Tunable molecular tension sensors reveal extension-based control of vinculin loading
- Duke University, United States
Figures
Figure 1 with 3 supplements
Design and characterization of tunable FRET-based molecular tension sensors.
(A) Schematic depiction of a generic TSMod and inverse relationship between FRET and force for molecular tension sensors under tensile loading. (B-D) TSMod function depends on the Förster radius of …
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Figure 1—source data 1
Measurements and models of the mechanical behavior of TSMods in vitro and in cellulo.
- https://doi.org/10.7554/eLife.33927.010
Figure 1—figure supplement 1
FRET efficiency measurements depend on the presence of unstructured residues in FPs, but are insensitive to fixation and sensor intensity.
(A) Quantification of FRET-polypeptide length relationship for (GPGGA)n extensible domains flanked by full-length Clover-mRuby2 FPs (containing unstructured residues, orange) as compared to model …
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Figure 1—figure supplement 1—source data 1
FRET-length relationships for TSMods in various conditions.
- https://doi.org/10.7554/eLife.33927.005
Figure 1—figure supplement 2
Increase in unloaded FRET efficiency with Clover-mRuby2 sensors in vitro.
(A, B) Representative images of quantitative spectral analysis of mTFP1-Venus (A) and Clover-mRuby2 (B) TSMod fluorescence in cell lysates using the (ratio)A method (Majumdar et al., 2005). (C) …
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Figure 1—figure supplement 2—source data 1
Fluorometric FRET measurements.
- https://doi.org/10.7554/eLife.33927.007
Figure 1—figure supplement 3
‘Minimal’ FPs exhibit spectral properties indistinguishable from full-length parent FPs.
(A) Schematic of donor and acceptor FPs highlighting 11 C-terminal residues (donor FP) and 2 N-terminal residues (acceptor FP), which do not contribute to beta barrel structure, but are highly …
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Figure 1—figure supplement 3—source data 1
Fluorescent protein spectra.
- https://doi.org/10.7554/eLife.33927.009
Figure 2 with 6 supplements
Predicting TSMod calibrations using a biophysical model.
(A, B) Model descriptions, at various persistence lengths (), of FRET-polypeptide length relationship (A) and FRET-force responses (B) of Cy3 and Cy5 dyes linked by SMCC linker + cysteine modified …
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Figure 2—source data 1
Measurements and models of the mechanical behavior of TSMod-like constructs in vitro.
- https://doi.org/10.7554/eLife.33927.020
Figure 2—figure supplement 1
Schematic depiction of biophysical model describing the mechanical sensitivity of TSMods.
(A-C) Mechanical sensitivity of FRET-based TSMods depends on the mechanical properties and length of the extensible domain (A), the physical separation of the chromophores within the FPs (B), and …
Figure 2—figure supplement 2
Verification of the proper implementation of a biophysical model describing the mechanical sensitivity of FRET-based TSMods.
(A) Probability distributions of polypeptide end-to-end distance (), normalized to contour length () such that calculated using the second Daniels approximation (Daniels, 1952; Yamakawa and …
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Figure 2—figure supplement 2—source data 1
Numerical simulations of the mechanical behavior of worm-like chains.
- https://doi.org/10.7554/eLife.33927.014
Figure 2—figure supplement 3
Parameter constraint has minimal effects on measurement of polypeptide persistence length () in vitro.
(A, B) Heatmaps of chi-squared error of model fits to in vitro ‘minimal’ Clover-mRuby2 based TSMod FRET-length measurements for various and for (GPGGA)n (A) and (GGSGGS)n (B) extensible …
Figure 2—figure supplement 4
Parameter constraint has minimal effects on measurement of polypeptide persistence length () in cellulo.
(A, B) Heatmaps of chi-squared error of model fits to in cellulo “minimal” Clover-mRuby2 based TSMod FRET-length measurements for various and for (GPGGA)n (A) and (GGSGGS)n (B) extensible …
Figure 2—figure supplement 5
Parameter constraint has minimal effects on measurement of polypeptide persistence length () for TSMod-like constructs in unloaded or loaded conditions.
(A, B) Heatmaps of chi-squared error of model fits to published fluorescence-force spectroscopy measurements of (GPGGA)5,8,10 extensible domains flanked by Cy3 and Cy5 fluorescent dyes in unloaded (A…
Figure 2—figure supplement 6
Experimental and theoretical examinations of other models (Brenner et al., 2016) of (GPGGA)n mechanical sensitivity.
