Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics.
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.
Grashoff
Carsten
C
Robert M. Berne Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia 22908, USA.
Hoffman
Brenton D
BD
Brenner
Michael D
MD
Zhou
Ruobo
R
Parsons
Maddy
M
Yang
Michael T
MT
McLean
Mark A
MA
Sligar
Stephen G
SG
Chen
Christopher S
CS
Ha
Taekjip
T
Schwartz
Martin A
MA
D000818:Animals; D015374:Biosensing Techniques; D002138:Calibration; D002417:Cattle; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D005456:Fluorescent Dyes; D022001:Focal Adhesions / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D009068:Movement; D052898:Optical Tweezers; D013050:Spectrometry, Fluorescence; D013314:Stress, Mechanical; D016596:Vinculin / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes; D016596:Vinculin