ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning

Fig. 1.. 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.…

Fig. 2.. The lamellipodial C5KO phenotype is mediated by isoform specificity and not by gene dose effects. (A to C) Representative epifluorescence micrographs of B16-F1 C5KO cells transfected with pCMV::empty_pCMV::RFP (A), pCMV::ArpC5L_pCMV::RFP (B), and pCMV::ArpC5_pCMV::RFP (C) visualizing the actin cytoskeleton using fluorescent phalloidin and verifying transfection via RFP fluorescence. (D) Quantitation of lamellipodium width of B16-F1 C5KO cells transfected with the same constructs as in (A) to (C). Measurements were done as shown in Fig. 1B. Kruskal-Wallis test combined with Dunn’s multiple comparison test, n = 30 cells per experimental condition, P values shown in the chart. Black lines indicate medians (1.143, 1.310, and 2.248 μm), and red lines indicate quartile ranges. (E to G) Representative epifluorescence micrographs of B16-F1 C5/C5LKO cells transfected with pCMV::empty_pCMV::RFP (E), pCMV::ArpC5L_pCMV::RFP (F), and pCMV::ArpC5_pCMV::RFP (G) visualizing the actin cytoskeleton using fluorescent phalloidin and verifying transfection via RFP fluorescence. (H) Quantitation of lamellipodium width of B16-F1 C5/C5LKO cells transfected with the same constructs as in (F) and (G). No…

Fig. 3.. 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,…

Fig. 4.. ArpC5 isoforms modulate random migration speed and lamellipodial actin dynamics. (A) Random migration speed of B16-F1 WT, C5KO, and C5LKO cells. Kruskal-Wallis test combined with Dunn’s multiple comparison test on pooled data from three independent experiments, n = 150 cells per experimental condition, P values shown in the chart. Black lines indicate overall medians (1.085, 0.8550, and 1.115 μm/min) and quartile ranges. (B) Representative time-lapse images of lamellipodia in B16-F1 WT, C5KO, and C5LKO cells expressing enhanced green fluorescent protein (EGFP)–actin, showing FRAP. Yellow rectangles indicate FRAP regions. (C to F) Protrusion speed (C), rearward flow (D), polymerization rate (E), and protrusion efficiency (F) as determined via FRAP from B16-F1 WT, C5KO, and C5LKO cells expressing EGFP-actin. Polymerization rate is calculated as the sum of the absolute protrusion speed and the absolute rearward flow. Protrusion efficiency is calculated by dividing protrusion speed by polymerization rate. Kruskal-Wallis test combined with Dunn’s multiple comparison test on pooled data from three independent experiments, n = 66, 71, and 72 cells for each experimental condition,…

Fig. 5.. ArpC5 isoforms modulate Mena/VASP recruitment to the leading edge. (A to L) Representative epifluorescence micrographs of B16-F1 WT (A, B, G, and H), C5KO (C, D, I, and J), and C5LKO (E, F, K, and L) cells visualizing the actin cytoskeleton (A, C, E, G, I, and K) and the localization of Mena (B, D, and F) and VASP (H, J, and L). Insets show magnified areas indicated in respective panels. Arrowheads annotate finger-like protrusions labeled by the anti-Mena antibody. (M) Zoom-in on the region indicated in (B), visualizing the measurements and calculations determining the relative accumulation of Mena/VASP at the leading edge. The leading edge was traced for at least 15 μm per measurement. To measure background and cell intensities, the leading edge region of interest was shifted by ~10 μm inside or outside cells while avoiding extremely bright secondary antibody aggregates. (N and O) Quantitative analysis of the relative Mena (N)/VASP (O) accumulation at the leading edges of B16-F1 WT, C5KO, and C5LKO cells. Kruskal-Wallis test combined with Dunn’s multiple comparison test, n = 30 cells for each experimental condition, for both Mena and VASP, P values shown in the chart. Black…

Fig. 6.. 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.