Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics

Journal name:
Nature
Volume:
466,
Pages:
263–266
Date published:
DOI:
doi:10.1038/nature09198
Received
Accepted

Mechanical forces are central to developmental, physiological and pathological processes1. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy2. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent3. We show that tension across vinculin in stable FAs is ~2.5pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

At a glance

Figures

  1. Vinculin tension sensor (VinTS) constructs.
    Figure 1: Vinculin tension sensor (VinTS) constructs.

    a, The tension sensor module (TSMod) consists of two fluorophores separated by a flagelliform linker sequence (GPGGA)8. b, When force across TSMod extends the elastic linker, FRET efficiency decreases (f, force). c, The vinculin tension sensor (VinTS) consists of TSMod inserted after amino acid 883 of vinculin. d, Vinculin–venus control (VinV). e, Vinculin tail-less control (VinTL). f, Localization of VinTS and VinV in vinculin−/− cells. Scale bar, 20μm. g, Normalized average fluorescence recovery rates of VinTS (open circles, n = 10) and VinV (closed circles, n = 8). Error bars represent standard error of the mean (s.e.m.). (Recovery half-time VinTS: 87.6±6.6s, VinV: 68.3±13.1s; mean±s.e.m., P = 0.205).

  2. Responses to mechanical force.
    Figure 2: Responses to mechanical force.

    a, FRET index in vinculin−/− cells expressing VinTS or VinTL seeded on poly-l-lysine (pL) or fibronectin (FN) (*P<0.05, Tukey-b test, n = 11–18). b, FRET measured by spectrofluorometry of lysates containing VinTS, VinTL or TSMod (n = 4, P>0.5, Tukey-b test). c, Fluorescence lifetime images of vinculin−/− cells expressing VinTS or VinTL. Scale bar, 2μm. d, Fluorescence lifetime histograms from FAs of VinTS (n = 11) or VinTL (n = 8) expressing vinculin−/− cells. eg, Multiple stretch/relax cycles of a single TSModCy using fluorescence force spectroscopy. e, Fluorescence intensity time traces for donor (green) and acceptor (red). f, Applied force versus time. g, FRET efficiency versus time. h, Single-molecule FRET histogram of TSModCy at zero force. The peak marked by a red Gaussian fit represents the TSModCy labelled with both donor and acceptor. i, Averaged FRET–force curves from n = 7 molecules show reversible stretching and relaxing of TSModCy between 0.25 and 19pN. All error bars represent s.e.m.

  3. Forces across vinculin during cell migration.
    Figure 3: Forces across vinculin during cell migration.

    a, Traction forces of VinTS-expressing cells (Con, n = 18), treated with Y-27632 (Inh, n = 15) or depleted of myosin IIa (IIa, n = 20) (*P<0.005, **P<0.05, Dunnet’s test). b, FRET index of VinTL and VinTS, or VinTS after IIa and Inh (n = 30, *P<0.005, Tukey-b test). c, Cells expressing VinCS on poly-l-lysine, fibronectin, or fibronectin after treatment with Y-27632 (Inh) (n = 30, *P<5×10−6, Tukey-b test). d, FRET index of VinTS in BAECs. P, protruding areas; R, retracting areas. e, FRET index of VinTL in BAECs. Scale bars, 20μm. f, g, FRET index of protruding (P) and retracting (R) areas normalized by FRET index of all FAs. f, VinTS (n = 4, *P<0.01). g, VinTL (n = 4, P>0.5). All error bars represent s.e.m.

  4. Tension on vinculin in dynamic FAs.
    Figure 4: Tension on vinculin in dynamic FAs.

    FAs were isolated, tracked and classified as assembling or disassembling. a, In assembling FAs (n = 78), FRET index (open circles) increased with normalized FA size index (defined in Supplementary Note 2, closed circles). b, In disassembling FAs (n = 92), FRET index was high and further increased at late stages. c, Lifetime of FAs visualized with EGFP–paxillin in vinculin−/− cells (vinc (−/−); n = 4 cells, 214 FAs) or cells re-expressing vinculin–Flag (vinc–Flag; n = 4 cells, 310 FAs). Difference at 870s was not significant (P = 0.24). d, FA lifetime in vinculin−/− cells (n = 7 cells, 408 FAs) and vinculin–Flag cells (n = 7 cells, 715 FAs) expressing MIIa (difference at 870s: P<0.005). e, Vinculin−/− cells (n = 3 cells, 250 FAs) or vinculin–Flag cells (n = 3 cells, 192 FAs) expressing active RhoA(G14V) (difference at 870s: P<0.05). All error bars represent s.e.m.

