Fluorescent proteins for two-photon FLIM analysis of presynaptic protein interactions

Abstract

Monitoring Förster Resonance Energy Transfer (FRET) between fluorescent proteins (FPs) fused to proteins of interest is a powerful approach to study dynamic interactions of cellular proteins in living cells. The choice of an appropriate FRET pair is still a challenging topic despite a large palette of existing FPs, where fluorescent properties of individual proteins, FRET properties of pairs and conformation of interacting constructs play a role. <br /> We compared FRET performance of 19 green/red and cyan/yellow pairs in identical conditions, FPs were fused via a short and flexible linker SRG4SG4S and tested in primary mouse cortical neurons using a two-photon FRET-FLIM (fluorescence lifetime imaging microscopy) technique. Most FRET donors displayed single exponential fluorescence decays in absence of acceptors. When fused to the acceptors, donor decays became double exponential. FRET efficiency was estimated as the reduction of the donor lifetime in presence of the acceptor. It ranged between 39% and 72% among the tested pairs, reaching the highest values of 39% in case of mNeon-mRFP and mNeon-mCherry2 and 72% in case of mTurquoise2-YPet among green/red and cyan/yellow pairs, respectively. These strong performances were confirmed in native neurons of hippocampal mouse brain slices after viral injections in vivo. <br /> Model-free graphical analysis showed presence of three fractions of molecules: with hi-FRET rate (~2.2 ns^-1), with low-FRET rate (~0.2 ns^-1) predicted by random orientation and non-FRET state consistent with immature and dark acceptors. Fractions of these populations defined the overall FRET efficiency and were primarily determined by the FRET acceptor. YPet had the largest hi-FRET population and the lowest non-interacting fraction. Red acceptors showed the worst performance due to their slow maturation and high dark state fraction. Further structural analysis of crystallized mTurquoise2-YPet protein demonstrated, that the nature of hi-FRET rate derived from close association of donor and acceptor FPs. <br /> Finally, fluorescent properties and FRET efficiencies were proved to be unaffected after targeting of the FPs to the presynaptic compartment by their fusion with vesicular proteins Synaptophysin1 and Synaptobrevin2. Interaction between these presynaptic proteins was detected in primary neurons. <br /> Overall, this work demonstrates and explains an outstanding FRET performance of mTurquoise2 / YPet FPs among the tested pairs and their applicability for the protein-protein interaction (PPI) studies using the two-photon FRET-FLIM approach in neurons. Our findings form the basis for further PPIs researches using the new pair with high FRET efficiency, as well as for the development and evolution of FRET-based sensors and reporters with high FRET efficiencies and large dynamic ranges.