Crosslinker identity and dynamics regulate F-actin network structure and mechanics

The coexistence of multiple actin crosslinking proteins in cells suggests that crosslinkers with distinct binding dynamics may cooperate to organize F-actin networks that support adhesion, motility, and division. Here, we examine how crosslinker identity and lifetime regulate actin-network structure and mechanics using a reconstituted system that combines $\alpha$ -actinin, a native dynamic actin crosslinker, with biotin-NeutrAvidin, a non-native model of long-lived, effectively persistent crosslinking. Using confocal fluorescence microscopy and optical-tweezers microrheology, we compared networks formed with $\alpha$ -actinin alone, biotin-NeutrAvidin alone, or a representative equimolar 50:50 mixture of the two crosslinking schemes at fixed total crosslinker-to-actin ratio, $R$ . Mixed crosslinking produced the strongest mesoscale structural heterogeneity at high $R$ , yielding larger characteristic structural features than either pure-crosslinker network. However, this structural coarsening did not translate into uniformly enhanced linear viscoelasticity: mixed networks generally exhibited moduli and viscosities intermediate between those of pure $\alpha$ -actinin and biotin-NeutrAvidin networks. Nonlinear microrheology further revealed that crosslinker identity and lifetime regulate force buildup, strain stiffening, and stress relaxation in an observable-dependent manner. Thus, the representative mixed-crosslinker network did not simply generate the strongest or most solid-like actin network. Instead, its effects were selective: it enhanced mesoscale heterogeneity at high $R$ , produced intermediate linear and total nonlinear force responses, and gave rise to distinct nonlinear stiffening and relaxation behavior. These results demonstrate that cooperation between dynamic and persistent crosslinks is deformation-regime dependent, providing a mechanism for tuning actin-network architecture and mechanics without uniformly increasing rigidity.