Journal: Biophysical Journal

Article Title: Vinculin Force-Sensitive Dynamics at Focal Adhesions Enable Effective Directed Cell Migration

doi: 10.1016/j.bpj.2018.02.019

Figure Lengend Snippet: Load across vinculin is strongly affected by the ability of vinculin to bind actin, but not talin. (A) TSMod was inserted into vinculin variants. (Left to right) Shown here is WT VinTS, mutation disrupting the vinculin binding to talin (A50I), and mutation disrupting the vinculin binding to actin (I997A). (B) Representative acceptor (top row) and masked FRET efficiency (bottom row) images are given of single Vinc −/− MEFs expressing each of the VinTS constructs. Scale bars, 30 μm. (C) Zoomed-in view is given of the regions indicated in (B). (D) A box-whisker plot is given of cell-averaged FRET efficiency (n = 150, 166, and 79 cells, respectively, from seven independent experiments) compared to previously established zero-load (dotted line). Differences between groups were detected using the Steel-Dwass test (∗∗∗p < 0.001); p values for all comparisons can be found in Table S1. (E) Line scans are shown of vinculin distribution and FRET efficiency across peripheral FAs from distal to proximal tip. Thin lines represent each individual adhesion (n = 27 FAs from three independent experiments); thick lines represent a smoothing spline fit to the collective FA data. To see this figure in color, go online.

Article Snippet: An analogous strategy was used to generate pcDNA3.1-VinTS-A50I using forward primer 5′-AAT AAG CTT GCC ATG CCC GTC TTC CAC AC-3′, reverse primer 5′-GCC GGA TCC GCA AGC CAG TTC-3′, template DNA pEGFPC1/GgVcl 1-851 A50I mutant (Plasmid No. 46269; Addgene, Cambridge, MA), and 5′- Hin dIII/3′- Bam HI restriction sites.

Techniques: Mutagenesis, Binding Assay, Expressing, Construct, Whisker Assay

Journal: Biophysical Journal

Article Title: Vinculin Force-Sensitive Dynamics at Focal Adhesions Enable Effective Directed Cell Migration

doi: 10.1016/j.bpj.2018.02.019

Figure Lengend Snippet: Vinculin dynamics are strongly affected by the ability of vinculin to bind talin, but not actin. (A) Representative FAs of cells expressing the VinTS and its mutant variants (VinTS A50I, VinTS I997A) are displayed in the acceptor channel during FRAP imaging. Scale bars, 3 μm. (B) Vinculin FRAP recovery curves are shown for cells expressing VinTS, VinTS A50I, or VinTS I997A (n = 34, 21, and 18 FAs, respectively, from seven independent experiments). (C) Box-whisker plots are given of the half-time of recovery for the FAs that were analyzed in (B). (D) Box-whisker plots are given of the mobile fraction for the FAs that were analyzed in (B). Differences between groups were detected using the Steel-Dwass test (∗∗p < 0.01, ∗∗∗p < 0.001); p values for all comparisons can be found in Table S2.

Article Snippet: An analogous strategy was used to generate pcDNA3.1-VinTS-A50I using forward primer 5′-AAT AAG CTT GCC ATG CCC GTC TTC CAC AC-3′, reverse primer 5′-GCC GGA TCC GCA AGC CAG TTC-3′, template DNA pEGFPC1/GgVcl 1-851 A50I mutant (Plasmid No. 46269; Addgene, Cambridge, MA), and 5′- Hin dIII/3′- Bam HI restriction sites.

Techniques: Expressing, Mutagenesis, Imaging, Whisker Assay

Journal: Biophysical Journal

Article Title: Vinculin Force-Sensitive Dynamics at Focal Adhesions Enable Effective Directed Cell Migration

doi: 10.1016/j.bpj.2018.02.019

Figure Lengend Snippet: Reduction of cytoskeletal tension via ROCK inhibition affects vinculin dynamics only in the presence of vinculin-talin interaction. (A) Representative FAs of cells expressing the VinTS and its mutant variants (VinTS A50I, VinTS I997A) treated with 25 μM Y-27632 are displayed in the acceptor channel during FRAP imaging. Scale bars, 3 μm. (B) Vinculin FRAP recovery curves are shown for cells expressing VinTS, VinTS A50I, or VinTS I997A (n = 24, 16, and 16 FAs, respectively, from four independent experiments). (C) Box-whisker plots are given of the half-time of recovery for the FAs analyzed in (B). No significant difference was detected between groups. (D) Box-whisker plots of the mobile fraction are given for the FAs analyzed in (B). Differences between groups were detected using the Steel-Dwass test (∗∗∗p < 0.001); p values for all comparisons can be found in Table S2.

Article Snippet: An analogous strategy was used to generate pcDNA3.1-VinTS-A50I using forward primer 5′-AAT AAG CTT GCC ATG CCC GTC TTC CAC AC-3′, reverse primer 5′-GCC GGA TCC GCA AGC CAG TTC-3′, template DNA pEGFPC1/GgVcl 1-851 A50I mutant (Plasmid No. 46269; Addgene, Cambridge, MA), and 5′- Hin dIII/3′- Bam HI restriction sites.

Techniques: Inhibition, Expressing, Mutagenesis, Imaging, Whisker Assay

Journal: Biophysical Journal

Article Title: Vinculin Force-Sensitive Dynamics at Focal Adhesions Enable Effective Directed Cell Migration

doi: 10.1016/j.bpj.2018.02.019

Figure Lengend Snippet: FRET-FRAP assay shows that force-sensitive vinculin dynamics depend on talin and actin interactions. (A) Correlation between recovery half-time and FRET efficiency for WT vinculin corresponds to the force-stabilized state (n = 32, p < 0.005). (B) Correlation between recovery half-time and FRET efficiency for VinTS A50I corresponds to the force-destabilized state (n = 21, p < 0.05). (C) No detectable correlation between recovery half-time and FRET efficiency was observed for VinTS I997A (n = 18, p = 0.88). (D) Y-27632 treatment reverses the relationship between recovery half-time and load for WT vinculin, corresponding to the force-destabilized state (n = 24, p < 0.05). (E) Y-27632 treatment does not affect the relationship between recovery and load for VinTS A50I, which remains in the force-destabilized state (n = 16, p < 0.01). (F) Y-27632 treatment does not affect the relationship between recovery and load for VinTS I997A, with turnover still insensitive to load (n = 16, p = 0.98). The p values indicate the results of a t-test comparing the regression slope to zero. (Vertical dotted lines) Previously established zero-load is shown.

Article Snippet: An analogous strategy was used to generate pcDNA3.1-VinTS-A50I using forward primer 5′-AAT AAG CTT GCC ATG CCC GTC TTC CAC AC-3′, reverse primer 5′-GCC GGA TCC GCA AGC CAG TTC-3′, template DNA pEGFPC1/GgVcl 1-851 A50I mutant (Plasmid No. 46269; Addgene, Cambridge, MA), and 5′- Hin dIII/3′- Bam HI restriction sites.

Techniques: FRAP Assay