FRET-based evaluation of Bid cleavage in a single primary cultured neuron.
FRET-based evaluation of Bid cleavage in a single primary cultured neuron.
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基于 FRET 的单个原代培养神经元中 Bid 裂解的评估。
DOI：
10.1016/j.neulet.2012.11.059
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Nakazawa H
中科院分区：
文献类型：
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作者：
Ida T;Sawa T;Ihara H;Tsuchiya Y;Watanabe Y;Kumagai Y;Motohashi H;Fujii S;Matsunaga T;Yamamoto M;Ono K;Fukuto J;Akaike T;Nakazawa H
细胞凋亡是一种由半胱天冬酶激活引发的细胞死亡方式。从理论上讲，荧光共振能量转移（FRET）分析应该是一个方便的工具，可视化的半胱天冬酶的激活。由于FRET探针不能在原代神经元培养物中有效转染，因此FRET信号不足以用于评估。我们开发了一种从荧光FRET图像中提取显著信号的方法，该方法能够分析细胞凋亡的启动。我们使用原代海马培养物转染与YFP和CFP融合的Bid编码载体。使用星形孢菌素（STS; 1μM）诱导细胞凋亡。使用倒置荧光显微镜观察CFP和YFP信号，并使用成像软件进行处理以进行分析。在减去背景信号之后，从源自线粒体的局部强信号中提取半胱天冬酶活化的区域和显著信号。将单个神经元中选定区域的CFP和YFP强度积分，并获得CFP/YFP比率。为了在类似的实验环境中确认半胱天冬酶活化，还进行了发光分析。来自培养的神经元的FRET信号局限于病灶，因为Bid接头特异性地定位于线粒体中。从病灶提取的CFP和YFP信号足够强以进行评价。STS刺激后，CFP/YFP比值由对照组的0.673±0.024增加到STS组的1.008±0.134（均数±标准差）（P<0.05）。我们的研究首次证明了在原代神经元中通过FRET表达的Bid裂解的定量。由于Bid位于线粒体中，因此感兴趣的区域被限制在特定区域，从而能够分析信号。这种方法可能是有用的FRET分析在原代培养细胞中的应用。
Apoptosis is a cell death modality that is initiated by the activation of caspases. Theoretically, fluorescence resonance energy transfer (FRET) analysis should be a convenient tool for visualizing the activation of caspase. Since the FRET probe cannot be transfected in primary neuronal cultures effectively, the FRET signal is not sufficiently strong for evaluations. We developed a method of extracting the significant signals from the fluorescent FRET images that enables the initiation of apoptosis to be analyzed. We used primary hippocampal cultures transfected with a vector encoding Bid fused with YFP and CFP. Apoptosis was induced using staurosporine (STS; 1μM). The CFP and YFP signals were observed using an inverted fluorescence microscope and were processed using imaging software for analysis. After the background signal was subtracted, the area of caspase activation and the significant signals were extracted from the localized intense signals originating from mitochondria. The CFP and YFP intensities of a selected area in a single neuron were integrated, and the CFP/YFP ratio was obtained. To confirm caspase activation in a similar experimental setting, a luminescence analysis was also performed. The FRET signals from the cultured neuron were confined to foci, since the Bid linker was specifically localized in the mitochondria. The extracted CFP and YFP signals from the foci were strong enough to be evaluated. The average CFP/YFP ratio in the neuron increased significantly after an STS challenge, from 0.673±0.024 (control) to 1.008±0.134 (STS) (mean±SD) (P<0.05). Our study demonstrated, for the first time, the quantification of Bid cleavage as expressed by FRET in a primary neuron. Since Bid is localized in the mitochondria, the region of interest was restricted to a specific area, enabling the signal to be analyzed. This methodology may be useful for the application of FRET analyses in primary cultured cells.