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...

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Autores principales:	Daniel Porto, Yohei Matsunaga, Barbara Franke, Rhys M Williams, Hiroshi Qadota, Olga Mayans, Guy M Benian, Hang Lu
Formato:	article
Lenguaje:	EN
Publicado:	eLife Sciences Publications Ltd 2021
Materias:	kinase FRET sarcomere Medicine R Science Q Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/3cbb59f548534da7ac17b2502b6e4447
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spelling	oai:doaj.org-article:3cbb59f548534da7ac17b2502b6e44472021-11-15T05:37:53ZConformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans10.7554/eLife.668622050-084Xe66862https://doaj.org/article/3cbb59f548534da7ac17b2502b6e44472021-09-01T00:00:00Zhttps://elifesciences.org/articles/66862https://doaj.org/toc/2050-084XThe 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).Daniel PortoYohei MatsunagaBarbara FrankeRhys M WilliamsHiroshi QadotaOlga MayansGuy M BenianHang LueLife Sciences Publications LtdarticlekinaseFRETsarcomereMedicineRScienceQBiology (General)QH301-705.5ENeLife, Vol 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	kinase FRET sarcomere Medicine R Science Q Biology (General) QH301-705.5
spellingShingle	kinase FRET sarcomere Medicine R Science Q Biology (General) QH301-705.5 Daniel Porto Yohei Matsunaga Barbara Franke Rhys M Williams Hiroshi Qadota Olga Mayans Guy M Benian Hang Lu Conformational changes in twitchin kinase in vivo revealed by FRET imaging of freely moving C. elegans
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	article
author	Daniel Porto Yohei Matsunaga Barbara Franke Rhys M Williams Hiroshi Qadota Olga Mayans Guy M Benian Hang Lu
author_facet	Daniel Porto Yohei Matsunaga Barbara Franke Rhys M Williams Hiroshi Qadota Olga Mayans Guy M Benian Hang Lu
author_sort	Daniel Porto
title	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_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_sort	conformational changes in twitchin kinase in vivo revealed by fret imaging of freely moving c. elegans
publisher	eLife Sciences Publications Ltd
publishDate	2021
url	https://doaj.org/article/3cbb59f548534da7ac17b2502b6e4447
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_version_	1718428545901920256

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

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