Multicolor FRET-FLIM Microscopy to Analyze Multiprotein Interactions in Live Cells.

The need to describe and understand signaling pathways in live cell is seen as a primary route to identifying and developing targeted medicines. Signaling cascade is also seen as a complex communication and involves interactions between multiple interconnecting proteins. Where subcellularly and how different proteins interact need to be preserved during investigation. Furthermore, these complex events occurring simultaneously may lead to a single or multiple end point or cell function such as protein synthesis, cell cytoskeleton formation, DNA damage repair, or autophagy. There is therefore a need of real-time noninvasive methods for protein assays to enable direct visualization of the interactions in their natural environment and hence overcome the limitations of methods that rely on invasive cell disruption techniques. Förster resonance energy transfer (FRET) coupled with fluorescence lifetime imaging microscopy (FLIM) is an advanced imaging method to observe protein-protein interactions at nanometer scale inside single living cells in real-time. Here we describe the development and use of two-channel pulsed interleave excitation (PIE) for multiple protein interactions in the mTORC1 pathway. The proteins were first tagged with multiple color fluorescent protein derivatives. The FRET-FLIM combination means that the information gained from using standard steady-state FRET between interacting proteins is considerably improved by monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the presence of the acceptor is evidence for a direct physical interaction.