Department of Anesthesiology, Kyorin University, School of Medicine, Japan.
Neurosci Lett. 2013 Mar 1;536:24-8. doi: 10.1016/j.neulet.2012.11.059. Epub 2012 Dec 20.
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.
细胞凋亡是一种由半胱氨酸天冬氨酸蛋白酶（caspases）激活引发的细胞死亡方式。理论上，荧光共振能量转移（FRET）分析应该是一种可视化 caspase 激活的便捷工具。由于 FRET 探针不能有效地转染原代神经元培养物，因此 FRET 信号不够强，无法进行评估。我们开发了一种从荧光 FRET 图像中提取显著信号的方法，使分析细胞凋亡的起始成为可能。我们使用转染编码 Bid 融合蛋白与 YFP 和 CFP 的载体的原代海马培养物。使用星形孢菌素（STS；1μM）诱导凋亡。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。在减去背景信号后，从起源于线粒体的局部强信号中提取 caspase 激活的区域和显著信号。在单个神经元的选定区域中整合 CFP 和 YFP 强度，并获得 CFP/YFP 比值。为了在类似的实验环境中确认 caspase 激活，还进行了发光分析。FRET 信号来自培养神经元的焦点，因为 Bid 接头特异性定位于线粒体中。从焦点中提取的 CFP 和 YFP 信号足够强，可以进行评估。STS 挑战后，神经元中的平均 CFP/YFP 比值显著增加，从 0.673±0.024（对照）增加到 1.008±0.134（STS）（平均值±SD）（P<0.05）。我们的研究首次在原代神经元中通过 FRET 定量表达了 Bid 切割。由于 Bid 位于线粒体中，因此感兴趣的区域仅限于特定区域，从而可以对信号进行分析。这种方法在原代培养细胞中的 FRET 分析应用中可能有用。
