PhD Scientific Days 2026

Molecular Medicine 3.

Flipping the Switch: An Inducible TurboID System for Mapping Interactome in C. elegans

Name of the presenter

Khatatneh, Saba

Institute/workplace of the presenter

Semmelweis University, Institute of Biochemistry and Molecular Biology, Department of Molecular Biology

Authors

Saba Khatatneh¹, Milán Somogyvári¹, Csaba Sőti¹
1: Semmelweis University, Institute of Biochemistry and Molecular Biology, Department of Molecular Biology

Text of the abstract

Introduction: Mapping protein-protein interactions in vivo with temporal control remains a major challenge. Proximity labelling with TurboID offers rapid biotinylation of neighbouring proteins, but current implementations in C. elegans rely on constitutive overexpression, introducing developmental artefacts and masking dynamic interactions. A controllable system that activates labelling only at a chosen time point would enable cleaner, temporally resolved interactome studies.

Aims: To develop the first doxycycline-inducible TurboID proximity-labelling system in C. elegans and validate it using HSP-90, a conserved molecular chaperone with a wide range of interactors involved in all types of cellular processes, making it a great model as a bait protein. Besides, this paradigm gives us the chance to fill gaps in HSP90 function in the proliferating and terminally differentiated tissues of C. elegans.

Methods: A bipartite system was engineered: a constitutive pDriver (Pprl-28::rtTA(Q)/tTS) and an inducible pResponder (TRE::HSP-90::GFP::TurboID), integrated via biolistic transformation. A homozygous double-transgenic strain was established through crossing and multi-generational selection. Induction, expression kinetics, and biotinylation efficiency were characterised by fluorescence microscopy, qPCR and Western blotting. The HSP-90 inhibitor 17-DMAG is being evaluated as a pharmacological negative control using the heat shock response marker hsp-70 qPCR as a preliminary readout.

Results: The system showed tight regulation: no detectable expression in the absence of doxycycline and robust induction peaking at 24 hours post-treatment with 2 ng/µL doxycycline. Biotin supplementation (1 mM, 2 h) significantly enhanced labelling yield. Uninduced animals showed no increase in biotinylation even with exogenous biotin, confirming the absence of leak. Mass spectrometry-based interactome analysis is ongoing.

Conclusion: We established a tightly controlled, inducible proximity-labelling platform in C. elegans. By eliminating overexpression artefacts and enabling temporal control, this method is applicable to any bait protein and compatible with tissue-specific promoters for targeted interactome studies.

Funding: Stipendium Hungaricum Scholarship Programme, and OTKA PD 142838 research grant.