National Research Center "Kurchatov Institute", Moscow 123182, Russia.
Moscow Institute of Physics and Technology, State University, Moscow 123182, Russia.
Int J Mol Sci. 2019 Jun 27;20(13):3138. doi: 10.3390/ijms20133138.
Hydrogen peroxide (HO) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of HO dynamics in live cells within a limited field of view. The visualization of HO within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the HO concentration over desired time scales. This would enable post hoc optical readouts in chemically fixed samples. Herein, we report the development and characterization of NeonOxIrr, a genetically encoded green fluorescent indicator, which rapidly increases fluorescence brightness upon reaction with HO, but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from . When compared in vitro to a standard in the field, HyPer3 indicator, NeonOxIrr showed 5.9-fold higher brightness, 15-fold faster oxidation rate, 5.9-fold faster chromophore maturation, similar intensiometric contrast (2.8-fold), 2-fold lower photostability, and significantly higher pH stability both in reduced (p of 5.9 vs. ≥7.6) and oxidized states (p of 5.9 vs.≥ 7.9). When expressed in the cytosol of HEK293T cells, NeonOxIrr demonstrated a 2.3-fold dynamic range in response to HO and a 44 min reduction half-time, which were 1.4-fold lower and 7.6-fold longer than those for HyPer3. We also demonstrated and characterized the NeonOxIrr response to HO when the sensor was targeted to the matrix and intermembrane space of the mitochondria, nucleus, cell membranes, peroxisomes, Golgi complex, and endoplasmic reticulum of HEK293T cells. NeonOxIrr could reveal endogenous reactive oxygen species (ROS) production in HeLa cells induced with staurosporine but not with thapsigargin or epidermal growth factor. In contrast to HyPer3, NeonOxIrr could visualize optogenetically produced ROS in HEK293T cells. In neuronal cultures, NeonOxIrr preserved its high 3.2-fold dynamic range to HO and slow 198 min reduction half-time. We also demonstrated in HeLa cells that NeonOxIrr preserves a 1.7-fold ex vivo dynamic range to HO upon alkylation with N-ethylmaleimide followed by paraformaldehyde fixation. The same alkylation-fixation procedure in the presence of NP-40 detergent allowed ex vivo detection of HO with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible HO indicator NeonOxIrr can be used for both the in vivo and ex vivo visualization of ROS. Expanding the family of fixable indicators may be a promising strategy to visualize biological processes at a single cell resolution within an entire organism.
过氧化氢 (HO) 在调节活生物体中的细胞信号和内稳态方面发挥着重要作用。HyPer 家族的基因编码指示剂允许在有限的视场中可视化活细胞中的 HO 动力学。如果有一种可以缓慢还原的荧光指示剂，可以在所需时间范围内整合 HO 浓度，那么就可以在整个生物体中以单个细胞的分辨率对 HO 进行可视化。这将能够在化学固定样本中进行光学后读取。本文报告了 NeonOxIrr 的开发和表征，NeonOxIrr 是一种基因编码的绿色荧光指示剂，与 HO 反应后荧光亮度迅速增加，但还原率较低。NeonOxIrr 由经循环排列的 mNeonGreen 荧光蛋白与从 中分离的截短 OxyR 转录因子融合而成。与该领域的标准指示剂 HyPer3 相比，NeonOxIrr 的亮度高 5.9 倍，氧化速率快 15 倍，发色团成熟速度快 5.9 倍，强度对比度相似（2.8 倍），光稳定性低 2 倍，在还原态（p 为 5.9 对≥7.6）和氧化态（p 为 5.9 对≥7.9）下的稳定性均较高。当在 HEK293T 细胞的细胞质中表达时，NeonOxIrr 在响应 HO 时具有 2.3 倍的动态范围，还原半衰期为 44 分钟，比 HyPer3 低 1.4 倍，长 7.6 倍。我们还证明并表征了当传感器靶向 HEK293T 细胞的线粒体基质和内膜空间、细胞核、细胞膜、过氧化物酶体、高尔基体和内质网时，NeonOxIrr 对 HO 的响应。NeonOxIrr 可以揭示 HeLa 细胞在用 staurosporine 诱导时产生的内源性活性氧 (ROS)，但不能用 thapsigargin 或表皮生长因子诱导。与 HyPer3 相反，NeonOxIrr 可以可视化 HEK293T 细胞中光遗传学产生的 ROS。在神经元培养物中，NeonOxIrr 保持对 HO 的高 3.2 倍动态范围和缓慢的 198 分钟还原半衰期。我们还在 HeLa 细胞中证明，在用 N-乙基马来酰亚胺进行烷基化，然后用多聚甲醛固定后，NeonOxIrr 在体外对 HO 的动态范围仍保持 1.7 倍。在 NP-40 去污剂存在的情况下，相同的烷基化-固定程序允许在神经元培养物和小鼠大脑皮层中以 1.5 倍的对比度检测 HO。这种缓慢可还原的 HO 指示剂 NeonOxIrr 可用于体内和体外 ROS 的可视化。扩展可固定指示剂的家族可能是在整个生物体中单细胞分辨率可视化生物过程的有前途的策略。
