FETCH enables fluorescent labeling of membrane proteins in vivo with spatiotemporal control in <i>Drosophila</i>

Fluorescent labeling approaches are crucial for elucidating protein function and dynamics. While robust methods to monitor gene transcription are widespread, the visualization of proteins in vivo is more elusive. To meet this challenge, we developed Fluorescent Endogenous Tagging with a Covalent Hook (FETCH) to label cell surface proteins (CSPs) in vivo through a stable covalent bond mediated by the DogTag-DogCatcher peptide partner system. FETCH leverages a spontaneous covalent isopeptide bond that forms between the 23-amino acid DogTag and the 15-kDa DogCatcher. Unlike most tags that work best at protein termini, DogTag functions well in protein loops, expanding the range of sites that can be targeted in proteins. In FETCH, DogTag is introduced into extracellular loops of CSPs through genome engineering, enabling covalent bond formation with a genetically encoded DogCatcher-GFP fusion protein that can be secreted from a variety of cell types in intact animals. To identify optimal DogTag insertions into CSPs, we describe a flow cytometry-based platform for rapidly screening candidates in vitro. We demonstrate the ability to tag and visualize three members of the immunoglobulin superfamily (IgSF) in vivo: the transmembrane protein mCD8 and two GPI-anchored proteins belonging to the DIP-Dpr interactome that interact biophysically to facilitate neuronal target recognition at <i>Drosophila</i> neuromuscular and brain synapses. FETCH enables precise temporal and spatial control to visualize tagged proteins in vivo, features that are adaptable to a multitude of applications for modifying any cell surface protein.