pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Imaging

Background:

• Subcellular pH gradients are critical for cellular processes like macromolecule degradation and secretion.
• Existing pH probes often lack the sensitivity or specificity required for precise measurements in acidic organelles.

Purpose of the Study:

• To engineer a novel, genetically encoded ratiometric pH biosensor for live imaging of acidic cellular compartments.
• To validate the pH-Lemon probe's performance in various cellular locations and conditions.

Main Methods:

• Constructed a ratiometric biosensor (pH-Lemon) by fusing pH-stable mTurquoise2 to pH-sensitive EYFP.
• Utilized Förster resonance energy transfer (FRET) for ratiometric pH sensing.
• Applied fluorescence lifetime imaging microscopy (FLIM) for pH measurements.
• Fused pH-Lemon to LC3B and a GPI anchor for targeting autolysosomes and the secretory pathway, respectively.

Main Results:

• pH-Lemon exhibits ratiometric fluorescence changes (decreased yellow, increased cyan) with decreasing pH due to altered FRET efficiency.
• The probe demonstrated pH-dependent fluorescence lifetime changes, suitable for FLIM.
• Targeting studies successfully localized pH-Lemon within autolysosomes and the secretory pathway.
• Visualized distinct acidic and neutral microenvironments within the endomembrane system.

Conclusions:

• pH-Lemon is a versatile and sensitive ratiometric fluorescent biosensor for studying local pH dynamics in subcellular compartments.
• The probe is suitable for live-cell imaging and offers an alternative method using FLIM.
• pH-Lemon facilitates the investigation of pH homeostasis and its role in cellular functions.