template pegfp c1 ggvcl 1 851 a50i mutant  (Addgene inc)

Journal: eLife

Article Title: Tunable molecular tension sensors reveal extension-based control of vinculin loading

doi: 10.7554/eLife.33927

Figure Lengend Snippet: ( A ) FA structural model consists of two parallel layers of proteins, which can be conceptualized as either ‘sensor’ (blue) or ‘linker’ (orange) elements. ( B ) Mimicking what was done in experiments , simulations involved three sensor elements with distinct stiffnesses ( k S 1 , k S 2 , k S 3 ), arranged in a stratified fashion with a single linker element (stiffness k L ), loaded by a bulk extension (or force) input. The forces and the extensions experienced by each sensor element were calculated, and a C o n t r o l M e t r i c relating the relative variation in forces to the variation in extensions was calculated (see Appendix 2 for details). ( C ) Schematic depiction of parameter space examined using this simple structural model of FAs, wherein the relative number of the sensor and linker element is varied (x-axis) along with their relative stiffness (y-axis); thicker springs indicate stiffer mechanics. ( D ) Summary of results from simulations quantifying force-controlled versus extension-controlled loading of the sensor element. C o n t r o l M e t r i c describes the ratio of variation in forces to the variation in extensions experienced by the sensor elements and will be positive for extension-controlled situations and negative for force-controlled situations. Following a bulk extension input, force-controlled loading of the sensor element occurs when the sensor element is stiff and in relatively high abundance, while extension-controlled loading of the sensor element occurs when the sensor element is soft and/or in relatively low abundance. Dashed contour lines are depicted that correspond to the measured C o n t r o l M e t r i c for VinTS, VinTS + Y-27632, VinTS-A50I, and VinTS on 10 kPa gels ( C o n t r o l M e t r i c = 3.2 , 3.1 , 2.6 , 1.7 , respectively). ( E-F ) FA structural model predictions of the relationship between sensor element stiffness (spring constant) and force ( E ) or extension ( F ). Dashed contour lines in panel ( D ) correspond to force-stiffness relationships in ( E ) and extension-stiffness relationships in ( F ).

Article Snippet: To generate A50I versions of the vinculin tension sensors, PCR was used to generate a fragment of the vinculin head domain containing the A50I mutation 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’, and template pEGFP-C1/GgVcl 1–851 A50I mutant (Addgene 46269).

Techniques:

Journal: eLife

Article Title: Tunable molecular tension sensors reveal extension-based control of vinculin loading

doi: 10.7554/eLife.33927

Figure Lengend Snippet: ( A-C ) Representative images of the localization of a trio of vinculin tension sensors containing A50I mutations to FAs. ( D ) Normalized histograms of acceptor intensities at FAs are indistinguishable between the three sensors. ( E-G ) Representative images of masked FRET efficiency and ( H ) normalized histograms of average FA FRET reported by each sensor. ( I-K ) Representative images of forces and ( L ) normalized histograms of average vinculin force in FAs reported by each sensor. ( M-O ) Representative images of extension and ( P ) normalized histograms of average vinculin extension in FAs reported by each sensor. Note that ~16% of FAs exhibited negative forces/extensions and were excluded from the analysis in panels ( L and P ). All normalized histograms depict data from individual FAs; n = 60, 58, 62 cells and n = 4759, 3393, 4147 FAs for (GGSGGS) 5,7,9 extensible domains, respectively; data pooled from three independent experiments; ****p<0.0001, n.s. not significant (p≥0.05), ANOVA. ( Q ) Quantification of cell-average FRET efficiency in control VinTS-expressing compared to VinTS-A50I -expressing cells; data represents ≥58 cells per condition, pooled from three independent experiments; red filled circle denotes sample mean; ****p<0.0001, Student’s t-test, two-tailed, assuming unequal variances. See for exact p-values and multiple comparisons test details.

Article Snippet: To generate A50I versions of the vinculin tension sensors, PCR was used to generate a fragment of the vinculin head domain containing the A50I mutation 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’, and template pEGFP-C1/GgVcl 1–851 A50I mutant (Addgene 46269).

Techniques: Control, Expressing, Two Tailed Test

Journal: eLife

Article Title: Tunable molecular tension sensors reveal extension-based control of vinculin loading

doi: 10.7554/eLife.33927

Figure Lengend Snippet: ( A-F ) Vin-/- MEFs expressing various version of VinTS-A50I show indistinguishable FA area ( A ), FA axis ratio ( B ), subcellular distributions of FAs quantified as normalized distance from cell edge ( C ), cell perimeter ( D ), cell axis ratio ( E ), number of FAs normalized by cell area ( F ). Different versions of the tension sensor were constructed with minimal Clover-mRuby2 modules flanking three distinct extensible domains, namely (GGSGGS) 5 (n = 60 cells), (GGSGGS) 7 (n = 58 cells), (GGSGGS) 9 (n = 62 cells), pooled from three independent experiments; n.s. not significant (p≥0.05), ANOVA. See for exact p-values.

Article Snippet: To generate A50I versions of the vinculin tension sensors, PCR was used to generate a fragment of the vinculin head domain containing the A50I mutation 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’, and template pEGFP-C1/GgVcl 1–851 A50I mutant (Addgene 46269).

Techniques: Expressing, Construct

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