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

Abstract

Two-photon fluorescence lifetime imaging microscopy (TPFLIM) enables the quantitative measurements of fluorescence resonance energy transfer (FRET) in small subcellular compartments in light scattering tissue. We evaluated and optimized the FRET pair of mEGFP (monomeric EGFP with the A206K mutation) and REACh (non-radiative YFP variants) for TPFLIM. We characterized several mutants of REACh in terms of their "darkness," and their ability to act as a FRET acceptor for mEGFP in Hela cells and hippocampal neurons. Since the commonly used monomeric mutation A206K increases the brightness of REACh, we introduced a different monomeric mutation (F223R) which does not affect the brightness. Also, we found that the folding efficiency of original REACh, as measured by the fluorescence lifetime of a mEGFP-REACh tandem dimer, was low and variable from cell to cell. Introducing two folding mutations (F46L, Q69M) into REACh increased the folding efficiency by ∼50%, and reduced the variability of FRET signal. Pairing mEGFP with the new REACh (super-REACh, or sREACh) improved the signal-to-noise ratio compared to the mEGFPmRFP or mEGFP-original REACh pair by ∼50 %. Using this new pair, we demonstrated that the fraction of actin monomers in filamentous and globular forms in single dendritic spines can be quantitatively measured with high sensitivity. Thus, the mEGFP-sREACh pair is suited for quantitative FRET measurement by TPFLIM, and enables us to measure protein-protein interactions in individual dendritic spines in brain slices with high sensitivity.

Studying Signal Transduction in Single Dendritic Spines

“…2). Thus, the recently developed resonance energy acceptor chromophore (REACh; a high-absorbing, dark EYFP variant), can be used as a FLIM acceptor (Ganesan et al 2006;Murakoshi et al 2008). Overall, whereas the ECFP-EYFP pairs are widely used for ratiometric imaging, the EGFP-mRFP, EYFP-mRFP and EGFP-REACh pairs are used for FLIM (Tramier et al 2006;Yasuda 2006;Murakoshi et al 2008).…”

“…To convert them to FLIM sensors, one can simply replace the ECFP-EYFP pair with EGFPmRFP or EGFP-sREACh (Murakoshi et al 2008). For example, the dynamics of actin polymerization in individual spines were first imaged by a ratiometric sensor (Okamoto et al 2004).…”

“…Using this method, Okamoto et al successfully used two-photon ratiometric imaging to visualize the polymerization of actin during spine structural changes induced by electric stimulation. Later the polymerization fraction was quantified with 2pFLIM in combination with a new actin polymerization sensor made of the mEGFP (monomeric EGFP: monomeric variant of EGFP) and sREACh (super REACh: high-folding REACh variant) pair (Murakoshi et al 2008). …”

“…More recently, the sensor was further optimized for 2pFLIM. In the new sensor called Green-Camuia, the fluorophores were replaced to the pair of mEGFP and REACh (Ganesan et al 2006;Murakoshi et al 2008), which allowed measurement of CaMKII activity in single spines during LTP with high temporal resolution (approximately seconds) ).…”

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

“…However, there are also possible avenues toward optimization of the biosensors for twophoton intravital imaging. One idea is to use a nonradiative mutant FP as an acceptor, such as sREACh, which has already been utilized in two-photon excitation FLIM-FRET (50). Another approach is to change the FP pair to an FP pair with a large gap between the excitation wavelength.…”