A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis.

Autor: Coïs J; Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France.; Laboratoire Neurosciences Paris Seine, Sorbonne Université, CNRS, INSERM, Paris 75005, France., Niepon ML; Laboratoire Neurosciences Paris Seine, Sorbonne Université, CNRS, INSERM, Paris 75005, France., Wittwer M; Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France., Sepasi Tehrani H; PASTEUR, Département de chimie, École normale supérieure, CNRS, PSL University, Sorbonne Université, Paris 75005, France., Bun P; Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, NeurImag Imaging Core Facility, Université Paris Cité, Paris 75014, France., Mallet JM; Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France., Vialou V; Laboratoire Neurosciences Paris Seine, Sorbonne Université, CNRS, INSERM, Paris 75005, France., Dumat B; Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France.
Jazyk: angličtina
Zdroj: ACS sensors [ACS Sens] 2024 Sep 27; Vol. 9 (9), pp. 4690-4700. Date of Electronic Publication: 2024 Aug 15.
DOI: 10.1021/acssensors.4c01057
Abstrakt: 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 analogues. It displays good pH sensitivity with a p K a 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.
Databáze: MEDLINE
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