A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range
| Autor: | Tatiana R. Simonyan, Elena A. Protasova, Anastasia V. Mamontova, Aleksander M. Shakhov, Konstantin A. Lukyanov, Eugene G. Maksimov, Alexey M. Bogdanov |
|---|---|
| Rok vydání: | 2022 |
| Předmět: |
Mammals
Organic Chemistry Green Fluorescent Proteins General Medicine Hydrogen-Ion Concentration Catalysis Fluorescence Computer Science Applications Inorganic Chemistry fluorescent indicators time-resolved spectroscopy fluorescence lifetime FLIM pH HEK293 Cells Microscopy Fluorescence Animals Humans Physical and Theoretical Chemistry Coloring Agents Molecular Biology Spectroscopy Fluorescent Dyes |
| Zdroj: | International Journal of Molecular Sciences; Volume 23; Issue 21; Pages: 12907 |
| ISSN: | 1422-0067 |
| Popis: | The real-time monitoring of the intracellular pH in live cells with high precision represents an important methodological challenge. Although genetically encoded fluorescent indicators can be considered as a probe of choice for such measurements, they are hindered mostly by the inability to determine an absolute pH value and/or a narrow dynamic range of the signal, making them inefficient for recording the small pH changes that typically occur within cellular organelles. Here, we study the pH sensitivity of a green-fluorescence-protein (GFP)-based emitter (EGFP-Y145L/S205V) with the alkaline-shifted chromophore’s pKa and demonstrate that, in the pH range of 7.5–9.0, its fluorescence lifetime changes by a factor of ~3.5 in a quasi-linear manner in mammalian cells. Considering the relatively strong lifetime response in a narrow pH range, we proposed the mitochondria, which are known to have a weakly alkaline milieu, as a target for live-cell pH measurements. Using fluorescence lifetime imaging microscopy (FLIM) to visualize the HEK293T cells expressing mitochondrially targeted EGFP-Y145L/S205V, we succeeded in determining the absolute pH value of the mitochondria and recorded the ETC-uncoupler-stimulated pH shift with a precision of 0.1 unit. We thus show that a single GFP with alkaline-shifted pKa can act as a high-precision indicator that can be used in a specific pH range. |
| Databáze: | OpenAIRE |
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