A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.

During eccentric contraction, muscle is lengthening so that the actin-myosin cross-bridges bear a load that exceeds the force they generate during isometric contraction. Using the optical trap technique, we simulated eccentric contraction at the single molecule level and investigated the effect of load on the skeletal actomyosin lifetime at different ATP concentrations. The range of the loads was up to 17 pN above the isometric level. We found that the frequency distribution of the lifetime of the actin-bound state of the myosin molecule was biphasic: it quickly rose and then decreased slowly. The rate of the slow phase of this distribution increased with both the load and the ATP concentration. The fast phase accelerated sharply with the load, but it was independent of ATP concentration. The presence of the fast phase demonstrates that some transition(s) in the actomyosin complex occur before the myosin head becomes able to bind ATP and detach from actin. Its high sensitivity to the load indicates that the transition is load-dependent.