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Author information

  1. These authors contributed equally to this work.

    • Carsten Grashoff &
    • Brenton D. Hoffman

Affiliations

  1. Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908, USA

    • Carsten Grashoff,
    • Brenton D. Hoffman &
    • Martin A. Schwartz
  2. Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA

    • Carsten Grashoff,
    • Brenton D. Hoffman &
    • Martin A. Schwartz
  3. Center for the Physics of Living Cells and Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

    • Michael D. Brenner,
    • Ruobo Zhou &
    • Taekjip Ha
  4. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

    • Michael D. Brenner &
    • Taekjip Ha
  5. Randall Division of Cell and Molecular Biophysics, King’s College London, London SE1 1UL, UK

    • Maddy Parsons
  6. Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, 19104, USA

    • Michael T. Yang &
    • Christopher S. Chen
  7. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA

    • Mark A. McLean &
    • Stephen G. Sligar
  8. Howard Hughes Medical Institute, Urbana, Illinois 61801, USA

    • Taekjip Ha
  9. Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia 22908, USA

    • Martin A. Schwartz

Contributions

M.A.S. conceived the general idea. C.G. conceived the use of the flagelliform sequence, designed and generated vinculin expression constructs, performed spectrofluorometry and all cell and imaging experiments. B.D.H. generated analysis tools and analysed the in vivo data. T.H., M.A.M. and S.G.S. conceived the sensor calibration scheme. M.D.B. designed and generated the tension sensor construct for the calibration. M.D.B. and R.Z. performed and analysed the single molecule experiments. T.H., R.Z., M.D.B and B.D.H. developed an algorithm to map in vitro and in vivo tension sensor data. M.P. performed and analysed FLIM experiments. M.T.Y and C.S.C. generated micropost arrays and analysed traction force data. C.G., B.D.H. and M.A.S. designed the in vivo experiments, discussed the results and wrote the paper with input from all authors.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Information (3.2M)

    This file contains Supplementary Figures 1-9 with legends, Supplementary Notes 1-3 and References.

Movies

  1. Supplementary Movie 1 (1.4M)

    This movie shows a bovine aortic endothelial cell expressing VinTS. The colour bar indicates FRET index. Notice lower FRET index in protruding regions but high FRET index in retracting areas of the cell. The time-lapse covers a period of 48 min. Frame rate: 90 s.

  2. Supplementary Movie 2 (1.1M)

    This movie shows a bovine aortic endothelial cell expressing VinTL. The colour bar indicates FRET index. Notice that the FRET index is uniformly high. The time-lapse covers a period of 45 min. Frame rate: 90 s.

  3. Supplementary Movie 3 (408K)

    This movie shows a vinculin-/- cell expressing paxillin-EGFP. The time-lapse covers a period of 30 min. Frame rate: 30 s.

  4. Supplementary Movie 4 (573K)

    This movie shows a vinculin-/- cell reconstituted with vinculin-flag expressing paxillin-EGFP. The time-lapse covers a period of 30 min. Frame rate: 30 s.

  5. Supplementary Movie 5 (316K)

    This movie shows a vinculin-/- cell expressing paxillin-EGFP and myosin IIa. The time-lapse covers a period of 30 min. Frame rate: 30 s.

  6. Supplementary Movie 6 (593K)

    This movie shows a vinculin-/- cell reconstituted with vinculin-flag expressing paxillin-EGFP and myosin IIa. The time-lapse covers a period of 30 min. Frame rate: 30 s.

  7. Supplementary Movie 7 (381K)

    This movie shows a vinculin-/- cell expressing paxillin-EGFP and RhoA-V14. The time-lapse covers a period of 30 min. Frame rate: 30 s.

  8. Supplementary Movie 8 (470K)

    This movie shows a vinculin-/- cell reconstituted with vinculin-flag expressing paxillin-EGFP and RhoA-V14. The time-lapse covers a period of 30 min. Frame rate: 30 s.

Additional data