FlnA levels affect actin node appearance and movement. (A) Immunoblot analysis showing the expression level of FlnA in samples corresponding to the images in C and D. (B) A FlnA (−/−) cell shows a hole in the cytoplasm (white box) during 400 nM LatA treatment as revealed in both differential interference contrast (DIC) and Lifeact-EGFP images. (C) The shape and movement of actin nodes are affected by the level of FlnA. Initial actin node formation was triggered by the addition of 400–800 nM LatA. Actin nodes moved in wild-type samples and FlnA (−/−) rescued samples, but were frozen in FlnA overexpressed cells. FlnA (−/−) cells demonstrated exaggerated dynamics of actin nodes. The boxed regions are enlarged on the right. The yellow arrows showed the merging of one pair of such nodes. The rightmost column shows merged images of actin nodes at 0 s (red) and 100 s (green). (D) MSD of the control and perturbed samples from MEFs or FlnA (−/−) MEFs corresponding to the images in C (summarized in Table 1; error bars indicate SD). Bars: (B, whole cell image) 10 µm; (enlarged image) 5 µm; (C, first column) 10 µm; (C, enlarged images) 5 µm.