Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP
Highly sensitive and quantitative FRET-FLIM imaging in single dendritic spines using improved non-radiative YFP
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DOI：
10.1007/s11068-008-9024-9
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发表时间：
2008-08-01
期刊：
影响因子：
--
通讯作者：
Yasuda, Ryohei
中科院分区：
文献类型：
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作者：
Murakoshi, Hideji;Lee, Seok-Jin;Yasuda, Ryohei
双光子荧光寿命成像显微镜(TPFLIM)能够定量测量光散射组织中小亚细胞室的荧光共振能量转移(FRET)。我们评估和优化了TPFLIM的mEGFP(带有A206K突变的单体EGFP)和REACH(非放射性YFP变体)的FRET对。我们从“黑暗”以及它们在HeLa细胞和海马神经元中作为mEGFP的FRET受体的能力方面对REACH的几个突变体进行了表征。由于常用的单体突变A206K增加了REACH的亮度，我们引入了不同的单体突变(F223R)，它不影响亮度。此外，我们还发现，原始REACH的折叠效率，通过mEGFP-REACH串联二聚体的荧光寿命来衡量，很低，并且在细胞之间是可变的。将两个折叠突变体(F46L和Q69M)引入REACH，使折叠效率提高了-50%，降低了FRET信号的变异性。与mEGFP-MRFP或mEGFP-原始REACH配对相比，mEGFP与新REACH(超级REACH或sREACh)配对的信噪比提高了-50%。使用这对新的肌动蛋白单体，我们证明了可以高灵敏度地定量测量单个树突棘中丝状和球状的肌动蛋白单体的比例。因此，mEGFP-sREACh对适合于TPFLIM的FRET定量测量，并使我们能够高灵敏度地测量大脑切片中单个树突棘中的蛋白质-蛋白质相互作用。
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 mEGFP-mRFP 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.