Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans
- Georgia Institute of Technology, United States
- Emory University, United States
- 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.
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FRET imaging and curvature data of freely moving C. elegansDryad Digital Repository, doi:10.5061/dryad.6wwpzgn09.
Article and author information
Author details
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.
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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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
