Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

Abstract

Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer . This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.Many cellular processes are governed by multi-component molecular machineries that rely on dynamic and highly coordinated protein-protein interactions. For example, assembly of protein complexes during signal transduction processes increases the speed of enzymatic reactions, ensures the specificity of signaling and targets signaling molecules to proper intracellular compartments. During recent years, fluorescence resonance energy transfer (FRET) has become a key method for the analysis of proteinprotein interactions during signal transduction in living cells [1][2][3] . FRET studies using proteins tagged with mutant derivatives of the green fluorescent protein (GFP) have demonstrated the formation of complexes of signaling proteins in various intracellular compartments 4-7 . FRET-based genetically encoded biosensors for second messengers, protein phosphorylation and activity of small GTPases have provided insights into the spatial and temporal regulation of signaling processes [8][9][10][11][12] .The combination of enhanced cyan (CFP) and yellow (YFP) fluorescent proteins has proven to be most effective in many FRET studies. Cloning of Anthozoa fluorescent proteins, such as red DsRed 13,14 and far-red HcRed 15 , has expanded the in vivo applications of FRET, but a major limitation of Anthozoa proteins for FRET applications is their obligate oligomerization 16 . Recent generation of a monomerized mutant of DsRed, monomeric red fluorescent protein 1 (mRFP) 17 , provides the opportunity to generate functional monomeric fusion proteins and to use mRFP as a FRET acceptor with proteins fused to GFP or its mutants.Various methods of FRET measurements have been used to visualize protein-protein interactions 18 . The general limitation of these methods is that they only permit the analysis of interactions between two proteins. To analyze multi-component signaling complexes, a method to measure interactions betwe...

Visualization of AP-1–NF-κB ternary complexes in living cells by using a BiFC-based FRET

“…The use of BiFC allows reconstitution of a fluorescent protein that can be used as a donor or an acceptor. This design would avoid the use of three chromophores for the visualization of ternary complexes (17). Because Cerulean and Venus are a better combination for conventional FRET analysis (7-11) and because Venus-based BiFC allows visualization of protein interactions under physiological conditions and shows higher BiFC efficiency than the YFP-based BiFC system (14), we chose Cerulean and Venus as a donor and an acceptor, respectively, and we used a well characterized Fos-Jun-NFAT system as a model to develop the BiFC-FRET assay.…”

“…Contrary to the availability of different fluorescent proteins for FRET-or BiFC-based analysis of individual protein-protein interactions, an easy-to-operate assay for the visualization of ternary complexes in living cells is still lacking. Recently, a threechromophore FRET system has been developed for visualization of ternary complexes (17). Because of the requirement of multiple filters and a complex data process, its application remains very limited.…”

“…Unfortunately, demonstration of a ternary complex in cells is a challenging task, and the methods available for visualization and identification of ternary complexes in living cells are extremely limited. Recently, a threechromophore-based FRET system has been developed for visualization of ternary complexes in cells (17). However, this assay has not been widely used because of its sophisticated optical setup and data process.…”

Enhanced Ubiquitylation and Accelerated Degradation of the Dopamine Transporter Mediated by Protein Kinase C

“…The method of sensitized FRET measurements used to examine FRET between CFP and YFP has been described previously (37,38). Briefly, images through YFP, CFP, and FRET filter channels were acquired using a Mariannas TM fluorescence imaging work station consisting of a Zeiss inverted microscope equipped with a cooled charged couple device CoolSnap HQ (Roper, CA), dual filter wheels, and a Xenon 175-watt light source, all controlled by SlideBook software.…”

“…Corrected FRET images were presented in a quantitative pseudocolor. The apparent FRET efficiency (E d ) was calculated as described previously (38). All calculations were performed using the FRET statistic module of SlideBook 4.0 or 4.1.…”