Figure 8.
Figure 8. FA growth rate linearly correlates with local F-actin flow velocity in FA, independently of vinculin. (A) Kymographs through the long axis of control and Vcl-KO MEF showing FA growth rate (EGFP-paxillin, green) and local F-actin flow velocity (X-rhodamine actin, purple) of individual FA. Distal end of FA at top and proximal end at bottom. SDC time-lapses, 10-s frame rate; D, distance; T, time. (B) Plot of FA growth rate versus local F-actin flow velocity for individual FA in control and Vcl-KO MEF; n = 85 (control) and n = 102 (Vcl-KO) FA. (C) Box and whisker plot of the ratio of FA growth rate and local F-actin flow velocity for individual FA in control and Vcl-KO MEF; n = 85 (control) and n = 102 (Vcl-KO) FA; means indicated; Student’s t test. (D) Kymographs through the long axis of FA in Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA showing FA growth (EGFP-paxillin, nontransfected/EGFP-vinculin variants, green) and local F-actin flow velocity (mApple-actin, purple) of individual FA. SDC time-lapses; 5-s frame rate; D, distance; T, time. (E) Plot of FA growth rate versus local F-actin flow velocity for individual FA 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. (F) Box and whisker plot of the ratio of FA growth rate and local F-actin flow velocity for individual FA 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; Student’s t test. (G) Model for the role of vinculin in FA maturation. Reduced traction stress at FA lacking vinculin is associated with an increased local F-actin flow velocity and a corresponding increase in FA growth rate. We propose that vinculin regulates FA maturation by an indirect mechanism and by engaging retrograde F-actin flow to FA, thus limiting F-actin flow velocity and hence reducing the F-actin flow velocity–dependent growth of FA.

FA growth rate linearly correlates with local F-actin flow velocity in FA, independently of vinculin. (A) Kymographs through the long axis of control and Vcl-KO MEF showing FA growth rate (EGFP-paxillin, green) and local F-actin flow velocity (X-rhodamine actin, purple) of individual FA. Distal end of FA at top and proximal end at bottom. SDC time-lapses, 10-s frame rate; D, distance; T, time. (B) Plot of FA growth rate versus local F-actin flow velocity for individual FA in control and Vcl-KO MEF; n = 85 (control) and n = 102 (Vcl-KO) FA. (C) Box and whisker plot of the ratio of FA growth rate and local F-actin flow velocity for individual FA in control and Vcl-KO MEF; n = 85 (control) and n = 102 (Vcl-KO) FA; means indicated; Student’s t test. (D) Kymographs through the long axis of FA in Vcl-KO MEF expressing the indicated EGFP-vinculin cDNA showing FA growth (EGFP-paxillin, nontransfected/EGFP-vinculin variants, green) and local F-actin flow velocity (mApple-actin, purple) of individual FA. SDC time-lapses; 5-s frame rate; D, distance; T, time. (E) Plot of FA growth rate versus local F-actin flow velocity for individual FA 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. (F) Box and whisker plot of the ratio of FA growth rate and local F-actin flow velocity for individual FA 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; Student’s t test. (G) Model for the role of vinculin in FA maturation. Reduced traction stress at FA lacking vinculin is associated with an increased local F-actin flow velocity and a corresponding increase in FA growth rate. We propose that vinculin regulates FA maturation by an indirect mechanism and by engaging retrograde F-actin flow to FA, thus limiting F-actin flow velocity and hence reducing the F-actin flow velocity–dependent growth of FA.

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