Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2
Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2
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使用线粒体钙荧光指示剂 mito-GCaMP2 检测不同细胞类型中不同的线粒体钙反应
DOI：
10.1093/abbs/gmr075
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发表时间：
2011-10-01
影响因子：
3.7
通讯作者：
Wang, Xianhua
中科院分区：
文献类型：
--
作者：
Chen, Min;Wang, Yanru;Wang, Xianhua
线粒体钙在线粒体代谢、细胞钙处理和细胞死亡中起着至关重要的作用。然而，有关线粒体钙调节的一些机制仍不清楚，特别是线粒体钙如何与细胞质钙偶联。在这项工作中，我们通过基因操作构建了一种新型线粒体钙荧光指示剂（mito-GCaMP2）。 Mito-GCaMP2 被高效导入线粒体，并且荧光信号与线粒体膜电位指示剂四甲基罗丹明甲酯的荧光信号共定位。线粒体抑制剂特异性降低 mito-GCaMP2 的信号。在成年大鼠心肌细胞中，在 pH 8.0 时，mito-GCaMP2 的表观 K-d 为 195.0 nmol/L。此外，我们观察到 mito-GCaMP2 更喜欢线粒体的碱性 pH 环境。在HeLa细胞中，我们发现线粒体钙（[Ca2+](mito)）对组胺或毒胡萝卜素诱导的胞质钙（[Ca2+](cyto)）的变化做出反应。此外，外部Ca2+（100 μmol/L）直接诱导透化HeLa细胞中[Ca2+](mito)的增加。然而，在大鼠心肌细胞中，[Ca2+](mito) 对电起搏器或咖啡因刺激的胞质钙瞬变没有反应。在透化的心肌细胞中，600 nmol/L游离Ca2+反复增强mito-GCaMP2的荧光信号，这排除了mito-GCaMP2在心肌细胞线粒体中丧失功能的可能性。这些结果表明，不同细胞谱系中线粒体钙的反应是不同的，这表明心肌细胞中的线粒体可能具有特殊的防御机制来控制钙通量。
Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K-d of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca2+](mito)) responded to the changes of cytosolic calcium ([Ca2+](cyto)) induced by histamine or thapasigargin. Moreover, external Ca2+ (100 mu mol/L) directly induced an increase of [Ca2+](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca2+](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca2+ repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.