PREM of HT1080 cells with and without CK-666 treatment. (A) Peripheral region of a control cell; dashed line marks the approximate boundary between protrusions containing branched actin networks and the cell body associated with other types of actin filament arrays. Boxed region is enlarged and rotated counterclockwise in B. (B) Lamellipodium (wide arrow) with embedded filopodia (thin arrows). Sparse cytoskeleton near filopodial bundles is highlighted in blue. (C) Relative cytoskeleton densities around filopodial roots expressed as percentage of the cytoskeleton density in adjacent lamellipodial regions in the same image (85 ± 13%; mean ± SD; P <0.0001; paired two-tailed t test; distribution normality was confirmed by Kolmogorov–Smirnov test; n = 24 region pairs). Box and whiskers encompass 50% and 100% of data, respectively. Thin line in the box indicates median, dashed line indicates mean. (D–G) Peripheral regions of cells treated with 200 µM CK-666 with filopodial (D and E) or blebbing (F and G) phenotype. (D) Wide arrows mark remaining lamellipodia. Boxed region is enlarged and rotated counterclockwise in E. (E) Yellow color marks a bleb formed between filopodial roots, as confirmed by 3D views of this region (not shown). Blue shade in D and E marks sparse cytoskeleton. (F) White arrowhead marks a bleb filled with a relatively uniform network. Stereo view of this bleb is shown in Fig. S1 D. Black arrow marks a crumpled bleb. Boxed region is enlarged and rotated counterclockwise in G. (G) Rounded blebs with a dense cortical cytoskeleton and a sparser internal network. An animated tilt series of this bleb is shown in Video 2. Scale bars: 2 µm (A, D, and F) and 500 nm (B, E, and G). See also Fig. S2.