Multiphoton-FLIM Quantification of the EGFP-mRFP1 FRET Pair for Localization of Membrane Receptor-Kinase Interactions

Abstract

We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer (FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM). The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) alpha in carcinoma cells for both live- and fixed-cell experiments. The CXCR4-EGFP: PKCalpha-mRFP1 complex was found to be localized precisely to intracellular vesicles and cell protrusions when imaged by multiphoton fluorescence-FLIM. A comparison of the FRET efficiencies obtained using mRFP1-tagged regulatory domain or full-length PKCalpha as the acceptor revealed that PKCalpha, in the closed (inactive) form, is restrained from associating with the cytoplasmic portion of CXCR4. Live-cell FLIM experiments show that the assembly of this receptor:kinase complex is concomitant with the endocytosis process. This is confirmed by experimental evidence suggesting that the recycling of the CXCR4 receptor is increased on stimulation with phorbol ester and blocked on inhibition of PKC by bisindolylmaleimide. The EGFP-mRFP1 couple should be widely applicable, particularly to live-cell quantitative FRET assays.

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…Another frequently used combination is EGFP and the red fluorescent protein (RFP). Studies of proteins tagged with EGFP and monomeric RFP (mRFP) have already shown the potential use of this pair for FRET-FLIM applications (Peter et al 2005;Tramier et al 2006;Albertazzi et al 2009). …”

Activated Ezrin Promotes Cell Migration through Recruitment of the GEF Dbl to Lipid Rafts and Preferential Downstream Activation of Cdc42

“…Time-domain fluorescence lifetime imaging microscopy (FLIM) was performed with a multiphoton microscope system, comprising a solid-statepumped (8 W Verdi, Coherent, Palo Alto, CA), femtosecond self-modelocked Ti:Sapphire (Mira, Coherent) laser system, an in-house developed scan-head and an inverted microscope (Nikon TE2000E, Melville, NY) as described previously (Peter et al, 2005). The presence/absence of FRET is determined by fitting of the experimental data to a single exponential decay.…”

Highly sensitive and quantitative FRET–FLIM imaging in single dendritic spines using improved non-radiative YFP

“…As a FRET donor for TPFLIM, enhanced green fluorescent protein (EGFP) or its monomeric variant (EGFP A206K or mEGFP) is superior to other GFP color variants, because it is bright and photostable under 2-photon microscopy and has a mono-exponential fluorescence lifetime decay. For the FRET acceptor, monomeric red fluorescent protein (mRFP) (Campbell et al, 2002) has often been used for FLIM, because of its high extinction coefficient and good spectral separation with EGFP (Peter et al, 2005;Tramier et al, 2006;Yasuda et al, 2006). Although mRFP is not the brightest red fluorescent protein, the brightness of the acceptor is not important for FLIM, because FRET-FLIM typically measures only the donor fluorescence.…”