Two Levels of Myosin-IIA Dynamics in Cells: Turnover of Filaments and Self-Organization of Filament Stacks

Dynamics and self-organization of myosin-II filaments in non-muscle cells are poorly understood. Here, using structured illumination microscopy (SIM), we visualized with high resolution bipolar myosin-II filaments in REF52 fibroblasts by marking myosin-II light chain (MLC), or myosin-IIA heavy chain (MHC-IIA). The filaments demonstrated a uniform length of 300 nm and formed characteristic periodic striations along actin filament bundles, such as ventral stress-fibers, or transverse arcs in spreading cells. Myosin IIA striations were alternating with domains of variable length enriched by actin cross-linking protein alpha-actinin. Free barbed ends of actin filaments (visualized by incorporation of labeled G-actin) were localized between myosin-II striations, while pointed ends of actin filaments (visualized by labeling tropomodulin-3) were enriched in the regions corresponding to myosin-II striations. The most striking feature of the myosin-IIA filament organization was in registry alignment of the myosin-IIA filaments with subsequent formation of several microns long filament “stacks” that apparently formed bridges between parallel stress-fibers or arcs. FRAP experiments were performed in SIM showed that the recovery time for both GFP-MLC and GFP-MHC-IIA in tens of seconds and did not depend on actin filament turnover. However, the process of self-organization of the individual filaments into stacks required much longer time (tens of minutes) and depended on both myosin II contractility and actin filament dynamics. Our observations reveal basic modes of such self-organization. A physical model considering myosin filaments as active force-dipoles embedded in a elastic medium shows that myosin filaments bound to neighboring actin bundles can interact mechanically through deformations of the intervening disordered cytoskeletal network leading to the observed stacking (registry) of these myosin filaments. Thus, myosin-IIA filament stacks are novel cytoskeletal elements that could maintain integrity and drive self-organization of the actin cytoskeleton.