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

Fluorescent protein based pH sensors are a useful tool for measuring protein trafficking through pH changes associated with endo-and exocytosis. However, commonly used pH sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering the ability to focus on specific trafficking pools of proteins. We developed a family of excitation-ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen activating protein, and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged {beta}2-adrenergic receptor (B2AR) at a single vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.