Gap-Junction-Dependent Labeling of Nascent Proteins in Multicellular Networks

Intercellular communication via gap junctions is crucial for orchestrating behaviors of multicellular systems. Imaging methods and electrophysiological techniques have been widely used to identify gap junctions and map the gap-junction-connected cell networks. However, analyzing gene expression within a gap-junction network remains challenging. Herein, we report the development of bio-orthogonal recording of translation in adjacent cells connected by gap junctions (BORTAC-GJ), a gap-junction-dependent protein tagging method based on local activation of clickable amino acid analogues that pass through gap junctions and are metabolically incorporated into nascent proteins. We demonstrated that BORTAC-GJ enabled selective labeling of nascent proteomes, thus recording translation, in cell networks connected by gap junctions, leaving unconnected cells not labeled. We further applied BORTAC-GJ to probe bystander STING activation triggered by gap-junction-mediated cGAMP transfer, an important process in innate immune response. BORTAC-GJ provides a means to investigate the gap-junction network at the proteome level and is broadly applicable for various cell types connected by gap junctions.