Whole worm transcriptomics after optogenetically induced mechanical muscle stress (OptIMMuS)
Mechanical stress during muscle contraction is a constant threat to proteome integrity. Experimental systems are needed to identify proteostasis regulators that are critical under mechanical stress conditions. Although Caenorhabditis elegans combines the advantages of existing organismal and cell culture models, an inducible method to stimulate muscle contraction in large populations has been lacking. Here, we present a versatile technique called OptIMMuS (Optogenetic Induction of Mechanical Muscle Stress) to study changes in the proteostasis network associated with mechanical forces. Repeated blue light exposure of a muscle-expressed Chlamydomonas rheinhardii channelrhodopsin-2 variant leads to sustained muscle contraction and mechanical stress. OptIMMuS combined with proximity labeling and mass spectrometry identified regulators that cooperate with the myosin-directed chaperone UNC-45 in muscle proteostasis. We show in genetic epistasis and co-immunoprecipitation experiments that the E3 ligase NHL-1 interacts with UNC-45 and muscle myosin, and we provide evidence that this TRIM32 orthologue affects myosin protein levels and function. Overall design: This project includes 16 samples of transgenic C. elegans whole worm lysate after mechanical stress in 4 replicates of 4 conditions. Transgenic C. elegans strains expressing the optogenetic channelrhodopsin mutant ChR2(C128S;H134R)-FLAG in body wall muscle cells (PP3189 hhIs251[myo-3p::ChR2(C128S,H134R)-FLAG::unc-54 3'UTR, Cbrunc-119(+)]) were used to contract the worms' muscles by blue light illumination to induce mechanical muscle stress. The optogenetic channel is activated by adding the cofactor all-trans retinal (ATR) to it's food source E. coli OP50. In this experiment, we compared the treatment condition (1) worms after 8 h of illumination on plates with ATR (light plus ATR) to three control conditions: (2) illumination on plates without ATR (light minus ATR), (3) worms kept in the dark on plates with ATR (dark plus ATR), and (4) worms kept in the dark on plates without ATR (dark minus ATR).