Improving FRET dynamic range with bright green and red fluorescent proteins

Abstract

A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of cyan and yellow fluorescent proteins (CFP and YFP), but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light, and complex photokinetic events such as reversible photobleaching and photoconversion. Here, we engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date, and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP in reporters of kinase activity, small GTPase activity, and transmembrane voltage significantly improves photostability, FRET dynamic range, and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action potential firing and RhoA activation in growth cones.

Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin-F-actin based oscillatory cell-cell interaction

“…The EGFP sequence of pT24xnrUAS:EGFP-UCHD was replaced by the sequence of mRuby2 (amplified from pcDNA3-mRuby2 was a gift from Michael Lin; Addgene plasmid #40260) 38 or by the sequence of mClav2 (amplified from pmClavGR2-NT; Allele Biotechnology) to generate the final plasmids pT24xnrUAS:mRuby2-UCHD and pT24xnrUAS:mClav2-UCHD respectively. These final plasmids were injected individually, together with tol2 RNA into Tg( fli1:Gal4ff ) ubs3 embryos and that were raised to adulthood and eventually stable transgenic fish lines Tg(UAS:mRuby2-UCHD) ubs20 and Tg(UAS:mClav2-UCHD) ubs27 were isolated and maintained.…”

Engineering Approaches to Illuminating Brain Structure and Dynamics

“…The latest generation of genetically encoded voltage indicators can now reliably report action potentials in cultured neurons and appear to be on the brink of gaining practical utility in mammalian brain tissue slices (Cao et al, 2013; Gong et al, 2013; Jin et al, 2012; Kralj et al, 2012; Lam et al, 2012). Ideally, improved voltage indicators should dovetail with concurrent advances in targeting proteins to particular cell types or subcellular compartments and would reveal neuronal spiking with millisecond-scale timing resolution, dendritic voltage dynamics, subthreshold inhibition and excitation, and high-frequency oscillations.…”

Fluorescent Protein-based Biosensors to Visualize Signal Transduction beneath the Plasma Membrane

“…With respect to several FRET-based biosensors, including that for RhoA (Raichu-RhoA), 29 that for PKA (AKAR2), 54 that for membrane potential (VSFP), 55 and that for CaMKIIα (Camuiα), 56 their FRET dynamic ranges and emission ratio changes have been improved up to 1.5-fold by substitution for the donor and acceptor fluorescent proteins. 57 One of the disadvantages of intramolecular FRET biosensors is that it takes substantial effort to optimize them for maximum sensitivity. To overcome this issue, Matsuda and his colleagues established a system for an optimized backbone consisting of a long and flexible peptide linker (EV linker) and an optimized pair of fluorescent proteins.…”