A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range
The real-time monitoring of the intracellular pH in live cells with high precision represents an important methodological challenge. Most of the genetically encoded fluorescent indicators are hindered by the inability to determine an absolute pH value and/or a narrow dynamic range of the signal. The team of scientists from the Laboratory of genetically encoded molecular tools IBCh RAS studied 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, authors proposed the mitochondria, which are known to have a weakly alkaline milieu, as a target for live-cell pH measurements. Using FLIM they 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. Thus the scientists showed that a single GFP with alkaline-shifted pKa can act as a high-precision indicator that can be used in a specific pH range. The work is published in the International Journal of Molecular Sciences. Learn more
november 17, 2022