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, 466 (7303), 263-6

Measuring Mechanical Tension Across Vinculin Reveals Regulation of Focal Adhesion Dynamics


Measuring Mechanical Tension Across Vinculin Reveals Regulation of Focal Adhesion Dynamics

Carsten Grashoff et al. Nature.


Mechanical forces are central to developmental, physiological and pathological processes. 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 spectroscopy. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. We show that tension across vinculin in stable FAs is approximately 2.5 pN 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.


Figure 1
Figure 1. Vinculin tension sensor (VinTS) construct
(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 aa 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: 87.6s +/− 6.6s, VinV: 68.3s +/− 13.1s, mean ± s.e.m., p=0.205).
Figure 2
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 spectrofluorimetry 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. (e–g) 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 vs. time. (g) FRET efficiency vs. 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 reveal reversible stretching and relaxing of TSModCy between 0.25 and 19 pN. All error bars represent s.e.m.
Figure 3
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, VinTS, IIa and Inh (n=30, *: p<0.005, Tukey-b Test). (c) Cells expressing VinCS on pL, FN, or FN treated with Y-27632 (Inh) (n=30, *: p<5×10−6, Tukey-b test). (d) FRET index of VinTS in BAECs. Protruding areas (P), retracting areas (R). (e) FRET index of VinTL in BAECs. (f, g) FRET index of protruding (P) and retracting (R) areas normalized by FRET index of interior FAs. (f) VinTS (n=4, *: p<0.01). (g) VinTL (n=4, p>0.5). Scale bar: 20µm. All error bars represent s.e.m.
Figure 4
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) increases with normalized FA size index (defined in Supplemental Note II, closed circles). (b) In disassembling FAs (n=92), FRET index is high and further increases at late stages. (c) Lifetime of FAs visualized with EGFP-paxillin in vinculin−/− cells (n=4 cells, 214 FAs) or cells re-expressing vinculin-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 RhoA-V14 (difference at 870s: p< 0.05). All error bars represent s.e.m.

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