Synergistic effects of 𝞪-actinin/neutravidin on the mechanics of F-actin networks

Actin, a globular protein, is one of the major cytoskeleton components of a cell. It is crucial for cell stability, movement, replication, and muscle contractions. Most of these structural changes in a cell are attributed to the intricate viscoelastic properties inherent in cross-linked actin filaments. Recent work suggests that distinct ABPs can act in concert to organize complex filament architectures, yet how mixed transient and permanent crosslinking modulates mechanics and structure remains incompletely understood. Here, we combine optical-tweezers microrheology with laser-scanning confocal microscopy to interrogate actin networks crosslinked simultaneously by a transient linker (𝛂-actinin) and a permanent linker (biotin-neutravidin). Specifically, we optically trap a 4.2 μm microsphere and translate it at constant speed through the network while recording the resistive force during loading and post-strain relaxation, systematically varying the relative and absolute crosslinker densities. In parallel, volumetric confocal imaging quantifies network architecture and heterogeneity under the same compositional conditions. By jointly resolving force response and microstructure, our measurements elucidate how mixed crosslinking tunes network elasticity, dissipation, and recovery, and reveal structure-mechanics couplings that are not captured by single-linker systems. These results clarify the synergistic roles of ABPs in shaping the emergent mechanics of crosslinked actin assemblies.