Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: Mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store
Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: Mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store
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DOI：
10.1016/j.ceca.2014.09.008
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发表时间：
2014-12-01
期刊：
影响因子：
4
通讯作者：
Ding, Shinghua
中科院分区：
文献类型：
--
作者：
Li, Hailong;Wang, Xiaowan;Ding, Shinghua
线粒体Ca2+在细胞能量代谢和信号传导中起着关键的生理作用，其超载有助于各种病理条件，包括神经系统疾病中的神经元凋亡死亡。活细胞线粒体Ca2+成像是了解线粒体Ca2+动力学的重要方法。最近开发的GCaMP基因编码Ca2+指标为高灵敏度/分辨率和细胞类型特异性线粒体Ca2+成像提供了独特的机会。在当前的研究中，我们实现了细胞特异性线粒体靶向GCaMP5G/6s (mitto -GCaMP5G/6s)，并使用双光子显微镜对培养的星形细胞和神经元线粒体Ca2+动力学进行了成像，揭示了这些细胞器在细胞刺激下摄取Ca2+的机制。利用这些线粒体Ca2+指标，我们的研究结果表明，分别在ATP和谷氨酸刺激下，培养的星形胶质细胞和神经元的单个线粒体中可以看到线粒体Ca2+摄取。我们进一步研究了ATP刺激培养星形胶质细胞后线粒体Ca2+动力学对胞质Ca2+变化的依赖性，分别使用mitto - gcamp5g和合成的有机Ca2+指示剂x-Rhod-1同时成像线粒体和胞质Ca2+增加。结合分子干预Ca2+信号通路，我们的研究结果表明，线粒体Ca2+摄取与肌醇1,4,5-三磷酸受体介导的内质网Ca2+释放和G蛋白偶联受体的激活紧密耦合。目前的研究提供了一种新的方法来成像线粒体Ca2+动力学以及Ca2+在内质网和线粒体之间的相互作用，这与健康和疾病中的神经元和星形细胞功能有关。(C) 2014 Elsevier Ltd.版权所有。
Mitochondrial Ca2+ plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca2+ imaging is an important approach to understand mitochondrial Ca2+ dynamics. Recently developed GCaMP genetically-encoded Ca2+ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca2+ imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca2+ dynamics in culture, revealing Ca2+ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca2+ indicators, our results show that mitochondrial Ca2+ uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca2+ dynamics on cytosolic Ca2+ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca2+ increase using mito-GCaMP5G and a synthetic organic Ca2+ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca2+ signaling pathway, our results demonstrated that the mitochondrial Ca2+ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca2+ dynamics as well as Ca2+ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease. (C) 2014 Elsevier Ltd. All rights reserved.