Pre-complexation of talin and vinculin without tension is required for efficient nascent adhesion maturation
- Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, United States
- Department of Biomedical Engineering, Michigan Technological University, United States
- Department of Cell Biology, University of Texas Southwestern Medical Center, United States
- School of Biosciences, University of Kent, United Kingdom
- Department of Cell Biology, University of Virginia, United States
- Department of Physics, University of California San Diego, United States
Figures
Figure 1 with 7 supplements
Experimental/computational pipeline to analyze heterogeneous adhesion dynamics in ChoK1 cells.
(a–c) High-resolution traction maps co-imaged with mGFP-tagged adhesion protein, talin (d), vinculin (e), and paxillin (f). 5 kPa silicone gel coated with high density beads was used as a TFM …
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Figure 1—source data 1
Source data for Figure 1j.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig1-data1-v1.xlsx
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Figure 1—source data 2
Source data for Figure 1k.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig1-data2-v1.xlsx
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Figure 1—source data 3
Source data for Figure 1l.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig1-data3-v1.xlsx
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Figure 1—source data 4
Source data for Figure 1m.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig1-data4-v1.xlsx
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Figure 1—source data 5
Source data for Figure 1n.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig1-data5-v1.xlsx
Figure 1—figure supplement 1
Simultaneous TFM-adhesion experimental approach.
(a) A schematic of the TFM-adhesion imaging using TIRF. Here, cells adhere to a fibronectin-coated, high-refractive index, mechanically defined substrate that is compatible with TIRF. 40 nm beads on …
Figure 1—figure supplement 2
Overall average traction per cell and cell spreading area did not change with expression of talin-GFP, vinculin-GFP, or paxillin-GFP.
(a) Bar plot of average traction quantified per cell. (b) Bar plot of cell spread area.
Figure 1—figure supplement 3
Validation of SVM-based machine learning.
(a) confusion matrix among nine different adhesion groups. (b) Feature space. (c) Similarity among features. (d) Similarity among trained data. White lined boxes represent similarity within each …
Figure 1—figure supplement 4
Boxplot of the fraction of G2 NAs that mature into FCs, FAs, or either FCs and FAs.
N is the number of movies, which includes data obtained from cells expressing tagged variants of talin, vinculin, or paxillin. In total 10,028 G2 NAs were analyzed. Importantly, owing to the limited …
Figure 1—figure supplement 5
Representative time series of fluorescence intensity, traction magnitude, edge protrusion speed, adhesion sliding speed, and distance to closest edge, for the five NA groups (G1–G5).
The names for all of the features are listed in Supplementary file 1A.
Figure 1—figure supplement 6
Differences in the feature values for NA subgroups in paxillin and talin time-lapse images.
NAs groups G1, G2, G3, G4, and G5, were classified with paxillin-mGFP and talin-mGFP, from which feature values were collected. The traction magnitude, which is a non-feature outcome (e.g. is not …
Figure 1—figure supplement 7
Classification shifts due to substrate stiffness.
ChoK1 cells, transfected with vinculin-GFP, were cultured and imaged on 18 kPa and 5 kPa silicone gel substrates with TIRF. (a) Color-coded classes of kinematically and kinetically different …
Figure 2 with 2 supplements
Talin and vinculin in non-maturing NAs are recruited in a sequential manner before traction development whereas in maturing NAs they are recruited concurrently, along with paxillin, briefly before the initial traction rise.
(a–l) Representative traces of protein recruitment and traction generation at non-maturing (a,b,c), or maturing (g,h,i) NAs. Each panel contains three views at different time points of mGFP-tagged …
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Figure 2—source data 1
Source data for Figure 2n.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig2-data1-v1.xlsx
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Figure 2—source data 2
Source data for Figure 2o.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
The time-series of fluorescence intensity and traction force within non-maturing (G1) and maturing (G2) NAs for talin, vinculin and paxillin show heterogeneity but ‘increasing’ trend within their lifetimes.
Images of GFP-tagged (a) talin, (b) vinculin, and (c) paxillin. Their corresponding traction maps are shown immediately below each image. (d–i) Individual time-series of fluorescence intensity (left …
Figure 2—figure supplement 2
Box plots of fractions of force-transmitting NAs for each classification group for (a) talin, (b) vinculin, and (c) paxillin.
