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A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in

Tatiana R Simonyan1,2, Elena A Protasova2,3, Anastasia V Mamontova1,2

  • 1Center of Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, 121205 Moscow, Russia.

International Journal of Molecular Sciences
|November 11, 2022
PubMed
Summary

This study introduces a modified green-fluorescence-protein (GFP) for precise live-cell pH monitoring. The enhanced GFP allows accurate measurement of mitochondrial pH changes in mammalian cells.

Keywords:
FLIMfluorescence lifetimefluorescent indicatorspHtime-resolved spectroscopy

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Real-time intracellular pH monitoring is crucial but challenging.
  • Existing genetically encoded fluorescent indicators lack precision and dynamic range for organelle pH changes.

Purpose of the Study:

  • To develop a high-precision pH indicator for live-cell imaging.
  • To investigate the pH sensitivity of an alkaline-shifted green-fluorescence-protein (GFP) variant.

Main Methods:

  • Utilized a modified EGFP (EGFP-Y145L/S205V) with an alkaline-shifted pKa.
  • Employed fluorescence lifetime imaging microscopy (FLIM) for live-cell imaging.
  • Targeted mitochondria in HEK293T cells.

Main Results:

  • Demonstrated a quasi-linear fluorescence lifetime change (~3.5-fold) of the EGFP variant in the pH range of 7.5-9.0.
  • Successfully determined absolute mitochondrial pH and recorded pH shifts with 0.1 unit precision.
  • Showcased the potential for precise pH measurements in weakly alkaline cellular compartments.

Conclusions:

  • A single GFP variant with an alkaline-shifted pKa can serve as a high-precision pH indicator within a specific range.
  • FLIM combined with the engineered GFP enables accurate live-cell mitochondrial pH measurements.
  • This method overcomes limitations of previous fluorescent indicators for organelle pH dynamics.