Locally-Activated Chemogenetic pH Probes for Monitoring Protein Exocytosis

12 April 2024, Version 2
This content is a preprint and has not undergone peer review at the time of posting.


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.


pH probes
Chemogenetic probes
fluorogenic probes

Supplementary materials

Supplementary Information
Materials and methods, synthetic procedures, additional figures and data


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