Justine Coïs, Marie-Laure Niepon, Manon Wittwer, Hessam Sepasi Tehrani, Philippe Bun, Jean-Maurice Mallet, Vincent Vialou, Blaise Dumat
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引用次数: 0
Abstract
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 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.
期刊介绍:
ACS Sensors is a peer-reviewed research journal that focuses on the dissemination of new and original knowledge in the field of sensor science, particularly those that selectively sense chemical or biological species or processes. The journal covers a broad range of topics, including but not limited to biosensors, chemical sensors, gas sensors, intracellular sensors, single molecule sensors, cell chips, and microfluidic devices. It aims to publish articles that address conceptual advances in sensing technology applicable to various types of analytes or application papers that report on the use of existing sensing concepts in new ways or for new analytes.