Note that nearly 100% of non-maturing (G1) and maturing (G2) NAs generate force, which is much higher than other adhesion classes (G3–G9). Statistical differences among G3-G9 are purposefully not …
Figure 3
Vinculin, but not talin and paxillin, is recruited significantly faster in maturing NAs than in non-maturing NAs.
(a) Assembly rate of talin, vinculin, and paxillin, to G1 (non-maturing) or to G2 (maturing) NAs, quantified by the slope of fluorescence intensity over the initial 10 s after detection. (b) …
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Figure 3—source data 1
Source data for Figure 3a.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig3-data1-v1.xlsx
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Figure 3—source data 2
Source data for Figure 3b.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig3-data2-v1.xlsx
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Figure 3—source data 3
Source data for Figure 3c.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig3-data3-v1.xlsx
Figure 4 with 1 supplement
Stabilizing the ‘threonine belt’ in the R8 domain of talin inhibits talin-vinculin interactions under tension-free conditions.
(a) Cartoon representation of talin R7R8 (pdb id 2X0C) showing the ‘threonine belt’, comprised of residues T1502, T1542, and T1562, labeled and shown as sticks (cyan), the VBS helix is colored red. …
Figure 4—figure supplement 1
Fluorescence polarization assay showing the binding affinities for R8 ligand peptides from (top) RIAM TBS1 and (bottom) DLC1 with WT and R7R8vvv.
Figure 5 with 4 supplements
Expression of the talin1 R8vvv mutant in ChoK1 cells with endogenous talin1 knocked down results in the formation of denser NAs, but lesser and smaller FAs, lower traction, more active protrusions, and less maturing adhesions compared to WT talin.
(a–j) Adhesion, traction, and protrusion phenotype of a representative ChoK1 cell on 5 kPa substrate expressing (a–e) talin R8vvv mutant or (f–j) WT talin. (a and f) inverted talin-mNeonGreen …
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Figure 5—source data 1
Source data for Figure 5k.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig5-data1-v1.xlsx
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Figure 5—source data 2
Source data for Figure 5l.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig5-data2-v1.xlsx
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Figure 5—source data 3
Source data for Figure 5m.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig5-data3-v1.xlsx
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Figure 5—source data 4
Source data for Figure 5n.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig5-data4-v1.xlsx
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Figure 5—source data 5
Source data for Figure 5o.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig5-data5-v1.xlsx
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Figure 5—source data 6
Source data for Figure 5p.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig5-data6-v1.xlsx
Figure 5—figure supplement 1
Western blot for talin in WT ChoK1 cells, ChoK1 with shRNA-mediated knockdown of talin, WT cells with ectopic expression of talin1-mNG, WT cells with ectopic expression of talin1-R8vvv-mNG, and ChoK1 cells with shRNA-mediated knockdown of talin and ectopic expression of talin1-R8vvv-mNG.
Actin is shown as a loading control. Note that the double bands in the third and fourth lanes is likely due to the presence of both endogenous and ectopic talin. The presence of a fluorescent …
Figure 5—figure supplement 2
Western blot of talin1 and talin 2 in WT and knockdown (shRNA) cells.
A blot of actin is shown as a control.
Figure 5—figure supplement 3
Traction forces generated by ChoK1 cells expressing the talin1 shRNA are smaller than GFP-talin1 and mNG-talin1 rescue cells.
(a–c) Traction map with force vectors for ChoK1 celsl with (a) GFP-talin1 overexpression, (b) talin1 shRNA, and (c) mNG-talin1 rescue. (d) The average traction at force blobs, that is, local forces …
Figure 5—figure supplement 4
Expression of the talin1 R8vvv mutant results in formation of denser NAs, smaller FAs, lower traction, and less maturing adhesions compared to expression of the talin WT.
(a–h) Adhesion and traction phenotype of a representative IMCD cell on 5 kPa gel substrates expressing talin R8vvv mutant (a–d) vs WT talin1 (e–h). (a,e) Inverted talin-mNeonGreen images. (b,f) …
Figure 6 with 1 supplement
Expression of talin1 R8vvv-mNG mutant does not change the recruitment timing of talin to NAs, but reduces the force growth rate in NAs.
