Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions

Afadin and its Drosophila homolog Canoe are key components of the network of proteins that help link cell-cell adherens junctions to the actomyosin cytoskeleton. They stabilize this connection as force is generated to drive cell shape change and migration during embryonic morphogenesis, organogenesis and tissue homeostasis. Some domains or regions have known binding partners, but the interactors for other well conserved domains remain unknown. We sought an unbiased approach to identify proteins within the Afadin protein network, including those that bind directly and those that are proximal. To do so, we made use of BioID-based proximity labeling, in which the promiscuous biotin ligase enzyme (BirA-R118G, referred to as BirA*) is fused to a protein of interest. Following the addition of exogenous biotin to live cells expressing the BirA*:bait fusion, proteins within a ~10-50nM sphere are biotinylated. Biotinylated proximal proteins are then purified with streptavidin for downstream identification and quantification.