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Proximity labelling of mitochondria and endosomes upon mtDNA replication stress.

ABSTRACT: This project aims to identify novel regulators of mtDNA turnover, for what we used TurboID. For specific detection of the mitochondria-endosome proteome, we fused the N-terminal part of Turbo ID to RAB5C (TurboID aa1-aa72; RAB5C-SplitTurboNt-V5) and, the C-terminal part to SAMM50 (TurboID aa73–aa246; SAMM50-SplitTurboCt-HA). Proximity biotinylation was performed in transduced HEK293 cells expressing Split TurboID constructs. Cells expressing only SAMM50-SplitC-HA were used as a negative control. For protein isolation, HEK293 cells were incubated for 4 h with 0,25 mM biotin. For inducing mtDNA damage, cells were previously transfected with Twinkle K319E-Cherry 24 h before the biotin incubation.

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Prerana Wagle

LAB HEAD: David Pla Martín

PROVIDER: PXD049081 | Pride | 2025-04-07

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.

Project description:Tau pathology is a major hallmark of Alzheimer’s disease (AD) and related diseases, called tauopathies. While Tau is normally enriched in axons, somatodendritic missorting of the microtubule-associated protein is a key event in early disease development. Tau missorting promotes synaptic loss and neuronal dysfunction but the mechanisms underlying both normal axonal sorting and pathological missorting remain unclear. Interestingly, the disease-associated Tau brain isoforms show different axodendritic distribution, but the distinct role of these isoforms in health and disease largely unknown. Here, we aimed to identify domains or motifs of Tau and cellular binding partners that are required for efficient axonal Tau sorting, and we studied the differences of the isoform-specific Tau interactome. By using human MAPT-KO induced pluripotent stem cell (iPSC)-derived glutamatergic neurons, we analyzed the sorting behavior of more than 20 truncated or phosphorylation-mutant Tau constructs, and we used TurboID-based proximity labelling and proteomics to identify sorting- and isoform-specific Tau interactors. We found that efficient axonal Tau sorting was independent of the N-terminal tail, the C-terminal repeat domains, and the general microtubule affinity of Tau. In contrast, the presence of the proline-rich region 2 (PRR2) was necessary for successful sorting. Our interactome data revealed peroxisomal accumulation of the Tau N-terminal half, while axonal Tau interacted with the PP2A activator HSP110. When we compared the interactome of 0N3R- and 0N4R-Tau, we observed specific interactions of 0N4R-Tau with regulators of presynaptic exocytosis and postsynaptic plasticity, which are partially associated with AD pathogenesis, such as members of the CDC42 pathway and the RAB11 proteins, while 0N3R-Tau bound to MAP4 and other cytoskeletal elements. In sum, our study postulates that axonal Tau sorting relies on the PRR2 domain but not on microtubule affinity, and unravels a potential isoform-specific role in synaptic function and AD-related dysfunction.

Project description:In this study, we used the in vivo proximity labeling biotin identification (BioID; Roux et al., 2012) method to capture proteins of the microenvironment of the striatin interacting kinase and phosphatase complex of the filamentous fungus Sordaria macrospora (SmSTRIPAK). For that, the TurboID biotin ligase (Branon et al., 2018) was fused to the STRIPAK complex interactor 1 (sci1; SMAC_05559), a subunit of the SmSTRIPAK complex (Hollstein et al., 2022). We applied two control strains for relative quantification to reduce false positive hits. The first control expressed a free unfused TurboID ligase under the control of the constitutive promoter of the clock controlled gene 1 (ccg1) from Neurospora crassa. For the second control, we crossed the strain expressing the sci1-TurboID fusion gene (native sci1 promoter) with the Δpro11 (Bloemendal et al., 2012) deletion strain. The pro11 gene encodes a striatin homolog, which serves as the main scaffold of the SmSTRIPAK complex. Therefore, the second control setup enables the comparison of the proxiome of SCI1-TurboID in the presence or absence of the assembled SmSTRIPAK complex (Δpro11). We generated additional input controls of the crude protein extracts with aliquots taken before biotin affinity purification. Roux, K. J., Kim, D. in, Raida, M., & Burke, B. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. The Journal of Cell Biology, 196(6), 801–810. Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N., & Ting, A. Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nature Biotechnology, 36(9), 880–887. Hollstein, L. S., Schmitt, K., Valerius, O., Stahlhut, G., & Pöggeler, S. (2022). Establishment of in vivo proximity labeling with biotin using TurboID in the filamentous fungus Sordaria macrospora. Scientific Reports, 12(1), 17727. Bloemendal, S., Bernhards, Y., Bartho, K., Dettmann, A., Voigt, O., Teichert, I., Seiler, S., Wolters, D. A., Pöggeler, S., & Kück, U. (2012). A homologue of the human STRIPAK complex controls sexual development in fungi. Molecular Microbiology, 84(2), 310–323.

			Action	DRS
	3316_msms.txt	Txt
	3316_proteinGroups.txt	Txt
	3344_RAB-SAM-TwinkleSEQ.fasta	Fasta
	3344_msms.txt	Txt
	3344_proteinGroups.txt	Txt

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Proximity labelling of mitochondria and endosomes upon mtDNA replication stress.

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