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Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans
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 activat...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8523150/ https://www.ncbi.nlm.nih.gov/pubmed/34569929 http://dx.doi.org/10.7554/eLife.66862 |
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author | Porto, Daniel Matsunaga, Yohei Franke, Barbara Williams, Rhys M Qadota, Hiroshi Mayans, Olga Benian, Guy M Lu, Hang |
author_facet | Porto, Daniel Matsunaga, Yohei Franke, Barbara Williams, Rhys M Qadota, Hiroshi Mayans, Olga Benian, Guy M Lu, Hang |
author_sort | Porto, Daniel |
collection | PubMed |
description | 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 Caenorhabditis 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 signaling 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). |
format | Online Article Text |
id | pubmed-8523150 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-85231502021-10-20 Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans Porto, Daniel Matsunaga, Yohei Franke, Barbara Williams, Rhys M Qadota, Hiroshi Mayans, Olga Benian, Guy M Lu, Hang eLife Physics of Living Systems 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 Caenorhabditis 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 signaling 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). eLife Sciences Publications, Ltd 2021-09-27 /pmc/articles/PMC8523150/ /pubmed/34569929 http://dx.doi.org/10.7554/eLife.66862 Text en © 2021, Porto et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Physics of Living Systems Porto, Daniel Matsunaga, Yohei Franke, Barbara Williams, Rhys M Qadota, Hiroshi Mayans, Olga Benian, Guy M Lu, Hang Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans |
title | Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans |
title_full | Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans |
title_fullStr | Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans |
title_full_unstemmed | Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans |
title_short | Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans |
title_sort | conformational changes in twitchin kinase in vivo revealed by fret imaging of freely moving c. elegans |
topic | Physics of Living Systems |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8523150/ https://www.ncbi.nlm.nih.gov/pubmed/34569929 http://dx.doi.org/10.7554/eLife.66862 |
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