Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling

Abstract

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A modified TurboID approach identifies tissue-specific centriolar components in C. elegans

“…Centrosomal pellets were then resolubilized under denaturing conditions and used for streptavidin pulldowns followed by LC-MS/MS analysis. As also noted by Artan et al [ 20 ] endogenously biotinylated carboxylases represent a significant proportion of the isolated peptides. However, they, along with other common contaminants including vitellogenins and mitochondrial proteins, are largely filtered out by considering only those proteins significantly enriched in experimental samples over controls (Log2 enrichment >1, p-value <0.05).…”

“…When we set out on this project TurboID had not yet been successfully applied to identify the proximity interactors of specific proteins in C . elegans (two such studies were recently published by the Feldman and de Bono labs, [ 13 , 20 ]). To explore the potential for TurboID to map centrosomal protein-protein interactions we chose two target proteins for our proof of principle experiments, the PCM scaffolding protein SPD-5 and the Polo-like kinase PLK-1.…”

“…Moreover, those two TurboID constructs will usually be expressed as transgenes. While endogenous tagging can be carried out [ 20 ], it is then difficult to generate a corresponding control matching the expression level of the TurboID-protein fusion, important for normalization of the MS data. Expression level is a particular concern when aiming to isolate proximity interactors of a protein in a specific cell type or tissue, since the relevant promoters will not necessarily match the expression level of the endogenous protein.…”

“…Significantly enriched proteins (Log2 enrichment >1, p-value <0.05) are indicated in pink, with selected proximity interactors highlighted. Note that these interactors are present at much lower levels in the sample compared to endogenously biotinylated proteins, most notably the carboxylases PCCA-1, PYC-1, POD-2 and MCCC-1 [ 20 ], highlighting the importance of proper sample normalization. ( E ) TurboID applied to SPD-5 in germ cell precursors.…”

Towards a Comprehensive Optical Connectome at Single Synapse Resolution via Expansion Microscopy

“…A fusion protein of SYD-2 and GFP allowed presynaptic puncta to be labeled in live C. elegans (Yeh et al, 2005 ). Furthermore, synaptic protein-protein interactions can now be probed in worms with Turbo ID, an enzyme-based proximity labeling strategy (Branon et al, 2018 ; Artan et al, 2021 ). TurboID was used to identify protein-protein interactions that a presynaptic protein, ELKS-1, was involved in Artan et al ( 2021 ), representing a blueprint for a potential strategy for synaptic proximity labeling in C. elegans .…”