The Establishment And Application Of Far-red Bimolecular Fluorescence Complementation System

Protein-protein interactions regulate almost all aspects of cellular biological processes,such as growth,development and apoptosis.The recently developed BiFC (bimolecular fluorescence complementation) assay is a new technique to study protein interactions inside living cells.Compared with FRET(fluorescence resonance energy transfer),the BiFC is relatively simple and straightforward to implement and more sensitive.To date,ten different fluorescent proteins(FPs) that cover a broad range of spectra are available for BiFC.Among those FPs,only three GFP variants,Cerulean(cyan FP variant),Citrine and Venus(yellow FP variants),have been demonstrated to support BiFC under physiological conditions(37℃).However,two red BiFC systems based on mRFP1 variants,mRFP1-Q66T and mCherry,only work at relatively low temperatures (≤30℃).Hence,identification of red FPs that could allow fluorescence complementation will increase the number of interactions that could be visualized simultaneously using multicolor BiFC.In this study,we tested the ability of fluorescence complementation of newly reported red FPs mKate,and found that mKate fragments could complement each other at 37℃. According to the crystal structure of the related eqFP611,we performed the site-directed mutagenesis at Ser158 of mKate and screened the brightest mKate mutant,mKate-S158A (named as mLumin).Using mLumin-based BiFC system,several tumor-related and Alzheimer-related protein interactions were visualized.The major results of this study are showed as following:1) Four red FPs including mRFP1-Q66T,mCherry,TagRFP and mKate at 37℃and 26℃,respectively,were examined for fluorescence complementation.Fluorescence results showed that mKate,but not TagRFP,could support BiFC at 37℃.Furthermore, quantification of fluorescence intensity revealed that the fragments split at 151 exhibited the highest BiFC efficiency among fragments of mKate.2) According to the eqFP611's crystal structure,we performed saturation mutagenesis of Ser158,screened this directed library and identified two bightest clones,mKate-S158A (named as mLumin) and mKate-S158C.Compared to mKate,mLumin and mKate-S158C exhibited almost identical excitation and emission spectra and comparable chromophore maturation rates.These two mutants have a lower pKa(4.7 and 4.2 for S158A and S158C, respectively) than mKate.The brightness of mKate-S158A and mKate-S158C are 1-and 0.3-fold higher than that of mKate.However,mLumin was approximately 3-fold less photostable than mKate,and mKate-S158C showed a very slow photobleaching rate, whose photostability is over 1.5-fold greater than that of mKate.3) Using this novel red BiFC system,we validate that unphosphorylated STAT5B existed as dimmer and that epidermal growth factor receptor(EGFR) directly interacted with STAT5B in living cells.Furthermore,by combination of mLumin with mCeruleanand mVenus- based BiFC systems,different subcellular localizations and interactions of six proteins(bFos-bJun,EGFR-Grb2 and STAT5B-STAT5B) involved in EGFR signaling pathway were simultaneously visualized in the same living cell.These results highlight its potential application in studies of the complicated signal transduction network in living cell.4) Using TagRFP- and mLumin-based on BiFC system,bFos and bJun interaction was visualized in C.elegans.The novel red BiFC systems will enrich the current BiFC toolbox and offer the potential to investigate multiple protein interactions under physiological conditions. mLumin-based BiFC system enables non-invasive whole-body imaging of protein interactions in live animals,due to the lower scattering and absorption of far-red fluorescent protein in living tissues.