A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis - Processus d'activation sélective par transfert d'énergie uni-électronique ou radiatif
Article Dans Une Revue ACS Sensors Année : 2024

A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis

Résumé

Fluorescent protein-based pH biosensors enable the tracking of pH changes during protein trafficking and in particular exocytosis. The recent development of chemogenetic reporters combining synthetic fluorophores with self-labeling protein tags offers a versatile alternative to fluorescent proteins that combines the diversity of chemical probes and indicators with the selectivity of the genetic-encoding. However this hybrid protein labeling strategy does not avoid common drawbacks of organic fluorophores such as the risk of off-target signal due to unbound molecules. Here, we describe a novel fluorogenic and chemogenetic pH sensor based on a cell-permeable molecular pH indicator called pHluo-Halo-1 whose fluorescence can be locally activated in cells by reaction with HaloTag ensuring excellent signal selectivity in wash-free imaging experiments. pHluo-Halo-1 was selected out of a series of four fluorogenic molecular rotor structures based on protein chromophore analogs. It displays a good pH sensitivity with a pKa of 6.3 well-suited to monitor pH variations during exocytosis and an excellent labeling selectivity in cells. It was applied to follow the secretion of CD63-HaloTag fusion proteins using TIRF microscopy. We anticipate that this strategy based on the combination of a tunable and chemically-accessible fluorogenic probe with a well-established protein tag will open new possibilities for the development of versatile alternatives to fluorescent proteins for elucidating the dynamics and regulatory mechanisms of proteins in living cells.
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hal-04675333 , version 1 (22-08-2024)

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Justine Coïs, Marie-Laure Niepon, Manon Wittwer, Hessam Sepasi Tehrani, Philippe Bun, et al.. A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis. ACS Sensors, inPress, ⟨10.1021/acssensors.4c01057⟩. ⟨hal-04675333⟩
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