Myosin activity drives entangled actin networks out-of-equilibrium - a quantitative approach

ATP-driven myosin II activity remodels actin networks and drives cytoskeletal matter out of thermal equilibrium, but how ATP concentration controls these dynamics remains difficult to isolate in vivo. Here, we reconstitute minimal actomyosin networks from purified components and combine passive microrheology with mean back relaxation (MBR) analysis to quantify ATP-dependent nonequilibrium fluctuations. Nonequilibrium activity is strongest at intermediate ATP concentrations (0.2-0.5 mM) and decreases at higher ATP. While single-bead van Hove distributions are approximately Gaussian, pooled distributions display apparent tails caused mainly by bead-to-bead heterogeneity rather than frequent active bursts. MBR, however, reveals clear time-irreversible dynamics by distinguishing restoring relaxation from persistent active motion. Comparing activity with network stiffness suggests a trade-off between ATP-dependent stiffening and myosin-driven remodeling. A minimal active Langevin simulation reproduces the observed MBR phenomenology, supporting a picture in which rare myosin-driven cage rearrangements generate detectable nonequilibrium signatures. These results establish MBR as a sensitive probe of active matter behavior in actomyosin networks. Significance StatementCells operate out of equilibrium, yet the specific role of ATP concentration in driving cytoskeletal activity remains difficult to isolate in vivo. By reconstituting minimal actomyosin networks and applying passive microrheology, we directly quantify how ATP levels modulate out-of-equilibrium fluctuations. The application of mean back relaxation (MBR) analysis thereby provides a clear and broadly accessible measure of broken time-reversal symmetry that surpasses conventional analysis methods. Our results reveal an inverse relationship between ATP concentration and network dynamics, arising from different modes of myosin activity and ATP-dependent network stiffening. This work provides a quantitative framework for linking biochemical energy supply to mechanical activity in reconstituted cytoskeletal systems, offering new insights into cellular self-organization and energy-dependent regulation.