Identifying mechanisms of non-muscle myosin II filament assembly and amplification

The majority of contractile forces in non-muscle cells are generated by non-muscle myosin 2-driven contraction of actin networks. High-resolution imaging in the lamella of migrating fibroblasts revealed myosin filaments appear and amplify by an actin-dependent partitioning process, feeding into larger actomyosin networks.Our goals were to delineate mechanisms of new filament establishment, and define how filament partitioning enables the formation of contractile networks. We fail to observe calcium- or Rho-mediated activation in the immediate vicinity of a new myosin filament, suggesting monomer activation occurs with low spatial precision. However, consistent with published work, we observe filament appearance following leading edge retraction. Appearance stalls when actin dynamics are pharmacologically halted, but rescued by myosin monomer release from posterior stress fibers via ROCK inhibition. This suggests that filament formation is a low-precision, stochastic event dependent on regional increases in available monomer. We then used a molecular counting method to determine the mechanism of partitioning after filaments are established. Our in vitro, fixed-cell, and live-cell imaging suggests that 1) in vitro single filaments are ~30 monomers, 2) live-cell bipolar structures can be upward of a single filament (>>30 monomers), and 3) multiple filaments are present during partitioning. Taken together, we propose a model whereby increased actin network density during retraction events acts as a kinetic trap for myosin monomers, resulting in filament nucleation. Then, established myosin filaments act as a diffusion trap for assembly-competent monomers and possibly other cytoplasmic filaments, resulting in sub-resolution stacks and clusters. Established filaments and filament clusters then mature into a contractile network to enable cell migration. We anticipate similar mechanisms exist in more complex contractile processes throughout cell and tissue biology.