Fluorogenic and genetic targeting of a red-emitting molecular calcium indicator

Bachollet, Sylvestre P. J. T.; Pietrancosta, Nicolas; Mallet, Jean‐Maurice; Dumat, Blaise

doi:10.1039/d2cc01792j

Cited by 4 publications

(3 citation statements)

References 29 publications

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“…We have previously shown that small viscosity-sensitive molecular rotors could be used as fluorogenic HaloTag probes and used as a plaftform to build a dual-input calcium sensor. [25][26][27][28] We have adapted this approach to design a dual-input pH probe by exploring GFP-based chromophore structures that already possess pH-sensitive properties due to their phenol group and are based on a flexible molecular rotor structure that should also be activated by reaction with HaloTag. However, the pKa of the isolated GFP chromophore is too high for the biological pH range of exocytosis events.…”

Section: Resultsmentioning

confidence: 99%

Locally-Activated Chemogenetic pH Probes for Monitoring Protein Exocytosis

Coïs,

Niepon,

Wittwer

et al. 2024

Preprint

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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.

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Section: Resultsmentioning

confidence: 99%

Locally-Activated Chemogenetic pH Probes for Monitoring Protein Exocytosis

Coïs,

Niepon,

Wittwer

et al. 2024

Preprint

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“…reported the sensor Ca‐DIP, based on a BAPTA‐functionalized fluorogenic molecular rotor, which also exploits fluorogenicity to gate sensor function (Figure 6d). [74] While the calcium sensitivity of this dye is similar to that of the JF and MaP‐based sensors, the brightness is lower, primarily due to the fluorophore scaffold.…”

Section: Functional Fluorophore Ligandsmentioning

confidence: 90%

HaloTag‐Based Reporters for Fluorescence Imaging and Biosensing

2023

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Visualizing the structure and dynamics of biomolecules is critical to understand biological function, and requires methods to fluorescently label targets of interest in their cellular context. Self‐labelling proteins, which combine a genetically encoded tag with a small‐molecule fluorophore, have attracted considerable attention for this purpose, as they can overcome limitations of fluorescent proteins. Among them, the HaloTag protein is the most broadly used, showing fast specific labelling with a small, easy to functionalize and cell‐permeant ligand. Synthetic chemistry and protein engineering have provided a portfolio of powerful imaging tools exploiting HaloTag, along with general methods to optimize and adapt them to specific applications. Here, we provide an overview of fluorescent reporters based on the HaloTag protein for imaging and biosensing, highlighting engineering strategies and general applications.

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“…Because of these advantages, fluorogenic reactions are ideal reactions for chemical biology applications. Additionally, after the fluorogenic reaction, a very strong fluorescence signal can be obtained; the starting materials do not need to be fluorescent or weakly fluorescent, which is of particular interest for in vivo labeling in bioconjugation, such as labeling and sequencing of nuclear acids − and labeling of proteins, lipids, − glycans, and others . In addition, enzyme-triggered bioorthogonal cleavage fluorogenic reactions have attracted wide attention because of their high biocompatibility, fast reaction rate, and robustness. − These fluorogenic reactions can also be applied in detecting and acting as no-washing probes for fluorescent imaging. , …”

Section: Applications Of Fluorogenic Reactions In Chemical Biologymentioning

confidence: 99%