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

Area of Science:

• Biophysics
• Cell Biology
• Molecular Imaging

Background:

• Single-molecule imaging is crucial for understanding protein dynamics and interactions within living cells.
• Simultaneous monitoring of diverse proteins requires orthogonal labeling strategies with photostable dyes.
• G-protein-coupled receptors (GPCRs), key drug targets, are often studied using single-molecule techniques.

Purpose of the Study:

• To develop a novel multicolor single-molecule imaging method for labeling and tracking cell-surface GPCRs.
• To create fluorescence-labeled HiBiT peptides (F-FiBiTs) for orthogonal labeling.
• To demonstrate the utility of F-FiBiTs in multicolor microscopy and bioluminescence assays.

Main Methods:

• Synthesized four F-FiBiTs, each conjugated with a different photostable dye (Setau-488, TMR, SaraFluor 650, SaraFluor 720).
• Developed a multicolor total internal reflection fluorescence microscopy system for simultaneous four-color imaging.
• Labeled N-terminally LgBiT-fused GPCRs (Lg-GPCRs) with F-FiBiTs for single-molecule tracking in cells.

Main Results:

• Successfully tracked individual F-FiBiT-labeled Lg-GPCRs in living cells at the single-molecule level using multicolor microscopy.
• Observed agonist-dependent changes in GPCR diffusion dynamics and accumulation into clathrin-coated pits.
• Demonstrated the applicability of F-FiBiTs in bioluminescence plate-reader assays via luciferase complementation.

Conclusions:

• The F-FiBiT method provides a versatile tool for multicolor single-molecule imaging of GPCRs.
• This technique enhances the understanding of GPCR signaling pathways at the molecular level.
• F-FiBiTs can be combined with existing labeling methods for advanced cellular studies.