|
ИСТИНА |
Войти в систему Регистрация |
ИСТИНА ИНХС РАН |
||
A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Rangeстатья
Статья опубликована в высокорейтинговом журнале
Статья опубликована в журнале из списка Web of Science и/или Scopus- Авторы: Simonyan Tatiana R., Protasova Elena A., Mamontova Anastasia V., Shakhov Aleksander M., Lukyanov Konstantin A., Maksimov Eugene G., Bogdanov Alexey M.
- Журнал: International Journal of Molecular Sciences
- Том: 23
- Номер: 21
- Год издания: 2022
- Издательство: MDPI
- Местоположение издательства: Basel, Switzerland
- Первая страница: 12907
- DOI: 10.3390/ijms232112907
- Аннотация: 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.
- Добавил в систему: Максимов Евгений Георгиевич