Genetically Encoded, pH-Sensitive mTFP1 Biosensor for Probing Lysosomal pH.

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Imaging

Background:

• Lysosomes are critical for cellular degradation, maintaining an acidic lumenal pH (around 4.5).
• Understanding lysosomal pH dynamics is crucial for studying cellular health and disease.
• Existing methods for lysosomal pH measurement have limitations.

Purpose of the Study:

• To engineer a novel, genetically encoded fluorescent protein (FP)-based biosensor for accurate lysosomal pH monitoring.
• To validate the biosensor's specificity, robustness, and versatility in cellular models.
• To enable high-content quantitative imaging of lysosomal pH.

Main Methods:

• Designed FIRE-pHLy (Fluorescence Indicator REporting pH in Lysosomes), a lysosome-targeted FP biosensor using mTFP1 and mCherry.
• Targeted the biosensor to lysosomes via lysosomal-associated membrane protein 1 (LAMP1).
• Utilized ratiometric quantification and high-content imaging to measure pH in cellular models.

Main Results:

• FIRE-pHLy accurately reported lysosomal lumenal pH within the 3.5–6.0 range.
• The biosensor demonstrated sensitivity to V-ATPase inhibition (bafilomycin A1), showing an alkalinizing response.
• Successful expression and application in various cellular models were achieved.

Conclusions:

• FIRE-pHLy is a specific, robust, and versatile biosensor for lysosomal pH.
• This tool offers broad applications for investigating pH dynamics in aging and lysosome-related diseases.
• FIRE-pHLy is valuable for lysosome-based drug discovery and understanding disease mechanisms.