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