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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- Supplementary Information (3.2M)
This file contains Supplementary Figures 1-9 with legends, Supplementary Notes 1-3 and References.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.