Fig 1: Schematic summary ArpC5 isoform–dependent regulation of lamellipodium characteristics and cell migration.Schematic representation of the phenotypes observed for C5KO and C5LKO cells and of how altered actin filament polymerization velocities caused by differential recruitment of Mena/VASP overcome the intrinsically different nucleation speeds of the associated Arp2/3 complexes.
Fig 2: ArpC5 isoforms govern lamellipodial actin network architecture and have differential effects on ArpC1 stabilization in the branch junction.(A to C) Sums of seven computational slices (~9.5 nm thickness) through cryo-electron tomograms of B16-F1 WT, C5KO, and C5LKO lamellipodia. Dashed yellow lines indicate the leading edge. Insets show enlarged views of representative branch junctions. (D to F) Segmentations of actin filaments contained in the full height of tomograms shown in (A) to (C). Color coding is given in (E). (G and H) Visualization of actin filament angular distribution with respect to the vector of protrusion, pooled from 13 tomograms of B16-F1 WT, C5KO, and C5LKO cells each. (G) Violin plot. (H) Histogram. Kruskal-Wallis test combined with Dunn’s multiple comparison test, n = 2819, 2195, and 2410 filaments, P values shown in the chart. Black lines indicate medians (43.93°, 53.27°, and 40.20°), and red lines indicate quartile ranges. (I) Branch density per tomogram in B16-F1 WT, C5KO, and C5LKO lamellipodia. Kruskal-Wallis test combined with Dunn’s multiple comparison test, n = 61, 97, and 61 tomograms, P values shown in the chart. Black lines indicate medians (252.9, 242.0, and 258.7 µm-2), and red lines indicate quartile ranges. (J to L) Isosurface representations of the actin filament–Arp2/3 complex branch junction derived from B16-F1 WT, C5KO, and C5LKO cells, low-pass filtered to 8.1 Å resolution. Isosurface coloring of Arp2/3 complex subunits is annotated in the figure. (M) The EM density map from B16-F1 WT cells (shown transparent) with the rigid body fitted model of the branch junction (PDB 7AQK). Color code as in (J) to (L). (N) Difference map (both side views shown) was calculated from the structures of C5KO and C5LKO branch junctions projected onto the map of the C5KO branch junction. Red coloring indicates differences in occupancy. Insets show the ArpC1 subunit ß-propeller (green dashed box) and protrusion helix (pink dashed box). Scale bars, 100 nm.
Fig 3: ArpC5 isoforms affect lamellipodium morphology.(A) Schematic representation of the branch junction highlighting the positions of the individual Arp2/3 complex subunits within this assembly. Subunit colors are annotated. (B to J) Analysis of lamellipodium width phenotype. (B to E and G to I) Representative epifluorescence micrographs of B16-F1 wild-type (WT) (B), C5KO (C to E), and C5LKO (G to I) cells visualizing the actin cytoskeleton using fluorescent phalloidin. For the knockout (KO) cells, three individual clones are shown. The right panel in (B) shows a zoom-in of the indicated area in the left panel and exemplifies which kind of areas were considered as lamellipodia for width measurements. Three such measurements were performed per cell, and their average was then used for statistical analysis. (F) Lamellipodium width of B16-F1 C5KO lines and B16-F1 WT cells. Kruskal-Wallis test combined with Dunn’s multiple comparison test, n = 50 cells for each experimental group, P values shown in the chart. Black lines indicate medians (3.020, 1.742, 1.830, and 1.972 µm), and red lines indicate quartile ranges. (J) Lamellipodium width of B16-F1 C5LKO lines and B16-F1 WT cells. Kruskal-Wallis test combined with Dunn’s multiple comparison test, n = 50 cells for each experimental group, P values shown in the chart. Black lines indicate medians (2.884, 4.237, 4.324, and 4.280 µm), and red lines indicate quartile ranges. Scale bars, 20 µm.
Supplier Page from Abcam for Anti-Arp2 antibody