Fluorogen-activating proteins: beyond classical fluorescent proteins

Xu, Shengnan; Hu, Hai-Yu

FAP-based fluoromodules have been successfully and widely utilized in selectively visualization of protein location, internalization and trafficking in mammalian and yeast cells since 2008. As discussed above, FAP technology has multiple advantages in biological imaging: 1) experimental flexibility-fluorescence is generated only upon addition of the fluorogen; 2) fast responses -FAPs can be visualized few seconds or minutes after addition of the fluorogen; 3) high spatial labeling discrimination-by playing with the cell-permeability of the fluorogen, fusion proteins in a given cell location can be selectively observed. For instance, comprehensive studies of plasma membrane G-protein coupled receptors (GPCRs), especially for β2-adrenergic receptor (β2-AR), expression, location, trafficking and quantification, as well as other membrane proteins, have been investigated by genetically encoding FAP tags in targeting molecules and imaging with modified TO or MG fluorogens 29,39-42 . Meanwhile, FAP-fluorogen technology was shown to be also suitable for labeling intracellular/cytosolic targets with the evolution of fluorogens. Based on FAPs and membrane permeable fluorogen MG-ester, Bruchez et al. 25 presented a new labeling technology for cytoplasmic compartments, which is nowash, far-red, highly fluorogenic, photostable, and nonphototoxic and functions in all organelles. Later, a two-color, Green-Inside Red-Outside (GIRO) (Fig. 9) , compartment selective FAP-based approach that generates distinct signals from surface and internal proteins in live cells for simultaneous detection is also demonstrated by Bruchez's group 28 . Lately, the same group established a threecolor labeling approach, allowing excitation-dependent visualization of extracellular, intracellular, and total protein pools in the same cells by using one fluorogenic tag that combines with distinct dyes to affect different spectral characteristics 34 .