Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking

Area of Science:

• Cell biology
• Biochemistry
• Microscopy

Background:

• Fluorescent protein pH sensors are vital for studying protein trafficking via endo- and exocytosis.
• Existing probes are ubiquitously expressed, limiting the focus on specific protein pools.
• Selective tracking of protein trafficking is crucial for understanding cellular processes.

Purpose of the Study:

• To develop novel, activatable, cell-excluded pH sensors for selective protein labeling.
• To enable real-time analysis of endocytosis and recycling using advanced microscopy.
• To investigate the pharmacological differences in β2-adrenergic receptor (B2AR) trafficking.

Main Methods:

• Development of excitation ratiometric, pH-responsive tandem dyes (Cy3 donor, malachite green acceptor).
• Genetic expression of fluorogen-activating protein for targeted dye activation.
• Application in live-cell confocal and superresolution microscopy for selective surface protein labeling.
• Single-vesicle level quantitative profiling of endocytosis and recycling.

Main Results:

• The developed dyes allow for targeted and activated labeling of specific surface proteins.
• Selective analysis of endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) was achieved.
• Differences in B2AR agonist effects on trafficking were quantitatively profiled at a single-vesicle level.

Conclusions:

• The novel cell-excluded pH sensors facilitate precise tracking of protein trafficking in live cells.
• This technology enhances the study of endocytosis and recycling with improved specificity.
• The findings provide a new tool for detailed pharmacological profiling of receptor dynamics.