Nanoscale organization and dynamics of myosin II filaments in the lamella. (A–C) Motor domains and tails of myosin II filaments resolved with SIM. (A) Schematic depicting the myosin II filament with mEGFP fused to the N terminus of the heavy chain. Examples of myosin II filaments imaged with wide-field, SIM, and PALM are shown. (B) Schematic and SIM imaging of a myosin II filament labeled with mEGFP on the N terminus of the heavy chain (green) and an antibody labeling the tail domains (red). (C) Schematic and SIM imaging of myosin II filaments with mEmerald fused to the N terminus (green) and mApple fused to the C terminus (red). (D) Low magnification of a U2OS cell expressing the mEmerald/mApple construct. Bar, 10 µm. (E and F) High-magnification views of boxes 1 and 2 from D. (E) mEmerald/mApple construct incorporated into myosin II filaments in newly formed actin arcs in box 1 from D showing single filaments (black arrowhead) and stacks of filaments (white arrowhead) with their long axis parallel to the edge. (F) Mature actin arcs in box 2 from D showing that myosin II filaments exist in stacks (arrowhead) with their long axes parallel to the edge. (G) High-magnification view of an actin arc from a cell expressing α-actinin–mApple (red) and myosin IIA–mEGFP–N-terminal (green). Fig. S4 D shows the entire cell. (H) Line scan from the broken line in G showing two myosin IIA head groups (green line) alternating with α-actinin (red line). Broken lines denote one sarcomere-like unit. (I) Schematic showing sarcomere-like contraction of actin arcs. Green arrows denote myosin II walking and black arrows denote α-actinin zones coming together (i.e., contraction). (J) Montage showing the expansion of two myosin II filaments into filament stacks at the edge of a cell. (K) Montage showing that α-actinin surrounds, but does not colocalize with, expanding stacks of myosin II filaments. Bars: (B and C) 400 nm; (D–F) 2 µm; (G) 400 nm.