FRET imaging and curvature data 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, s tretch - induced unfolding is postulated to be the activation mechanism of the t witchin/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 tra nsgenic C. elegans expressing tw itchin 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 c ontraction states in each frame , in order to obtain fluorescence and body curvature measurements with spatial and temporal precision in vivo . The data reveal ed statistically significant periodic changes in FRET signals during muscle activity, consistent with a periodic change in the conforma tion of twitchin kinase. We conclude that stretch - unfolding of twitchin kinase occur s in the active muscle , whereby mechanical activity titrates the signalling pathway of th is 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 tran sparency (e.g zebrafish).

Methods
All data was processed and saved using Matlab.