Abstract

Mechanical forces are integral to many biological processes; however, current techniques cannot map the magnitude and direction of piconewton molecular forces. Here, we describe molecular force microscopy, leveraging molecular tension probes and fluorescence polarization microscopy to measure the magnitude and 3D orientation of cellular forces. We mapped the orientation of integrin-based traction forces in mouse fibroblasts and human platelets, revealing alignment between the organization of force-bearing structures and their force orientations.

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Acknowledgements

The authors thank A. Garcia (Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University) for providing NIH-3T3 fibroblasts stably transfected with GFP-paxillin; E. Bartle and T. Urner for assistance with optical systems; and Y. Liu and J. Kindt for helpful discussion. The work was supported through NIGMS R01 GM124472 (K.S.), NSF 1350829 (K.S.), NSF CAREER 1553344 (A.L.M.), NSF IDBR 1353939 (K.S. and A.L.M.), NSF GRFP DGE-1444932 (J.M.B., A.T.B., W.D.D., and M.E.F.), NCI 1F99CA223074-01 (V.P.-Y.M.), Alfred P. Sloan Foundation (F.A.E.), NSF CAREER 1150235 (W.A.L.), NIH Grants RO1HL121264 (W.A.L.), RO1 HL130918 (W.A.L.), U01-HL117721 (W.A.L.), and U54HL112309 (W.A.L.). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

Author information

Author notes

    • Wallace D Derricotte
    •  & Alexa L Mattheyses

    Present addresses: Department of Chemistry, Morehouse College, Atlanta, Georgia, USA (W.D.D.) and Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA (A.L.M.).

    • Joshua M Brockman
    •  & Aaron T Blanchard

    These authors contributed equally to this work.

Affiliations

  1. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

    • Joshua M Brockman
    • , Aaron T Blanchard
    • , Meredith E Fay
    • , Wilbur A Lam
    •  & Khalid Salaita
  2. Department of Chemistry, Emory University, Atlanta, Georgia, USA.

    • Victor Pui-Yan Ma
    • , Wallace D Derricotte
    • , Yun Zhang
    • , Francesco A Evangelista
    •  & Khalid Salaita
  3. Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA.

    • Alexa L Mattheyses

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Contributions

A.L.M. and K.S. conceived the general principle of MFM. J.M.B., A.T.B., A.L.M., and K.S. conceived of applying excitation-resolved fluorescence microscopy. J.M.B., A.T.B., A.L.M., and K.S. designed experiments. J.M.B. performed experiments. J.M.B. performed anisotropy simulations. A.T.B. derived tilt-angle measurement and performed error simulations. J.M.B., Y.Z., and V.P.-Y.M. synthesized reagents and prepared DNA-modified surfaces. J.M.B. and A.T.B. performed data analysis and wrote the required programs. J.M.B., M.E.F., and W.A.L. designed platelet aggregate experiments. M.E.F. assisted with platelet handling. W.D.D. and F.A.E. calculated the Cy3B transition dipole moment. J.M.B., A.T.B., A.L.M., and K.S. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Alexa L Mattheyses or Khalid Salaita.

Integrated supplementary information

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–14, Supplementary Notes 1–4 and Supplementary Tables 1–4

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    Life Sciences Reporting Summary

    Life Sciences Reporting Summary

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    Supplementary Software 1

    MFM GUI

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    Supplementary Software 2

    Analysis Script

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DOI

https://doi.org/10.1038/nmeth.4536

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