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 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).
Highlights
Cells and tissues invariably require mechanical stimulation for development, differentiation and physiological maintenance
In order to monitor the unfolding of NL and/or CRD sequences induced by sarcomeric stretch and, thereby, the freeing of twitchin kinase, we constructed a multi-domain kinase chimera carrying fluorescent proteins (FP) inserted N- and C-terminally to the NL-Kin-CRD assembly, to act as a FRET stretch sensor in vivo
The consistency between the fluorescence signals and curvature dynamics of freely moving animals suggests that the relationship between these variables is causal. 322 A conformational change occurs in the twitchin kinase region during muscle activity 325 We investigated the relationship between the state of muscle contractions and twitchin 326 kinase conformational changes
Summary
Cells and tissues invariably require mechanical stimulation for development, differentiation and physiological maintenance. Accumulating evidence suggests that proteins can act as mechanotransductors by undergoing repeated cycles of unfolding and refolding when subjected to directional forces [1, 2]. This mechanism appears to apply in particular to multi-domain proteins that can bear the unfolding of mechanolabile segments of their chains, while maintaining the structural integrity of their other functional regions [2]. The feasibility of such stretch-unfolding in proteins is evidenced by numerous in vitro and computational studies that use atomic force microscopy (AFM), optical tweezers and steered molecular dynamic simulations (SMDS) (reviewed in [3]). Force-induced unfolding is emerging as a possible general and central mechanism in mechanical sensing
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
