Fluorescent protein with environmentally-sensitive fluorescence lifetime for quantitative pH measurement
Tatiana R Simonyan1, Elena A Protasova2, Anastasia V Mamontova1
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, 117997, Russia.
Archives of Biochemistry and Biophysics
|February 19, 2025
Summary
Researchers developed a new pH indicator using yellow fluorescent protein EYFP-G65T for live-cell imaging. This tool accurately measures pH in organelles and tracks shifts in response to stimuli.
Area of Science:
- Cell Biology
- Biochemistry
- Biophysics
Background:
- Intracellular pH is crucial for cellular homeostasis and biochemical pathways.
- Monitoring organelle pH is essential for understanding cell function.
- Existing pH indicators have limitations in live-cell imaging.
Purpose of the Study:
- To develop and validate a time-resolved pH indicator based on yellow fluorescent protein (EYFP-G65T).
- To evaluate different EYFP-G65T constructs for live-cell pH imaging.
- To measure and map intracellular pH dynamics in organelles.
Main Methods:
- Constructing and testing EYFP-G65T variants (unmodified, circular permutant, chimeric).
- Utilizing two-photon excitation for fluorescence lifetime measurements.
- Performing live-cell imaging to assess indicator performance in organelles.
- Stimulating cells to observe dynamic pH changes.
Main Results:
- The unmodified EYFP-G65T demonstrated superior performance for live-cell imaging.
- Purified EYFP-G65T showed significant changes in fluorescence lifetime across physiological pH ranges (pH 5.5-8).
- The indicator successfully measured pH (6-8) in various organelles and mapped dynamic pH shifts in mitochondria and Golgi apparatus.
Conclusions:
- Unmodified EYFP-G65T is a robust and effective core for time-resolved pH indicators.
- This novel indicator enables precise measurement of intracellular pH dynamics.
- The tool facilitates the study of cellular responses to stimuli through pH monitoring.
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