Dataset Information

Whole worm proteomics after optogenetically induced mechanical muscle stress (OptIMMuS)

ABSTRACT: 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).

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Caenorhabditis Elegans

TISSUE(S): Whole Body

SUBMITTER: Prerana Wagle

LAB HEAD: Thorsten Hoppe

PROVIDER: PXD045078 | Pride | 2024-07-16

REPOSITORIES: Pride

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Project description:This project includes two proximity labeling experiments, one to establish the vicinity of UNC-45 under non-stress conditions and one to examine changes in the vicinity of UNC-45 under optogenetically induced mechanical muscle stress. The first non-stress experiment consists of 15 samples after biotin-streptavidin pull-down in 5 replicates of 3 conditions (3 transgenic C. elegans strains). Transgenic C. elegans strains expressing the mutant BirA biotin ligases miniTurbo and TurboID in body wall muscle cells were used to compare a proximity-labeled strain expressing an UNC-45-miniTurbo-2xHA fusion protein in the muscle cells (PP3135 unc-119(ed4)III; hhIs241[unc-54p::unc-45::miniTurbo::2xHA; unc-119(+)]) with a strain expressing the biotin ligase TurboID-2xHA without fusion protein in muscle cells (PP3138 unc-119(ed4)III; hhIs242[unc-54p::TurboID::2xHA; unc-119(+)]) to find transient interactors of the myosin chaperone UNC-45 in muscle. As a control, we included a strain expressing a transgenic UNC-45-FLAG protein in the body wall muscle (PP1017 unc-119(ed4)III; hhIs84[unc-119(+); unc-54p::unc-45::FLAG]). Biotinylated proteins in 2.5 mg lysates of these strains were pulled down with streptavidin sepharose beads and identified by mass spectrometry. The second mechanical stress experiment consists of 10 samples after biotin-streptavidin pull-down in 5 replicates of 2 conditions. A transgenic C. elegans strain expressing the biotin ligase miniTurbo fused to UNC-45 in body wall muscle cells was crossed with a transgenic strain expressing the optogenetic channelrhodopsin mutant ChR2(C128S;H134R)-FLAG in body wall muscle cells (PP3358 hhIs241[unc-54p::unc-45::miniTurbo::2xHA; unc-119(+)]; hhIs251[myo-3p::ChR2(C128S,H134R)-FLAG::unc-54 3'UTR, Cbrunc-119(+)]). The latter transgene is 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 its food source E. coli OP50. In this experiment, we compared proximity-labeling in the UNC-45-miniTurbo-2xHA expressing strain under conditions with ATR (L+, treatment, contraction) with proximity-labeling in the UNC-45-miniTurbo-2xHA expressing strain under conditions without ATR (L-, control, no contraction) to find transient interactors of the candidate UNC-45 in muscle under mechanical stress. Biotinylated proteins in 2.5 mg of lysates of these strains were pulled down using streptavidin sepharose beads and identified by mass spectrometry.

Dataset Information

Whole worm proteomics after optogenetically induced mechanical muscle stress (OptIMMuS)

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