(a–h) Representative talin (top) and traction force (bottom) images of talin1 R8vvv-mNG expressing cells (a–d) and WT talin-mNG rescue cells (e–h) within non-maturing (a,c,e,g) and maturing (b,d,f,h)…
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Figure 6—source data 1
Source data for Figure 6i.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig6-data1-v1.xlsx
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Figure 6—source data 2
Source data for Figure 6j.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig6-data2-v1.xlsx
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Figure 6—source data 3
Source data for Figure 6k.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig6-data3-v1.xlsx
Figure 6—figure supplement 1
An example of a nascent adhesion in an R8vvv-expressing ChoK1 cell exhibiting high assembly rate.
(a) Representative talin images within a non-maturing NA of three different time points, that is, at initial nucleation, at maximum fluorescence intensity, and at the end of the NA portion of the …
Figure 7
Vinculin recruitment is reduced in talin1 R8vvv mutant cells.
(a,d,g,j) Representative two-channel time-lapse images of talin-mNeonGreen (top) and vinculin-SnapTag-TMR-Star (bottom) of G1 NA in a Talin1 R8vvv mutant cell (a), G1 NA in WT talin1 rescue cell (d),…
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Figure 7—source data 1
Source data for Figure 7m.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig7-data1-v1.xlsx
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Figure 7—source data 2
Source data for Figure 7n.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig7-data2-v1.xlsx
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Figure 7—source data 3
Source data for Figure 7o.
- https://cdn.elifesciences.org/articles/66151/elife-66151-fig7-data3-v1.xlsx
Figure 8
A suggested mechanism of differential recruitment of talin and vinculin determining maturation of nascent adhesions.
(Top) For non-maturing NAs, talin binds to integrin before vinculin recruitment. Talin stretching might be limited to a shorter level, which limits the exposure of vinculin-binding-sites. …
Author response image 1
Histogram of mGFP molecules in one paxillin-mGFP time lapse images.
Mean value was 1.7 with 1.08 standard deviation. Up to 16 mGFP molecules were able to be resolved.
Videos
Video 1
Time-lapse images of GFP-tagged vinculin in a ChoK1 cell, overlaid with adhesion trajectories and classification state from the support vector machine (SVM)-based machine-learning.
Different colors represent different classes: G1 (light green), G2 (dark blue), G3 (red), G4 (light blue), G5 (yellow), G6 (cyan), G7 (pink), G8 (dark green), and G9 (brown). The time interval per …
Video 2
Time-lapse images of GFP-tagged talin in a ChoK1 cell.
The time interval per frame: 1.644 s. Playing speed: 25 frames/s. Duration of the movie: 8 min 13 s. Scale bar: 5 μm.
Video 3
Time-lapse images of traction force magnitude for a ChoK1 cell expressing GFP-talin.
Traction forces are reconstructed from the bead images using high-resolution, L1-regularized, TFM software (Han et al., 2015). The color scale is the same as one at Figure 1a, that is, 0 (blue) – …
Video 4
Time-lapse images of GFP-tagged talin in a ChoK1 cell, overlaid with adhesion trajectories and classification state from the support vector machine (SVM)-based machine-learning.
The color coding is the same as in the legend of Video 1. The time interval per frame: 1.644 s. Playing speed: 25 frames/s. Duration of the movie: 8 min 13 s. Scale bar: 5 μm.
Video 5
Time-lapse images of GFP-tagged vinculin in a ChoK1 cell.
The time interval per frame: 2 s. Playing speed: 25 frames/s. Duration of the movie: 12 min. Scale bar: 5 μm.
Video 6
Time-lapse images of traction force magnitude for a ChoK1 cell expressing GFP-vinculin.
Traction forces are reconstructed from the bead images using high-resolution, L1-regularized, TFM software (Han et al., 2015). The color scale is the same as one at Figure 1b, that is, 0 (blue) – …
Video 7
Time-lapse images of GFP-tagged paxillin in a ChoK1 cell.
The time interval per frame: 1.644 s. Playing speed: 25 frames/s. Duration of the movie: 8 min 13 s. Scale bar: 5 μm.
