Acid-brightening fluorescent protein (abFP) for imaging acidic vesicles and organelles

Abstract Acidic organelles and vesicles, such as endosomes, lysosomes, autophagosomes, trans-Golgi network, and synaptic vesicles, are known to play important roles in a broad range of cellular events. To facilitate studying these multifunctional systems, we describe here an acid-brightening fluorescent protein (abFP), which fluoresces strongly at acidic pH, but is almost nonfluorescent at or above physiological pH, making it well suited for imaging molecules residing in acidic microenvironment in live cells. Specifically, a quinoline-containing unnatural amino acid Qui is incorporated into the chromophore of EGFP via genetic code expansion to generate the abFP. When being exposed to acidic environment, protonation of Qui results in a cationic chromophore and fluorescence increase. Protocols are presented to express abFP in E. coli and mammalian cells, and to fluorescently image the endocytosis of δ opioid receptor-abFP fusion protein in mammalian cells. This strategy may be similarly applicable to other fluorescent proteins to enable acidic imaging.