Four-color single-molecule imaging system for tracking GPCR dynamics with fluorescent HiBiT peptide

Single-molecule imaging provides information on diffusion dynamics, oligomerization, and protein-protein interactions in living cells. To simultaneously monitor different types of proteins at the single-molecule level, orthogonal fluorescent labeling methods with different photostable dyes are required. G-protein-coupled receptors (GPCRs), a major class of drug targets, are prototypical membrane receptors that have been studied using single-molecule imaging techniques. Here we developed a method for labeling cell-surface GPCRs inspired by the HiBiT system, which utilizes the high affinity complementation between LgBiT and HiBiT fragments of the NanoLuc luciferase. We synthesized four fluorescence-labeled HiBiT peptides (F-FiBiTs) with a different color dye (Setau-488, TMR, SaraFluor 650 and SaraFluor 720). We constructed a multicolor total internal reflection fluorescence microscopy system that allows us to track four color dyes simultaneously. As a proof-of-concept experiment, we labeled an N-terminally LgBiT-fused GPCR (Lg-GPCR) with a mixture of the four F-FiBiTs and successfully tracked each dye within a cell at the single-molecule level. The F-FiBiT-labeled Lg-GPCRs showed agonist-dependent changes in the diffusion dynamics and accumulation into the clathrin-coated pits as observed with a conventional method using a C-terminally HaloTag-fused GPCR. Taking advantage of luciferase complementation by the F-FiBiT and Lg-GPCRs, the F-FiBiT was also applicable to bioluminescence plate-reader-based assays. By combining existing labeling methods such as HaloTag, SNAP-tag, and fluorescent proteins, the F-FiBiT method will be useful for multicolor single-molecule imaging and will enhance our understanding of GPCR signaling at the single-molecule level.