Video 8
Time-lapse images of traction force magnitudes generated by a ChoK1 cell expressing GFP-paxillin.
Traction forces are reconstructed from the bead images using high-resolution, L1-regularized, TFM software (Han et al., 2015). The color scale is the same as one at Figure 1c, that is, 0 (blue) – …
Video 9
Time-lapse images of GFP-tagged paxillin in a ChoK1 cell, overlaid with adhesion trajectories and classification state from the support vector machine (SVM)-based machine-learning.
The color coding is the same as in the legend of Video 1. The time interval per frame: 1.644 s. Playing speed: 25 frames/s. Duration of the movie: 8 min 13 s. Scale bar: 5 μm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| E. coli | BL21(DE3) | Sigma-Aldrich | CMC0016 | Electrocompetent cells |
| Cell line (Cricetulus griseus) | CHO-K1 | ATCC | CCL-61 | Courtesy of Dr.Horwitz, Allen Institute |
| Cell line Mus musculus | IMCD Talin1/2 KO | This paper | Courtesy of Dr. Zent, Vanderbilt University | |
| Lentiviral expression vector | pLVX-shRNA1 | Clontech | Lentiviral construct for stable expression of shRNA. | |
| Lentiviral expression vector | pLVX-CMV-100 | Addgene | Catalog # 110718 | Lentiviral construct for stable expression of ectopic proteins. |
| Transient expression vector | pCDNA3.1(+) | ThermoFisher Scientific | Catalog # V79020 | DNA construct for transient expression of ectopic proteins. |
| Primary antibody | Anti-talin 1 | Abcam | Catalog # 71333 | WB 1:1000 |
| Primary antibody | Anti-talin 2 | Abcam | Catalog # 105458 | WB 1:1000 |
| Secondary antibody | Goat anti-Mouse IgG (H+L) Cross-Adsorbed- Perosidaxe Antibody | ThermoFisher Scientific | Catalog # G-21040 | WB 1:1000 |
| Secondary antibody | Goat anti-Rabbig IgG (H+L) Cross-Adsorbed-Peroxidase Antibody | ThermoFisher Scientific | Catalog # G-21234 | WB 1:1000 |
| Transient expression vector | pCDNA-mNG-Talin-shRNA-R8 | R8 mutant of Talin1 resistant to shRNA. | ||
| Transient expression vector | pCDNA-mNG-shRNA-Talin | WT Talin1 resistant to shRNA | ||
| Transient expression vector | pCDNA-mNG-Talin | WT Talin1 | ||
| Lentiviral expression vector | pLVXCMV100-mNG-Talin-shRNA-R8 | R8 mutant of Talin1 resistant to shRNA. | ||
| Lentiviral expression vector | pLVXCMV100-mNG-Talin-shRNA | WT Talin1 resistant to shRNA | ||
| Lentiviral expression vector | pLVXCMV100-mNG-Talin | WT Talin1 | ||
| Transient expression vector | Paxillin-GFP | Original construct from Horwitz Lab | ||
| Transient expression vector | Vinculin-GFP | Original construct from Horwitz Lab | ||
| Transient expression vector | Talin-GFP | Original construct from Horwitz Lab | ||
| Recombinant expression vector | pet151-Talin1-R7R8-WT (murine) | GeneArt | Original construct from Goult Lab | |
| Recombinant expression vector | pet151-Talin1-R7R8-VVV (murine) | GeneArt | Original construct from Goult Lab | |
| Recombinant expression vector | pet151-Vinculin-Vd1 (murine) | Original construct from Goult Lab | ||
| Synthetic peptide | RIAM-TBS1 peptide (RIAM_6_30-C) DIDQMFSTLLGEMDLLTQSLGVDT-C | GLBiochem | ||
| Synthetic peptide | DLC1 peptide (DLC1_465_489-C) IFPELDDILYHVKGMQRIVNQWSEK-C | GLBiochem |
Additional files
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Supplementary file 1
Tables for information used in machine learning of adhesions.
(A) Features used for classification of adhesions. (B) Nine adhesion groups defined heuristically. (C) Automatic labeling criteria.
- https://cdn.elifesciences.org/articles/66151/elife-66151-supp1-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66151/elife-66151-transrepform-v1.pdf
