Perturbation of myosin altered the actin node dynamics. (A) Actin node movement is reduced by myosin II inhibition. Cells were treated with 200 nM LatA (0 s) until the cell entered phase 2. When active aggregation of actin nodes occurred, 25 µM blebbistatin was added at 35 min. (B) The 3D kymograph of Lifeact-Ruby was created from the marked area (yellow box, 5 × 5 µm²) in A. Initial LatA treatment triggered the appearance and merging of actin nodes. Subsequent blebbistatin treatment decelerated the lateral movement of actin nodes and prevented further aggregation events. (C) MSD of the control and perturbed samples from MEFs (summarized in Table 1, error bars indicate SD). (D) Actin nodes formed in myosin IIA knockdown cells but did not actively move compared with the control. The last frame is the merged images of nodes at 0 s (in red) and 90 s (in green). Node position was strongly altered in control cells but not in myosin IIA knockdown cells. (E) Histogram of the actin node size detected in control and myosin IIA knockdown cells. Total actin nodes n = 2 × 10⁴ for two experiments in control cells and n = 1.6 × 10⁴ for two experiments in myosin IIA knockdown cells. (F) Knockdown of mouse myosin IIA by siRNA was confirmed by Western blotting. Bars: (A) 10 µm; (B) 5 µm; (D) 5 µm.