Effect of Load on Non‐Muscle Myosin 2 Paralog Filaments in a Biomimetic Contractile Actin Array

Abstract Cells express three non‐muscle myosin 2 (NM2) paralogs that form bipolar filaments of ≈30 motors each. Of these, NM2A and NM2B are best studied and show distinct enzymatic and mechanical properties. Although they can colocalize in cells, they also have unique localization patterns, suggesting functional differences. Most studies have examined these proteins interacting with actin under very low loads, but in cells they likely contribute to generating and maintaining cytoskeletal tension. Inspired by sarcomere‐like tension generation in non‐muscle cells, a minimal platform is reconstituted to study NM2 activity under load. Using micropatterned formin to align actin filaments in anti‐parallel bundles, phosphorylated NM2 filaments are introduced to observe their interactions via TIRF (Total Internal Reflection Fluorescence) microscopy. NM2B contracts and bundles the actin filaments to form a tensed system. The rate of myosin filament movement slows, and many filaments completely stall. In contrast, NM2A moved faster but disrupted bundles via severing and retraction. FRET‐based tension sensors revealed that both paralogs generate comparable tension despite these differing behaviors. This suggests that NM2A and NM2B play distinct mechanical roles in cells, with NM2B better suited for sustained tension and NM2A contributing to dynamic remodeling.