Actin node formation triggered by LatA-mediated F-actin depolymerization. (A) Schematic diagram of the experimental setup. Transfected cells were placed on fibronectin-patterned glass coverslips. The “zone of observation” (green) represents the area of the cell observed in all experiments and corresponds to the actin network that spanned the nonadhesive areas. (B) Representative images of LatA treatment results. HeLa JW cells transfected with Lifeact-Ruby were treated with 800 nM LatA. The yellow dotted lines mark the adhesive areas patterned with fibronectin. In phase 1, stress fibers shortened and eventually disappeared (red arrows). Actin nodes appeared in phase 2. (C) Actin node aggregation. In phase 2, actin nodes merged and became bigger. In the event where two actin nodes merge together (yellow arrows), only one track is continued. At time 1,060 s, the red track ends and the green track continues, while at time 1,088 s, the blue track ends and the green track continues. (D) The histogram was obtained from the measurements of 10 s displacement in one of the control samples that contained 100-s long trajectories of ∼100 nodes. Distribution analysis of the angle (θ) between direction of movement (yellow arrow) of a given node (red dot) at a given moment, and the neighboring nodes within the distance of R. Blue region: R < 2 µm. Pink region: 2 µm ≤ R < 4 µm. Orange region: 4 µm ≤ R < 6 µm. The histograms plot the distribution of θ in the respected color-coded regions. In the range of 0–4 µm, θ has a biased distribution toward small angles. When the distance is >4 µm, the distribution of θ is uniform. (E) STORM images of Alexa Fluor 647–phalloidin staining of actin structures before (left) and after treatment with 200 nM LatA for 20 min (middle and right). The image on the right represents the same field as the middle panel visualized by TIRF. Blue arrowheads indicate some of the actin nodes. The boxed regions are enlarged below. Bars: (A) 20 µm; (B) 10 µm; (C) 2 µm; (D) 5 µm; (E, top) 5 µm; (E, bottom) 1 µm.