Brain Cell Biology

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 approximately 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 approximately 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.

Hideji Murakoshi

Seok-Jin Lee

Ryohei Yasuda

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

高灵敏度定量FRET-FLIM成像在单个树突棘中使用改进的非辐射YFP

Cantagrel

DJ Lefeber

J Silhavy

S Bielas

B Ng

Z Guan

R Houliston

D Swistun

B Ali

TH Lidner

D Babovic-Vulksanovic

C Raetz

RA Wevers

L Lehle

H Freeze

Eva Morava

L Al-Gazali

J Gleeson

SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation

SRD5A3 是多萜醇转化为多萜醇所必需的，对N-连接蛋白糖基化至关重要

George Goodbye

脑细胞生物学

出版信息	出版商: Springer Publishing Company，出版周期: ，期刊类型: journal，开源期刊: 否
基本数据	创刊年份: 2008，原创研究文献占比: ，自引率:， Gold OA占比:

Brain Cell Biology

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

SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation

SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation

SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation

SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation

Brain Cell Biology

SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation

SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation

SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation

SRD5A3 is required for the conversion of polyprenol to dolichol, essential for N-linked protein glycosylation