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pH Regulation in Cells

pH Regulation in Cells

pH plays a critical role in maintaining normal cellular activities. It helps maintain the structure and function of various proteins, dictates the charge on cellular membranes, and is crucial for metabolic reactions inside the cell. Moreover, cells use the energy from the proton motive force to generate ATP.
Cytosolic pH
Under physiological conditions, the cytosolic pH is slightly more acidic than the extracellular pH. However, cells must prevent further acidification of their cytosol to...

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Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

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A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis.

Justine Coïs^1,2, Marie-Laure Niepon², Manon Wittwer¹

¹Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France.

|August 15, 2024

Summary

We developed a novel chemogenetic pH sensor, pHluo-Halo-1, for precise tracking of pH changes during cellular processes like exocytosis. This wash-free imaging method ensures high signal selectivity, overcoming limitations of traditional fluorescent probes.

Keywords:

HaloTag chemogenetic sensors exocytosis fluorogenic probes pH probes

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Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Published on: August 11, 2017

Area of Science:

Cellular Biology
Biochemistry
Molecular Imaging

Background:

Fluorescent protein biosensors are crucial for tracking pH dynamics in cellular events like exocytosis.
Chemogenetic reporters offer a versatile alternative, combining chemical probe diversity with genetic encoding.
Existing methods face challenges with off-target signals from unbound organic fluorophores.

Purpose of the Study:

To develop a novel fluorogenic and chemogenetic pH sensor with enhanced signal selectivity for live-cell imaging.
To create a wash-free imaging solution for monitoring pH variations during exocytosis.
To establish a versatile alternative to fluorescent proteins for studying protein dynamics.

Main Methods:

Screening of four fluorogenic molecular rotor structures based on protein chromophore analogues.
Selection and characterization of pHluo-Halo-1, a cell-permeable molecular pH indicator.
Application of pHluo-Halo-1 with HaloTag for wash-free imaging of CD63-HaloTag fusion protein secretion via TIRF microscopy.

Main Results:

pHluo-Halo-1 exhibits good pH sensitivity (pKa of 6.3), suitable for monitoring exocytosis-related pH changes.
The sensor demonstrates excellent labeling selectivity in cells through local fluorescence activation via HaloTag reaction.
Successful application in tracking CD63-HaloTag secretion dynamics using TIRF microscopy.

Conclusions:

The pHluo-Halo-1 sensor provides a highly selective, wash-free method for studying pH dynamics in live cells.
This chemogenetic strategy offers a powerful alternative to fluorescent proteins for elucidating protein trafficking and regulatory mechanisms.
The combination of tunable fluorogenic probes and protein tags opens new avenues for advanced cellular imaging.