New Techniques to Study Intracellular Receptors in Living Cells: Insights Into RIG-I-Like Receptor Signaling.
New Techniques to Study Intracellular Receptors in Living Cells: Insights Into RIG-I-Like Receptor Signaling.
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研究活细胞内受体的新技术：深入了解 RIG-I 样受体信号传导。
DOI：
10.1007/5584_2018_297
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发表时间：
2019
影响因子：
--
通讯作者：
Frick,DavidN
中科院分区：
文献类型：
--
作者：
Corby,MJ;Raicu,Valerica;Frick,DavidN
本文综述了福斯特共振能量转移（FRET）显微镜及其应用于细胞受体的新发展。该方法是基于动力学理论的FRET，它可以用来预测FRET不仅在二聚体，但也更高阶的低聚体的供体和受体的荧光团。基于这种FRET预测的模型可以拟合到观察到的FRET效率直方图（也称为FRET谱图），并用于估计细胞内结合常数、自由能值和化学计量。这些“FRET光谱法”方法已被用于分析由细胞信号传导途径中的各种受体形成的寡聚体，但直到最近，此类研究仅限于驻留在细胞表面上的受体。为了研究细胞内的复合物，开发了一种称为定量显微光谱成像（Q-MSI）的技术。Q-MSI结合了从像素级表观FRET光谱图确定四级结构，以及在细胞器水平确定供体和受体浓度。这是通过解析和分析不参与FRET的第三种荧光标记物的光谱来完成的。Q-MSI首先用于研究一类细胞质受体的相互作用，这些受体结合病毒RNA并通过主要在线粒体膜上形成的复合物发出抗病毒反应的信号。Q-MSI揭示了以前未知的RNA线粒体受体方向，以及称为LGP 2的病毒RNA受体与肝炎病毒编码的RNA解旋酶之间的相互作用。这些新的观察结果的生物学意义进行了讨论。
This review discusses new developments in Förster resonance energy transfer (FRET) microscopy and its application to cellular receptors. The method is based on the kinetic theory of FRET, which can be used to predict FRET not only in dimers, but also higher order oligomers of donor and acceptor fluorophores. Models based on such FRET predictions can be fit to observed FRET efficiency histograms (also called FRET spectrograms) and used to estimate intracellular binding constants, free energy values, and stoichiometries. These “FRET spectrometry” methods have been used to analyze oligomers formed by various receptors in cell signaling pathways, but until recently such studies were limited to receptors residing on the cell surface. To study complexes residing inside the cell, a technique called Quantitative Micro-Spectroscopic Imaging (Q-MSI) was developed. Q-MSI combines determination of quaternary structure from pixel-level apparent FRET spectrograms with the determination of both donor and acceptor concentrations at the organelle level. This is done by resolving and analyzing the spectrum of a third fluorescent marker, which does not participate in FRET. Q-MSI was first used to study the interaction of a class of cytoplasmic receptors that bind viral RNA and signal an antiviral response via complexes formed mainly on mitochondrial membranes. Q-MSI revealed previously unknown RNA mitochondrial receptor orientations, and the interaction between the viral RNA receptor called LGP2 with the RNA helicase encoded by the hepatitis virus. The biological importance of these new observations is discussed.