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Proteomics after labeling the vicinity of UNC-45 using miniTurbo and TurboID biotin ligases followed by streptavidin pull-down under non-stress and optogenetically induced mechanical muscle stress (OptIMMuS).

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Caenorhabditis Elegans

TISSUE(S): Body Wall Muscle

SUBMITTER: Prerana Wagle

LAB HEAD: Thorsten Hoppe

PROVIDER: PXD045081 | Pride | 2024-07-16

REPOSITORIES: Pride

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Project description:modENCODE_submission_2430 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP77(made_by : R. Waterston description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The UNC-130::EGFP fusion protein is expressed in the correct unc-130 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the UNC-130 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc-119(ed3) III; wgIs77 [unc-119(+) unc-130::TY1::EGFP::3xFLAG] official name : OP77 ); Developmental Stage: fed L1; Genotype: unc-119(ed3) III; wgIs77 [unc-119(+) unc-130::TY1::EGFP::3xFLAG]; Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage fed L1; Target gene unc-130; Strain OP77(made_by : R. Waterston description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The UNC-130::EGFP fusion protein is expressed in the correct unc-130 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the UNC-130 transcription factor. tags : GFP::3xFlag mutagen : Bombard outcross : 3 genotype : unc-119(ed3) III; wgIs77 [unc-119(+) unc-130::TY1::EGFP::3xFLAG] official name : OP77 ); temp (temperature) 20 degree celsius Series_type: CHIP-seq

Project description:modENCODE_submission_3385 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP218(official name : OP218 genotype : unc-119(ed3); wgIs218(R02D3.7:TY1 EGFP FLAG; unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The R02D3.7::EGFP fusion protein is expressed in the correct R02D3.7 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the RO2D3.7 transcription factor. made_by : ); Developmental Stage: L2; Genotype: unc-119(ed3); wgIs218(R02D3.7:TY1 EGFP FLAG; unc-119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L2; Target gene R02D3.7; Strain OP218(official name : OP218 genotype : unc-119(ed3); wgIs218(R02D3.7:TY1 EGFP FLAG; unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The R02D3.7::EGFP fusion protein is expressed in the correct R02D3.7 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the RO2D3.7 transcription factor. made_by : ); temp (temperature) 20 degree celsius

Project description:modENCODE_submission_3600 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP212(official name : OP212 genotype : unc-119(ed3); wgIs212(F45C12.2:TY1 EGFP FLAG; unc-119(+)) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Dresden using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The F45C12.2::EGFP fusion protein is expressed in the correct F45C12.2 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the F45C12.2 transcription factor. made_by : Mihail Sarov ); Developmental Stage: fed L1; Genotype: unc-119(ed3); wgIs212(F45C12.2:TY1 EGFP FLAG; unc-119(+)); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage fed L1; Target gene F45C12.2; Strain OP212(official name : OP212 genotype : unc-119(ed3); wgIs212(F45C12.2:TY1 EGFP FLAG; unc-119(+)) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Dresden using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The F45C12.2::EGFP fusion protein is expressed in the correct F45C12.2 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the F45C12.2 transcription factor. made_by : Mihail Sarov ); temp (temperature) 20 degree celsius

Project description:modENCODE_submission_3586 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP218(official name : OP218 genotype : unc-119(ed3); wgIs218(R02D3.7:TY1 EGFP FLAG; unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The R02D3.7::EGFP fusion protein is expressed in the correct R02D3.7 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the RO2D3.7 transcription factor. made_by : Mihail Sarov ); Developmental Stage: L3; Genotype: unc-119(ed3); wgIs218(R02D3.7:TY1 EGFP FLAG; unc-119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L3; Target gene R02D3.7; Strain OP218(official name : OP218 genotype : unc-119(ed3); wgIs218(R02D3.7:TY1 EGFP FLAG; unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The R02D3.7::EGFP fusion protein is expressed in the correct R02D3.7 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the RO2D3.7 transcription factor. made_by : Mihail Sarov ); temp (temperature) 20 degree celsius

Project description:modENCODE_submission_3077 This submission comes from a modENCODE project of Michael Snyder. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We are identifying the DNA binding sites for 300 transcription factors in C. elegans. Each transcription factor gene is tagged with the same GFP fusion protein, permitting validation of the gene's correct spatio-temporal expression pattern in transgenic animals. Chromatin immunoprecipitation on each strain is peformed using an anti-GFP antibody, and any bound DNA is deep-sequenced using Solexa GA2 technology. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: OP218(official name : OP218 genotype : unc-119(ed3); wgIs218(R02D3.7:TY1 EGFP FLAG; unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The R02D3.7::EGFP fusion protein is expressed in the correct R02D3.7 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the RO2D3.7 transcription factor. made_by : ); Developmental Stage: L4; Genotype: unc-119(ed3); wgIs218(R02D3.7:TY1 EGFP FLAG; unc-119); Sex: Hermaphrodite; EXPERIMENTAL FACTORS: Developmental Stage L4; Target gene R02D3.7; Strain OP218(official name : OP218 genotype : unc-119(ed3); wgIs218(R02D3.7:TY1 EGFP FLAG; unc-119) outcross : 3 mutagen : Bombard tags : GFP::3xFlag description : This strain's transgene was constructed by Mihail Sarov at the Max Planck Institute for Cell Biology in Tubiginen using Tony Hyman's recombineering pipeline. The resulting plasmid was used for biolistic transformation of an unc-119(ed3) strain. The R02D3.7::EGFP fusion protein is expressed in the correct R02D3.7 spatio-temporal expression pattern. This strain was used for ChIP-seq experiments to map the in vivo binding sites for the RO2D3.7 transcription factor. made_by : ); temp (temperature) 20 degree celsius

			Action	DRS
	PRIDE_2359_2655.zip	Other
	Q1_ColID_280_2655_01.raw	Raw
	Q1_ColID_280_2655_02.raw	Raw
	Q1_ColID_280_2655_03.raw	Raw
	Q1_ColID_280_2655_04.raw	Raw

Dataset Information

Proteomics after labeling the vicinity of UNC-45 using miniTurbo and TurboID biotin ligases followed by streptavidin pull-down under non-stress and optogenetically induced mechanical muscle stress (OptIMMuS).

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