(A) Measured relationship between average polypeptide end-to-end distance as a function of the number of residues in the (GPGGA)n extensible domain shows behavior characteristic of …
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Figure 2—figure supplement 6—source data 1
Comparitive analysis of models of flagelliform polypeptide mechanics.
- https://doi.org/10.7554/eLife.33927.019
Figure 3 with 2 supplements
Optimized tension sensor reveals sub-FA gradients in vinculin tension.
(panels A and B) Representative images of subcellular distribution of VinTS (Grashoff et al., 2010) (A, A’) along with representative (A’’) and aggregate (A’’’) line scans of single FAs of size >0.5 …
Figure 3—figure supplement 1
Selection of optimal (GGSGGS)n extensible domain length in a Clover-mRuby2 based TSMod for measuring ~1-6 pN loads borne by vinculin.
(A) Predicted force sensitivities of TSMods containing “minimal” Clover-mRuby2 FRET pair and (GGSGGS)n extensible domains (n =1 to 16). (B) Schematic of metrics used to quantify and compare …
Figure 3—figure supplement 2
FA morphologies, cell morphologies, and sensor localization to FAs are indistinguishable between different versions of VinTS.
(A-F) Vin-/- MEFs expressing various version of VinTS show indistinguishable FA area (A), FA axis ratio (B), subcellular distributions of FAs quantified as normalized distance from cell edge (C), …
Figure 4 with 9 supplements
Using tension sensors with distinct mechanical sensitivities to test force-based versus extension-based control of vinculin loading.
(A-C) Representative images of the localization of a trio of vinculin tension sensors to FAs. (D) Normalized histograms of acceptor intensities at FAs are indistinguishable between the three …
Figure 4—figure supplement 1
Schematic depiction of force-extension relationships and potential force- and extension-control paradigms.
(A, B) A force-control paradigm cannot be detected if only a single sensor is used (A), but can be discerned if multiple sensors report the same forces, but distinct extensions (B). (C, D) An …
Figure 4—figure supplement 2
Evaluating gradients in vinculin extension and force at the sub-FA length scale.
(A-D) Line scans of single FAs of size >0.5 μm2 and axis ratio >1.5 were compiled and averaged to quantify the spatial distribution of acceptor intensity (A), as well as corresponding FRET …
Figure 4—figure supplement 3
Effect of Y-27632 treatment on vinculin extension-control.
(A-C) Representative images of the localization of a trio of vinculin tension sensors to FAs formed in cells treated with Y-27632. (D) Normalized histograms of acceptor intensities at FAs are …
Figure 4—figure supplement 4
Three versions of VinTS respond similarly to Y-27632 treatment.
(A-F) Vin-/- MEFs expressing various version of VinTS, treated with Y-27632, show indistinguishable FA area (A), FA axis ratio (B), subcellular distributions of FAs quantified as normalized distance …
Figure 4—figure supplement 5
Effect of disrupting vinculin-talin interactions on vinculin extension-control.
(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 …
Figure 4—figure supplement 6
Three versions of VinTS-A50I exhibit similar FA morphologies, cell morphologies, and sensor localization to FAs.
(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)…
Figure 4—figure supplement 7
Effect of substrate stiffness on vinculin extension-control.
(A-C) Representative images of the localization of a trio of vinculin tension sensors to FAs formed in cells plated on fibronectin-coated 10 kPa gels. (D) Normalized histograms of acceptor …
Figure 4—figure supplement 8
Three versions of VinTS respond similarly to softer substrates.
(A-F) Vin-/- MEFs expressing various version of VinTS plated on 10 kPa gels show indistinguishable FA area (A), FA axis ratio (B), subcellular distributions of FAs quantified as normalized distance …
Figure 4—figure supplement 9
Various structural arrangements within FAs could lead to force-based or extension-based control.
(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 (F…
Figure 5 with 1 supplement
Roadmap to enable the rational design of FRET-based molecular tension sensors.
(A-C) Representative plots of relationships between FRET efficiencies, forces, and extensions reported by a single sensor, highlighting (A), (B), and polypeptide extension (C) required to …
Figure 5—figure supplement 1
Roadmaps to guide the rational design of FRET-based molecular tension sensors for some commonly used FRET-pairs.
