Locally-Activated Chemogenetic pH Probes for Monitoring Protein Exocytosis

Genetically encoded biosensors based on fluorescent proteins are valuable tools for imaging biological processes with high selectivity. In particular, pH-sensitive fluorescent proteins such as the GFP-derived superecliptic pHluorin (SEP) or pHuji allow tracking of protein trafficking through acidic and neutral compartments. Recently, chemogenetic indicators combining synthetic fluorophores with genetically encoded self-labeling protein tags (SLP-tag) offer a versatile alternative that combines the diversity of chemical probes and indicators with the selectivity of the genetic-encoding. Here, we describe a novel fluorogenic and chemogenetic pH sensor consisting of a cell-permeable molecular pH indicator called pHluo-Halo-1 whose fluorescence can be locally activated in cells by reaction with the SLP-tag HaloTag ensuring high signal selectivity in wash-free imaging experiments. pHluo-Halo display a good pH sensitivity and a suitable pKa of 5.9 to monitor biological pH variations. This hybrid chemogenetic pH probe was applied to follow the exocytosis of a CD63-HaloTag fusion proteins enabling the visualization of exosomes released from acidic vesicles into the extracellular media using TIRF microscopy. This chemogenetic platform is expected to be a powerful and versatile tool for elucidating the dynamics and regulatory mechanisms of proteins in living cells.