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Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1

Nature Methods volume 14, pages 10901096 (2017) | Download Citation

Abstract

Förster resonance energy transfer (FRET)-based tension sensor modules (TSMs) are available for investigating how distinct proteins bear mechanical forces in cells. Yet, forces in the single piconewton (pN) regime remain difficult to resolve, and tools for multiplexed tension sensing are lacking. Here, we report the generation and calibration of a genetically encoded, FRET-based biosensor called FL-TSM, which is characterized by a near-digital force response and increased sensitivity at 3–5 pN. In addition, we present a method allowing the simultaneous evaluation of coexpressed tension sensor constructs using two-color fluorescence lifetime microscopy. Finally, we introduce a procedure to calculate the fraction of mechanically engaged molecules within cells. Application of these techniques to new talin biosensors reveals an intramolecular tension gradient across talin-1 that is established upon integrin-mediated cell adhesion. The tension gradient is actomyosin- and vinculin-dependent and sensitive to the rigidity of the extracellular environment.

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Acknowledgements

The authors thank R. Fässler (MPI of Biochemistry, Department of Molecular Medicine) for providing talin-deficient cells, C. Schweizer for help with programming, the MPIB core facility for technical support, and the Microscopy Rennes Imaging Centre (MRic), where multiplex FRET experiments were performed. C.G. is supported by the German Research Council (DFG, GR3399/5-1 and GR3399/6–1), the Collaborative Research Centre SFB863 (B9), and the Max Planck Förderstiftung. M.R. acknowledges support from the German Research Council through the Collaborative Research Centre SFB863 (A2), and M.T. acknowledges funding by the Ligue Régionale Contre le Cancer. A.F. was supported by the Boehringer Ingelheim Fonds.

Author information

Affiliations

  1. Group of Molecular Mechanotransduction, Max Planck Institute of Biochemistry, Martinsried, Germany.

    • Pia Ringer
    • , Anna-Lena Cost
    • , Andrea Freikamp
    •  & Carsten Grashoff
  2. Physics Department E22, Technical University of Munich, Garching, Germany.

    • Andreas Weißl
    • , Alexander Mehlich
    •  & Matthias Rief
  3. ICS-2/IAS-2, Forschungszentrum Jülich, Jülich, Germany.

    • Benedikt Sabass
  4. CNRS, UMR 6290, Rennes, France.

    • Marc Tramier
  5. Institut de Génétique et Développement de Rennes, Université de Rennes, Rennes, France.

    • Marc Tramier
  6. Microscopy Rennes Imaging Centre, Université de Rennes, Rennes, France.

    • Marc Tramier
  7. Munich Centre for Integrated Protein Science, Munich, Germany.

    • Matthias Rief

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Contributions

P.R. generated expression constructs and cell lines, performed experiments, wrote data analysis software, and analyzed data. A.W. performed calibration experiments, data analysis, and theoretical modeling with A.M. under the supervision of M.R. A.-L.C. wrote FLIM analysis software and advanced data analysis algorithms with assistance from B.S. A.F. generated tools and performed initial experiments. M.T. provided multiplexing fluorophores and supervised dual-colour FLIM experiments and data analysis. C.G. purified TSMs, coordinated experiments, and wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Carsten Grashoff.

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DOI

https://doi.org/10.1038/nmeth.4431

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