In vivo photo-crosslinked protein assemblies in C.elegans

The Caenorhabditis elegans worm, a multicellular eukaryotic organism, offers a unique opportunity for in-depth investigations into molecular mechanisms governing DNA repair, replication, and metabolic or rare genetic disorders like the Bloom Syndrome (BS). The transparency of C. elegans facilitates the study of cell differentiation and other developmental processes within the intact organism. Additionally, the gonads of C. elegans serve as a valuable model for exploring fundamental biological processes, including chromosome segregation, DNA repair, morphogenesis, cell cycle control, and programmed cell death. While single-cell molecular genetics and cell biology have provided an extraordinary depth of analysis for this system, the molecular machinery, comprised of protein complexes, within the gonads remains relatively unexplored. Protein complexes and protein- protein interactions play a crucial role in understanding dynamic processes of cell division and DNA repair, but stabilizing and studying them within the native living worm has proven challenging. Over the past two decades, researchers have developed and refined advanced bioengineering strategies to introduce non-natural amino acids (unAAs) into various organisms, including C. elegans. These specialized amino acids can be administered to the worm and incorporated into cellular proteins. Consequently, proteins can be directly labeled within the living worm. Through UV irradiation, these specific amino acids can be activated, enabling them to interact with complex partners and direct interactors. This interaction remains preserved during sample preparations, allowing the analysis of proteins within their natural environment, whether in specific cell types, cell cycle phases, or subcellular regions. In the context of this research project, biochemical methods such as pull-down assays, protein labeling, and mass spectrometry are modified and further developed using this innovative method of stabilizing protein complexes and their interactors. This modification aims to investigate protein complexes that are challenging to access or only occur under specific cellular conditions. One such complex is the BTR complex, whose study, interaction partners, and function are of particular significance. Dysfunctions and mutations within this complex can lead to Bloom Syndrome in humans. Insights into the dynamics of BTR complex formation during meiosis may lead to innovative therapeutic approaches and a better understanding of the disease. This novel approach offers the possibility of systematically generating and stabilizing protein-protein interaction networks within germ cells and beyond in a living organism. Moreover, this project will incorporate artificial intelligence into data analysis to validate newly acquired insights and create 3D models of protein complexes.

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