Journal: iScience

Article Title: Direct Arp2/3-vinculin binding is required for pseudopod extension, but only on compliant substrates and in 3D

doi: 10.1016/j.isci.2025.112623

Figure Lengend Snippet: The Arp2/3-vinculin hybrid complex is required for pseudopod extension in 3D (A and B) Vinculin and kindlin-2, but not FAK knockout U2OS cells have spreading defects in 3D. Cells embedded in collagen gels were stained for F-actin, imaged and quantified as described in . PEI is normalized to wild-type control. Data are shown as mean ± s.d. Kruskal-Wallis test: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001 (C) Direct Arp2/3-binding by vinculin is required for vinculin function during pseudopod extension in 3D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt), Arp3-binding mutant (Δ-Arp3) or Actin-binding mutants (Δ-IA or Δ-RE) in full-length vinculin. PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. Seven data points are outside the y axis limit, and the full graph is presented in E. Data are shown as mean ± s.d. Kruskal-Wallis test: ns = not significant, ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001. (D) Arp2/3-binding by vinculin is not essential for vinculin function during pseudopod extension in 2D. Vinculin KO cells were reconstituted with empty vector (ev), wild-type (wt) or indicated vinculin mutants (Arp3-binding mutant (Δ-Arp3), Actin-binding mutants (Δ-IA or Δ-RE)). PEI values are normalized to the mean of the ev-distribution. Gray dotted line designates the spread levels of wild-type U2OS cells. All vinculin constructs except the Δ-RE mutant were able to restore pseudopod formation of the vinculin KO cells comparable to that of wild-type U2OS cells. All data were collected from two to five independent experiments (N) and are shown as mean ± s.d. Each data point represents the median of the PEI of all cells within one image field of view (n) and normalized to the mean of the ev-distribution. Total number of quantified individual cells are indicated in brackets, but not used to compute any statistics. Kruskal-Wallis test: ns = not significant, ∗ p < 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. (E) Hybrid Arp2/3-vinculin complex formation is not abrogated in 2D. p34-ARC-vinculin PLA density was detected in cells seeded on collagen-coated glass coverslips, background subtracted (see ) and normalized to wild-type cells (wt). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗ p < 0.05, ∗∗∗∗ p ≤ 0.0001. (F) Formation of the hybrid Arp2/3-vinculin complex is sensitive to the extracellular environment. p34-ARC-vinculin PLA densities were analyzed as in (E) but from cells seeded on soft substrate (∼8 kPa). Each data point represents a cell. Data were collected from three independent experiments (N). Total number of quantified cells are indicated in brackets. Mean ± s.e.m. One-way ANOVA: ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: Subsequently, the samples were stained with rabbit anti-p34-ARC/ARPC2 (Millipore; 07–227; 1:250) and mouse anti-vinculin (Sigma-Aldrich; V9264; 1:500) for 1 h at RT, followed by 1 h RT incubation with the Duolink PLA probes, 30 min ligation and 200 min amplification at 37°C in a humidified chamber.

Techniques: Knock-Out, Staining, Control, Binding Assay, Plasmid Preparation, Mutagenesis, Construct