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Proteomics after co-immunoprecipitation of myosin/MHC B/UNC-54 (with a Gly387Arg mutation) from C. elegans worms grown on nhl-1 or F40A3.6 RNAi compared to control RNAi

ABSTRACT: This project includes 20 samples after immunoprecipitation of C. elegans myosin heavy chain B (MHC B/UNC-54/F11C3.3, UniProt P02566 with a Gly387Arg mutation) in 4 replicates of 4 conditions. Lysates from a myosin-misfolding C. elegans strain (unc-54(e1301) with a Gly387Arg substitution in MHC B) grown on (A) control RNAi, (B) RNAi against nhl-1 (F54G8.4), (C) RNAi against F40A3.6, and (D) RNAi against nhl-1 and F40A3.6 together were used for immunoprecipitation. Samples from groups A, B, C and D were incubated over night at 4°C with 1 µg of a mouse monoclonal hybridoma antibody against MHC B (mAb 5-8 from the Developmental Studies Hybridoma Bank, DSHB), while 4 lysate samples from unc-54(e1301) worms on control RNAi (Z) were incubated WITHOUT antibody addition. The next day, co-immunoprecipitated proteins were collected on magnetic Dynabeads Protein A for 2 h at 4°C, washed, on-bead digested, and subjected to identification by mass spectrometry.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Caenorhabditis Elegans

TISSUE(S): Body Wall Muscle

SUBMITTER: Prerana Wagle

LAB HEAD: Thorsten Hoppe

PROVIDER: PXD045076 | 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.

			Action	DRS
	ColID_527_Q1_3303_01.raw	Raw
	ColID_527_Q1_3303_02.raw	Raw
	ColID_527_Q1_3303_03.raw	Raw
	ColID_527_Q1_3303_04.raw	Raw
	ColID_527_Q1_3303_05.raw	Raw

Dataset Information

Proteomics after co-immunoprecipitation of myosin/MHC B/UNC-54 (with a Gly387Arg mutation) from C. elegans worms grown on nhl-1 or F40A3.6 RNAi compared to control RNAi

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