcement of actin/actin interaction is an important regulatory mechanism underlying cytoskeletal dynamic rearrangement by affecting the actin depolymerization kinetics.