(A) Live MVD7shEVL cells expressing mRuby2-PCARE B with GFP, GFP-ENAH, GFP-dEVH1-ENAH, GFP-EVL, or GFP-VASP, imaged using total internal reflection fluorescence (TIRF) microscopy. Scale bar = 25 μm. (B) Enrichment ratio of mRuby2-PCARE B at GFP-positive focal adhesions over cytosolic signal under indicated overexpression conditions. n = 30 focal adhesions, N = 3 biological replicates. (C) Normalized fluorescence intensity of GFP signal (left axis) and mRuby2-PCARE B signal (right axis) along a line drawn through focal adhesions, indicated by the red arrow in (A). (D) Live MVD7shEVL cells expressing Mito-mRuby2-PCARE B with GFP, GFP-ENAH, GFP-dEVH1-ENAH, GFP-EVL, or GFP-VASP. Image is a maximum intensity projection of z-stack acquired using widefield fluorescence microscopy. Scale bar = 25 μm. (E) Enrichment ratio of GFP-tagged constructs to Mito-mRuby2-PCARE B-positive mitochondria over cytosolic signal under indicated overexpression conditions. n = 30 mitochondria, N = 3 biological replicates. (F) Magnified region of interest indicated by the box in (D), showing colocalization of Mito-mRuby2-PCARE B and GFP-ENAH at increasing depths in the cell. Scale bar = 5 μm. (G) Immunofluorescence labeling of MCF7 cells expressing nontargeting LKO control, ENAH-targeting shRNA, mRuby2-PCARE B, or Mito-mRuby2-PCARE B. Cells were fixed 8 hr after plating and immunolabeled for focal adhesion marker paxillin and endogenous ENAH, and additionally stained with phalloidin for F-actin. Box indicates positions of magnified regions of interest (ROI). Scale bar = 25 μm, magnified ROI scale bar = 5 μm. (H) Box-and-whisker plot of total paxillin-positive area per cell normalized to the total cell area, for indicated conditions. N = 3 biological replicates, n = 45–49 cells. (I) Violin plot of individual focal adhesion size (for adhesions greater than 0.25 μm2) for indicated conditions. The central black line indicates the median, peripheral gray lines indicate interquartile ranges. N = 3 biological replicates, n = 1452–2159 individual adhesions. In panels B, E, H, and I, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, Kruskal–Wallis test.