Mechanical Force Rather Than Strong Binding Intermediates Extends Activation of Regulated Actin

Abstract

Recent evidence demonstrates that the gliding time of thin filaments in a motility assay is extended by force but questions arise about the mechanism. To address whether the force is mechanical, we varied the density of myosin immobilized on the surface in contact with actin filaments regulated by native complex of tropomyosin and troponin. Compared with low myosin density, we expected the average run times to be longer at high myosin density because previous results showed that the measured gliding velocity was greater. However, persistence of strong binding crossbridge intermediates could extend average run time by delaying deactivation. Competing chemical binding and mechanical force mechanisms can be distinguished in the probability density distributions of run time frequencies. Because chemical potential is constant in the motility assay, chemical states turn over at constant rate and, hence, have probability given by a Poisson distribution. in this scenario, Poisson rate is given by the odds of a pause, and hence, run times between pauses fit an exponential distribution that slopes negatively for all observable run times. However, we determined that relative density of observed run times fits an exponential only at low myosin density. At the higher myosin density, the relative frequency of short run times fit the positive slope of a gamma distribution, which derives from waiting times between Poisson events. Reaction intermediates including strong binding myosin respond to a thermodynamic potential and, hence, cannot explain the gamma distributed response observed. Cumulative densities of run-time frequencies at both myosin densities fit a model in which mechanical force contributes to the activation of thin filaments. We propose that the integral force of myosin delays the transition state during activation. Work supported by NIH R01-HL128683 (JRP) and leave supported by UWG (HGZ).