Imaging pH Dynamics Simultaneously in Two Cellular Compartments Using a Ratiometric pH-Sensitive Mutant of mCherry

Area of Science:

• Cell Biology
• Biochemistry
• Physiology

Background:

• Organelle pH regulation is crucial for cellular functions, impacting signaling and metabolism.
• Mitochondrial energy production relies on pH gradients between the matrix and cytosol.
• Current pH sensors lack spectral compatibility for simultaneous multi-compartment imaging.

Purpose of the Study:

• To develop a novel ratiometric red fluorescent protein pH sensor for multicolor imaging of organelle pH dynamics.
• To enable quantitative, live-cell analysis of spatially resolved pH changes.
• To investigate interorganelle pH coupling and regulation at organelle boundaries.

Main Methods:

• Engineering a mutant of the red fluorescent protein mCherry (I158E/Q160A) as a ratiometric pH sensor.
• Characterizing the sensor's pKa and signal change.
• Utilizing the mCherryEA mutant with a green fluorescent sensor (ratiometric-pHluorin) for dual-compartment imaging in cultured neurons and neuroblastoma cells.

Main Results:

• The mCherryEA mutant functions as an effective ratiometric pH sensor with a pKa of 7.3 and a >3-fold ratio signal change.
• Demonstrated activity and metabolism-dependent pH dynamics in cultured primary neurons and neuroblastoma cells.
• Successfully performed simultaneous imaging of pH changes in the cytosol and mitochondria.

Conclusions:

• The developed mCherryEA sensor facilitates the study of interorganelle pH dynamics in live cells.
• This new tool broadens the applicability of pH sensors in understanding cellular metabolism and signaling.
• Quantitative, multicolor imaging of pH dynamics is now feasible for studying organelle function.