Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow

Area of Science:

• Cellular Biology
• Biochemistry
• Biophysics

Background:

• Bimolecular fluorescence complementation (BiFC) is a powerful technique for visualizing protein-protein interactions (PPIs) in living cells.
• Existing BiFC methods face limitations in simultaneously detecting multiple PPIs due to spectral crosstalk.
• Advancements in fluorescent proteins have enabled deep tissue and super-resolution imaging, but multicolor BiFC remains challenging.

Purpose of the Study:

• To develop novel BiFC assays capable of simultaneously detecting and visualizing multiple PPIs in living cells.
• To overcome spectral crosstalk limitations inherent in traditional multicolor BiFC approaches.
• To expand the fluorescent protein toolbox for advanced BiFC applications.

Main Methods:

• Development of new BiFC assays utilizing large-Stokes-shift fluorescent proteins (LSS-FPs).
• Implementation of multicolor BiFC imaging by combining LSS-FPs with standard fluorescent proteins.
• Demonstration of BiFC rainbow assays for visualizing up to four PPI pairs.
• Integration of LSS-FP BiFC with intensity-based Förster resonance energy transfer (FRET) for ternary complex detection.

Main Results:

• Novel LSS-FP-based BiFC assays were successfully developed for multicolor imaging in living cells.
• The LSS-FP BiFC assays allow for simultaneous visualization of multiple PPIs with reduced spectral crosstalk.
• BiFC rainbow assays enabled the detection of four distinct PPIs within the same cell.
• Combined LSS-FP BiFC and FRET assays effectively detected ternary protein complex formation.

Conclusions:

• LSS-FP-based BiFC assays significantly enhance the capability for multicolor PPI visualization in living cells.
• These new assays overcome spectral crosstalk, facilitating the study of complex protein interaction networks.
• The developed methods provide a robust platform for investigating multiple protein complex dynamics and formation in real-time.