A Fluorogenic\nChemogenetic pH Sensor for Imaging Protein\nExocytosis

Fluorescent protein-based pH biosensors enable the tracking\nof\npH changes during protein trafficking and, in particular, exocytosis.\nThe recent development of chemogenetic reporters combining synthetic\nfluorophores with self-labeling protein tags offers a versatile alternative\nto fluorescent proteins that combines the diversity of chemical probes\nand indicators with the selectivity of the genetic encoding. However,\nthis hybrid protein labeling strategy does not avoid common drawbacks\nof organic fluorophores such as the risk of off-target signal due\nto unbound molecules. Here, we describe a novel fluorogenic and chemogenetic\npH sensor based on a cell-permeable molecular pH indicator called <b>pHluo-Halo-1</b>, whose fluorescence can be locally activated\nin cells by reaction with HaloTag, ensuring excellent signal selectivity\nin wash-free imaging experiments. <b>pHluo-Halo-1</b> was selected\nout of a series of four fluorogenic molecular rotor structures based\non protein chromophore analogues. It displays good pH sensitivity\nwith a p<i>K</i><sub>a</sub> of 6.3 well-suited to monitor\npH variations during exocytosis and an excellent labeling selectivity\nin cells. It was applied to follow the secretion of CD63-HaloTag fusion\nproteins using TIRF microscopy. We anticipate that this strategy based\non the combination of a tunable and chemically accessible fluorogenic\nprobe with a well-established protein tag will open new possibilities\nfor the development of versatile alternatives to fluorescent proteins\nfor elucidating the dynamics and regulatory mechanisms of proteins\nin living cells.