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).