Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans

  1. Daniel Porto
  2. Yohei Matsunaga
  3. Barbara Franke
  4. Rhys M Williams
  5. Hiroshi Qadota
  6. Olga Mayans
  7. Guy M Benian
  8. Hang Lu  Is a corresponding author
  1. Georgia Institute of Technology, United States
  2. Emory University, United States
  3. University of Konstanz, Germany

Abstract

The force-induced unfolding and refolding of proteins is speculated to be a key mechanism in the sensing and transduction of mechanical signals in the living cell. Yet, little evidence has been gathered for its existence in vivo. Prominently, stretch-induced unfolding is postulated to be the activation mechanism of the twitchin/titin family of autoinhibited sarcomeric kinases linked to the mechanical stress response of muscle. To test the occurrence of mechanical kinase activation in living working muscle, we generated transgenic C. elegans expressing twitchin containing FRET moieties flanking the kinase domain and developed a quantitative technique for extracting FRET signals in freely moving C. elegans, using tracking and simultaneous imaging of animals in three channels (donor fluorescence, acceptor fluorescence, and transmitted light). Computer vision algorithms were used to extract fluorescence signals and muscle contraction states in each frame, in order to obtain fluorescence and body curvature measurements with spatial and temporal precision in vivo. The data revealed statistically significant periodic changes in FRET signals during muscle activity, consistent with a periodic change in the conformation of twitchin kinase. We conclude that stretch-unfolding of twitchin kinase occurs in the active muscle, whereby mechanical activity titrates the signalling pathway of this cytoskeletal kinase. We anticipate that the methods we have developed here could be applied to obtaining in vivo evidence for force-induced conformational changes or elastic behavior of other proteins not only in C. elegans but in other animals in which there is optical transparency (e.g zebrafish).

Data availability

All data generated or analyzed during this study will be deposited to Dryad.

The following data sets were generated

Article and author information

Author details

  1. Daniel Porto

    Bioengineering, Georgia Institute of Technology, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Yohei Matsunaga

    Pathology, Emory University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Barbara Franke

    Biology, University of Konstanz, Konstanz, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Rhys M Williams

    Biology, University of Konstanz, Konstanz, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1982-2632
  5. Hiroshi Qadota

    Pathology, Emory University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Olga Mayans

    Biology, University of Konstanz, Konstanz, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Guy M Benian

    Pathology (and Cell Biology), Emory University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8236-3176
  8. Hang Lu

    Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, United States
    For correspondence
    hang.lu@gatech.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6881-660X

Funding

Human Frontier Science Program (RGP0044/2012)

  • Olga Mayans
  • Guy M Benian
  • Hang Lu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Olga Boudker, Weill Cornell Medicine, United States

Publication history

  1. Received: January 24, 2021
  2. Preprint posted: March 1, 2021 (view preprint)
  3. Accepted: September 24, 2021
  4. Accepted Manuscript published: September 27, 2021 (version 1)
  5. Version of Record published: October 18, 2021 (version 2)

Copyright

© 2021, Porto et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Daniel Porto
  2. Yohei Matsunaga
  3. Barbara Franke
  4. Rhys M Williams
  5. Hiroshi Qadota
  6. Olga Mayans
  7. Guy M Benian
  8. Hang Lu
(2021)
Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans
eLife 10:e66862.
https://doi.org/10.7554/eLife.66862

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