Parameter space highlighting the predicted at the 5% FRET noise floor (A, D, G), as well as corresponding target force, (B, E, H), and target extensions (C, F, I), for a variety of Cy3-Cy5 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus) | Vinculin -/- mouse embryonic fibroblast | PMID: 20181946 | NA | |
| Cell line (Homo sapiens) | HEK293, Human embryonic kidney cells | ATCC Cat# CRL-1573 | RRID:CVCL_0045 | |
| Transfected construct | pcDNA3.1(+) | Invitrogen, Carlsbad, CA | ||
| Transfected construct | VinculinTS | Addgene, Cambridge, MA; PMID: 20613844 | Plasmid #26019 | |
| Transfected construct | tCRMod-GGSGGS5 | Addgene, Cambridge, MA; this work | Plasmid #111760 | |
| Transfected construct | tCRMod-GGSGGS7 | Addgene, Cambridge, MA; this work | Plasmid #111761 | |
| Transfected construct | tCRMod-GGSGGS9 | Addgene, Cambridge, MA; this work | Plasmid #111762 | |
| Transfected construct | VinTS- tCRMod-GGSGGS5 | Addgene, Cambridge, MA; this work | Plasmid #111763 | |
| Transfected construct | VinTS- tCRMod-GGSGGS7 | Addgene, Cambridge, MA; this work | Plasmid #111764 | |
| Transfected construct | VinTS- tCRMod-GGSGGS9 | Addgene, Cambridge, MA; this work | Plasmid #111765 | |
| Sequence-based reagent | Oligonucleotides detailed in Supplementary file 2 | this work | NA | |
| Chemical compound, drug | Y-27632 | Sigma Aldrich, St. Louis, MO | Y0503; PubChem Substance ID 24277699 | Used at 25 μM |
| Software, algorithm | ImageJ | US National Institutes of Health, Bethesda, MD | RRID:SCR_003070 | http://imagej.nih.gov/ij/ |
| Software, algorithm | Image Corrections | PMID: 25640429; doi.org/ 10.1007/s12195-015-0404-9 | NA | https://gitlab.oit.duke.edu/HoffmanLab- Public/image-preprocessing (Rothenberg et al., 2018b; copy archived at https://github.com/elifesciences-publications/HoffmanLab-image-preprocessing) |
| Software, algorithm | FRET calculations from 3-cube imaging | PMID: 16815904; doi.org/ 10.1007/s12195-015-0404-9 | NA | https://gitlab.oit.duke.edu/HoffmanLab- Public/fret-analysis (Rothenberg et al., 2018a; copy archived at https://github.com/elifesciences-publications/HoffmanLab-fret-analysis) |
| Software, algorithm | FRET calculations from spectrofluorometry | PMID: 16055154 | NA | https://gitlab.oit.duke.edu/HoffmanLab- Public/fluorimetry-fret (LaCroix et al., 2018c; copy archived at https://github.com/elifesciences-publications/HoffmanLab-fluorimetry-fret) |
| Software, algorithm | TSMod calibration model | this work | NA | https://gitlab.oit.duke.edu/HoffmanLab- Public/tsmod-calibration-model (LaCroix et al., 2018b; copy archived at https://github.com/elifesciences-publications/HoffmanLab-tsmod-calibration-model) |
| Software, algorithm | FA structural model | this work | NA | https://gitlab.oit.duke.edu/HoffmanLab- Public/FA-structural-model (LaCroix and Hoffman, 2018a; copy archived at https://github.com/elifesciences-publications/HoffmanLab-FA-structural-model) |
Additional files
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Supplementary file 1
Persistence length () for a variety of published polypeptides.
Overall, persistence lengths < 0.2 and > 5.0 nm are rarely observed. Synthetic homo-polymers (ex. poly-proline) can achieve larger persistence lengths.
- https://doi.org/10.7554/eLife.33927.036
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Supplementary file 2
Primers used in this study.
- https://doi.org/10.7554/eLife.33927.037
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Supplementary file 3
Spectral bleed-through coefficients and G factors for mTFP1-Venus and Clover-mRuby2 based tension sensor FRET efficiency calculations.
- https://doi.org/10.7554/eLife.33927.038
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Supplementary file 4
Statistical test details including exact p-values for ANOVAs and post-hoc tests.
Note, individual comparisons were made only when statistical significance was detected in ANOVAs.
- https://doi.org/10.7554/eLife.33927.039
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Transparent reporting form
- https://doi.org/10.7554/eLife.33927.040
