Fig 1. The fluorogen-activating protein (FAP) biosensor system.

CXCR4 is expressed as a fusion protein with α-FAP and US27 is expressed as a fusion protein with β-FAP, which each bind with high affinity to distinct membrane impermeable fluorogen dyes. When extracellular fluorogen is not bound to the FAP, neither component exhibits fluorescence, and intracellularly localized FAP-tagged receptors are not exposed to membrane impermeable fluorogens. Upon addition of fluorogens to cell expressing FAP-tagged receptors, binding occurs rapidly and fluorescence can be measured using flow cytometry or fluorescence microscopy. Endocytosed receptors retain the bound fluorogen labels they acquired while present on the cell surface, allowing tracking of receptors from the surface to the cell’s interior.