Live-cell imaging study of mitochondrial morphology in mammalian cells irradiated
Live-cell imaging study of mitochondrial morphology in mammalian cells irradiated
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DOI：
10.1093/jrr/rrt167
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发表时间：
2014-03
影响因子：
2
通讯作者：
Yokoya A
中科院分区：
文献类型：
--
作者：
Kanari Y;Noguchi M;Kaminaga K;Sakamoto Y;Yokoya A
最近的报道表明，细胞质中的核外靶点可能在电离辐射暴露的哺乳动物细胞中介导辐射效应中起作用。由于线粒体是广泛存在于细胞质中的一种细胞器，因此我们将线粒体作为电离辐射特别是重离子辐射的靶点。它们通过位于膜上的电子传递链的运作对ATP的形成起着至关重要的作用，并在ATP的产生过程中产生活性氧作为副产物。由于线粒体根据细胞周期进行融合或分裂，其形态也在不断变化[]。这些过程的缺陷有助于神经退行性疾病的发病机制。然而，高LET照射（如重离子轰击）对线粒体形态的影响仍有待完全阐明。本研究的目的是揭示高LET辐射对线粒体的影响。我们已经用x射线进行了初步的测试实验。我们使用NMuMG（正常小鼠乳腺）-FUCCI2细胞系[]。利用表达fucci2的细胞，我们可以通过观察细胞核内红色或绿色荧光来区分G1期或S/G2/M期细胞。我们还用另一种荧光探针Mitotracker Red标记线粒体。利用荧光显微镜活细胞成像技术分析线粒体形态动力学。照射后一定时间采集线粒体图像，并将其分为管状、中间体和碎片状三大类（图1）。未辐照（对照）和线粒体形态载体显示每种形态的细胞的相对比例。(1) 0Gy, (2) 6gy和(2)8gy辐照。细胞周期也用实线表示。(a)大多数线粒体看起来是管状的。(b)在细胞中管状和碎片状两种形态的发生率相同。(c)线粒体呈点状，在整个细胞质中散乱分布。我们发现，x射线照射细胞导致线粒体断裂，线粒体断裂的细胞数量随着照射剂量的增加而增加，照射后的时间也随着时间的延长而增加（图1）。虽然从细胞核颜色可以看出，照射后48 h左右细胞开始融合，但线粒体形态仍在发生变化。特别是，在8 Gy x射线照射后96 h，带有碎片的细胞数量达到最大值。在本研究的基础上，我们将进一步研究高LET粒子辐照对线粒体形态的影响。
Recent reports suggest that extranuclear targets in cytoplasm may have a role in mediating radiation effects in mammalian cells exposed to ionizing radiation. We have focused mitochondria as a target of ionizing radiation, particularly heavy ions, because mitochondria are a kind of organelles existing widely in cytoplasm. They play a vitally important role of ATP formation through the operation of electron transport chain located in the membranes, and generate reactive oxygen species as a by-product in the process of ATP production. As mitochondria are fusing or dividing depended on cell cycle, their morphology is continuously changing [ ]. Defects of these processes contribute to the pathogenesis of neurodegenerative disease. Effects of high LET irradiation, such as heavy ion bombardment, on mitochondrial morphology, however, remain to be fully elucidated. The object of this study is to reveal effects of high LET radiation on mitochondria. We have performed preliminary test experiments using X-rays. We used NMuMG (Normal murine mammary gland)-FUCCI2 cell line [ ]. Using the FUCCI2-expressing cells, we can distinguish G1 or S/G2/M phase cells as observed red or green fluorescence in their nucleus. We also labeled mitochondria by another fluorescence probe, Mitotracker Red. Kinetics of mitochondrial morphology was analyzed by the live-cell imaging technique using a fluorescence microscope. Mitochondrial images were captured at certain hours after irradiation, and classified them into three categories, namely tubes, intermediates and fragments (Fig. 1). Relative fraction of cells showing each morphology for non-irradiated (control) and charectors of mitochondrial morphology. Irradiated with (1) 0Gy, (2) 6 Gy and (2) 8 Gy. The cell cycles are also shown by solid lines. (a) Mostly mitochondria look tubular. (b) Both tubular and fragment forms are observed with the same rate in a cell. (c) Mitochondria are visible as dots, and straggle in whole cytoplasm. We found that X-ray irradiation of cells caused mitochondrial fragmentation, and cell population with fragmented mitochondria increased with increasing dose, and also with time after irradiation (Fig. 1). Although the cells became confluent around 48 h after irradiation as indicated by the cell-nucleus color, the mitochondrial morphology was still changing. Particularly, the population of the cells with fragments showed a maximum at 96 h after irradiation when they were exposed to 8 Gy X-rays. Based on the present study, we further investigate morphological changes of mitochondria by high LET particle irradiation in future.