Figure 7. Vinculin activation and... | Rockefeller University Press

Figure 7.

$Figure 7. Vinculin activation and F-actin binding promote nascent FA formation and turnover, whereas vinculin–F-actin binding slows FA growth and vinculin activation is limiting for FA stabilization. (A) TIRF micrographs of live Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA and mApple-actin (Videos 9–10), showing FA at t = 0 s (green) and t = 90 s (purple); 3-s frame rate. Bar, 2 µm. (B) Box and whisker plot of nascent FA formation density in protruding lamellipodia of Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA; n = 19 (nontransfected), n = 20 (WT), n = 12 (PA), n = 12 (ΔAB), and n = 12 (PA-ΔAB) protrusion sequences (6–10 cells/genotype); means indicated; *, P < 0.002, Student’s t test. (C) Maturation fraction among >200 nascent FA in protruding lamellipodia of Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA; n = 19 (nontransfected, EGFP-paxillin), n = 20 (WT), n = 12 (PA), n = 12 (ΔAB), and n = 12 (PA-ΔAB) protrusion sequences (6–10 cells/genotype); means indicated; *, P < 0.0001, Student’s t test. (D) Box and whisker plot of FA growth rate in Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA; n = 41 (nontransfected, EGFP-paxillin), n = 32 (WT), n = 22 (PA), n = 30 (ΔAB), and n = 22 (PA-ΔAB) FA of 9–16 cells/condition; means indicated; *, P < 0.002, Student’s t test. (E) Box and whisker plot of FA persistence (fraction of FA persistent for >1 h) of Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA, recorded by laser scanning confocal microscopy. EGFP-vinculin (variants) as FA marker; frame rate of 2 min; n = 9 (nontransfected, i.e., EGFP-paxillin transfected), n = 9 (WT), n = 8 (PA), n = 7 (ΔAB), n = 8 (PA-ΔAB), and n = 9 (Vh) cells/condition; means indicated; *, P < 0.005, Student’s t test.$

Vinculin activation and F-actin binding promote nascent FA formation and turnover, whereas vinculin–F-actin binding slows FA growth and vinculin activation is limiting for FA stabilization. (A) TIRF micrographs of live Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA and mApple-actin (Videos 9–10), showing FA at t = 0 s (green) and t = 90 s (purple); 3-s frame rate. Bar, 2 µm. (B) Box and whisker plot of nascent FA formation density in protruding lamellipodia of Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA; n = 19 (nontransfected), n = 20 (WT), n = 12 (PA), n = 12 (ΔAB), and n = 12 (PA-ΔAB) protrusion sequences (6–10 cells/genotype); means indicated; *, P < 0.002, Student’s t test. (C) Maturation fraction among >200 nascent FA in protruding lamellipodia of Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA; n = 19 (nontransfected, EGFP-paxillin), n = 20 (WT), n = 12 (PA), n = 12 (ΔAB), and n = 12 (PA-ΔAB) protrusion sequences (6–10 cells/genotype); means indicated; *, P < 0.0001, Student’s t test. (D) Box and whisker plot of FA growth rate in Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA; n = 41 (nontransfected, EGFP-paxillin), n = 32 (WT), n = 22 (PA), n = 30 (ΔAB), and n = 22 (PA-ΔAB) FA of 9–16 cells/condition; means indicated; *, P < 0.002, Student’s t test. (E) Box and whisker plot of FA persistence (fraction of FA persistent for >1 h) of Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA, recorded by laser scanning confocal microscopy. EGFP-vinculin (variants) as FA marker; frame rate of 2 min; n = 9 (nontransfected, i.e., EGFP-paxillin transfected), n = 9 (WT), n = 8 (PA), n = 7 (ΔAB), n = 8 (PA-ΔAB), and n = 9 (Vh) cells/condition; means indicated; *, P < 0.005, Student’s t test.