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 ∼2.5 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.
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Orr, A. W., Helmke, B. P., Blackman, B. R. & Schwartz, M. A. Mechanisms of mechanotransduction. Dev. Cell 10, 11–20 (2006)
Hohng, S. et al. Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the Holliday junction. Science 318, 279–283 (2007)
Bershadsky, A. D., Balaban, N. Q. & Geiger, B. Adhesion-dependent cell mechanosensitivity. Annu. Rev. Cell Dev. Biol. 19, 677–695 (2003)
Ballestrem, C., Hinz, B., Imhof, B. A. & Wehrle-Haller, B. Marching at the front and dragging behind: differential αVβ3-integrin turnover regulates focal adhesion behavior. J. Cell Biol. 155, 1319–1332 (2001)
Bakolitsa, C. et al. Structural basis for vinculin activation at sites of cell adhesion. Nature 430, 583–586 (2004)
Ziegler, W. H., Gingras, A. R., Critchley, D. R. & Emsley, J. Integrin connections to the cytoskeleton through talin and vinculin. Biochem. Soc. Trans. 36, 235–239 (2008)
Galbraith, C. G., Yamada, K. M. & Sheetz, M. P. The relationship between force and focal complex development. J. Cell Biol. 159, 695–705 (2002)
Riveline, D. et al. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an Mdia1-dependent and ROCK-independent mechanism. J. Cell Biol. 153, 1175–1186 (2001)
Xu, W., Baribault, H. & Adamson, E. D. Vinculin knockout results in heart and brain defects during embryonic development. Development 125, 327–337 (1998)
Alenghat, F. J. et al. Analysis of cell mechanics in single vinculin-deficient cells using a magnetic tweezer. Biochem. Biophys. Res. Commun. 277, 93–99 (2000)
Mierke, C. T. et al. Mechano-coupling and regulation of contractility by the vinculin tail domain. Biophys. J. 94, 661–670 (2008)
Hu, K. et al. Differential transmission of actin motion within focal adhesions. Science 315, 111–115 (2007)
Ji, L., Lim, J. & Danuser, G. Fluctuations of intracellular forces during cell protrusion. Nature Cell Biol. 10, 1393–1400 (2008)
Balaban, N. Q. et al. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nature Cell Biol. 3, 466–472 (2001)
Shemesh, T., Geiger, B., Bershadsky, A. D. & Kozlov, M. M. Focal adhesions as mechanosensors: a physical mechanism. Proc. Natl Acad. Sci. USA 102, 12383–12388 (2005)
Tan, J. L. et al. Cells lying on a bed of microneedles: an approach to isolate mechanical force. Proc. Natl Acad. Sci. USA 100, 1484–1489 (2003)
Meng, F., Suchyna, T. M. & Sachs, F. A fluorescence energy transfer-based mechanical stress sensor for specific proteins in situ. FEBS J. 275, 3072–3087 (2008)
Day, R. N., Booker, C. F. & Periasamy, A. Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. J. Biomed. Opt. 13, 031203 (2008)
Becker, N. et al. Molecular nanosprings in spider capture-silk threads. Nature Mater. 2, 278–283 (2003)
Humphries, J. D. et al. Vinculin controls focal adhesion formation by direct interactions with talin and actin. J. Cell Biol. 179, 1043–1057 (2007)
Chen, H. et al. Spatial distribution and functional significance of activated vinculin in living cells. J. Cell Biol. 169, 459–470 (2005)
Cohen, D. M. et al. A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions. J. Biol. Chem. 281, 16006–16015 (2006)
Parsons, M. et al. Quantification of integrin receptor agonism by fluorescence lifetime imaging. J. Cell Sci. 121, 265–271 (2008)
Cai, Y. et al. Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow. Biophys. J. 91, 3907–3920 (2006)
Pertz, O., Hodgson, L., Klemke, R. L. & Hahn, K. M. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006)
Kolega, J. Asymmetric distribution of myosin IIB in migrating endothelial cells is regulated by a rho-dependent kinase and contributes to tail retraction. Mol. Biol. Cell 14, 4745–4757 (2003)
Periasamy, A., Wallrabe, H., Chen, Y. & Barroso, M. Chapter 22: Quantitation of protein-protein interactions: confocal FRET microscopy. Methods Cell Biol. 89, 569–598 (2008)
Zamir, E. et al. Molecular diversity of cell-matrix adhesions. J. Cell Sci. 112, 1655–1669 (1999)
Zaidel-Bar, R., Milo, R., Kam, Z. & Geiger, B. A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions. J. Cell Sci. 120, 137–148 (2007)
Crocker, J. C. & Hoffman, B. D. Multiple-particle tracking and two-point microrheology in cells. Methods Cell Biol. 83, 141–178 (2007)
We thank R. Horwitz, M. Vicente-Manzanares, D. Schafer, L. K. Tamm and K. A. DeMali for reagents and M. Gardel for critical reading of the manuscript. This work was supported by USPHS grant U54 GM64346 to M.A.S., C.G. was supported by a Research Fellowship from the Deutsche Forschungsgemeinschaft (DFG, GR3399/1-1). B.D.H. was supported by USPHS training grant 5T32-HL007284 and an AHA Postdoctoral Fellowship. M.D.B., R.Z. and T.H. were supported by the US National Science Foundation Physics Frontier Center grant 0822613 and by USPHS grant R21 RR025341. T.H. is an investigator with the Howard Hughes Medical Institute. M.P. was supported by a Royal Society University Research Fellowship (UK). M.T.Y. was supported by an IGERT fellowship from the National Science Foundation (DGE-0221664).
The authors declare no competing financial interests.
This file contains Supplementary Figures 1-9 with legends, Supplementary Notes 1-3 and References. (PDF 3336 kb)
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. (MOV 1447 kb)
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. (MOV 1226 kb)
This movie shows a vinculin-/- cell expressing paxillin-EGFP. The time-lapse covers a period of 30 min. Frame rate: 30 s. (MOV 408 kb)
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. (MOV 573 kb)
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. (MOV 316 kb)
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. (MOV 593 kb)
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. (MOV 381 kb)
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. (MOV 470 kb)
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Grashoff, C., Hoffman, B., Brenner, M. et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466, 263–266 (2010). https://doi.org/10.1038/nature09198
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