Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics

Abstract

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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Figure 1: Vinculin tension sensor (VinTS) constructs.
Figure 2: Responses to mechanical force.
Figure 3: Forces across vinculin during cell migration.
Figure 4: Tension on vinculin in dynamic FAs.

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Acknowledgements

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).

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Authors

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.

Corresponding authors

Correspondence to Taekjip Ha or Martin A. Schwartz.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9 with legends, Supplementary Notes 1-3 and References. (PDF 3336 kb)

Supplementary Movie 1

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)

Supplementary Movie 2

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)

Supplementary Movie 3

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)

Supplementary Movie 4

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)

Supplementary Movie 5

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)

Supplementary Movie 6

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)

Supplementary Movie 7

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)

Supplementary Movie 8

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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