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

Elife. 2021 Sep 27:10:e66862. doi: 10.7554/eLife.66862.

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).

Keywords: C. elegans; FRET; kinase; molecular biophysics; physics of living systems; sarcomere; structural biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Biophysical Phenomena
  • Caenorhabditis elegans
  • Caenorhabditis elegans Proteins / chemistry*
  • Calmodulin-Binding Proteins / chemistry*
  • Connectin
  • Fluorescence Resonance Energy Transfer / methods
  • Muscle Contraction / physiology*
  • Muscle Proteins / chemistry*
  • Protein Conformation*

Substances

  • Caenorhabditis elegans Proteins
  • Calmodulin-Binding Proteins
  • Connectin
  • Muscle Proteins
  • unc-22 protein, C elegans

Associated data

  • Dryad/10.5061/dryad.6wwpzgn09

Grants and funding

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.