Proximity‐dependent sensors for signaling

Abstract

Understanding dynamic subcellular organization in living cells is key to unravelling mechanisms of intracellular signaling that goes awry in disease. Using proximity‐dependent biotinylation (BioID), we first established a reference map for the human cell at steady state (Go et al., Nature, 2021; humancellmap.org) that serves to identify protein baits that can report on the recruitment of proteins to selected organelles or subcellular structures. These BioID “sensors” can then be applied to look at dynamic changes in signaling. For example, exploiting late endosome/lysosome BioID sensors (VAMP7 and VAMP8), we recently revealed new intricacies in the regulation of amino acid sensing pathways (Hesketh et al., Science, 2020). We also coupled BioID sensor profiling to CRISPR‐mediated ablation of pathway components to investigate the consequences on the environment detected by the sensor. Using these approaches, we recently used fast‐acting biotinylation enzymes, including miniTurbo, to provide a space and time‐resolved analysis of EGFR pathway activation, revealing previously uncharacterized associations. This presentation will revisit key principles of dynamic organelle mapping in living cells, and the utilization of coincidence detection for dynamic proximal interactomes.