Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer
Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer
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DOI：
10.1016/j.abb.2003.12.031
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发表时间：
2004-02-15
影响因子：
3.9
通讯作者：
Lemasters, JJ
中科院分区：
文献类型：
--
作者：
Elmore, SP;Nishimura, Y;Lemasters, JJ
线粒体去极化促进凋亡和坏死细胞死亡以及可能的其他细胞事件。极化的线粒体吸收去极化后释放的阳离子四甲基罗丹明甲酯（TMRM）。因此，TMRM不标记去极化的线粒体。为了鉴定活细胞中的极化和去极化线粒体，将培养的大鼠肝细胞和窦状隙内皮细胞与发绿色荧光的MitoTracker绿色FM（MTG）和发红色荧光的TMRM共加载，用于通过激光扫描共聚焦显微镜成像。与TMRM一样，MTG是一种阳离子荧光团，其在极化线粒体中聚集。与TMRM不同，MTG与线粒体内蛋白硫醇共价结合，并在去极化后保持结合。在仅用MTG标记的细胞中，用蓝光（488 nm）激发产生绿色荧光，但几乎没有红色荧光。随后用TMRM加载后，绿色MTG荧光被淬灭。相反，蓝色激发产生红色荧光。线粒体脱髓鞘恢复了绿色荧光，消除了红色荧光。相反，当将MTG加入TMRM标记的细胞中时，蓝光激发的红色荧光增强，这种效应再次被去荧光素逆转。供体荧光的可逆淬灭和受体荧光的增强的这些观察表明荧光共振能量转移（FRET）。在未受干扰的肝细胞中，线粒体亚组分的自发去极化是一种持续的现象。总之，共聚焦FRET歧视个别去极化线粒体对数百个极化线粒体的背景。(C)2004年爱思唯尔公司All rights reserved.
Mitochondrial depolarization promotes apoptotic and necrotic cell death and possibly other cellular events. Polarized mitochondria take up cationic tetramethylrhodamine methylester (TMRM), which is released after depolarization. Thus, TMRM does not label depolarized mitochondria. To identify both polarized and depolarized mitochondria in living cells, cultured rat hepatocytes, and sinusoidal endothelial cells were co-loaded with green-fluorescing MitoTracker Green FM (MTG) and red-fluorescing TMRM for imaging by laser scanning confocal microscopy. Like TMRM, MTG is a cationic fluorophore that accumulates electrophoretically into polarized mitochondria. Unlike TMRM, MTG binds covalently to intramitochondrial protein thiols and remains bound after depolarization. In cells labeled only with MTG, excitation with blue (488 nm) light yielded green but almost no red fluorescence. After subsequent loading with TMRM, green MTG fluorescence became quenched. Instead, blue excitation yielded red fluorescence. Mitochondrial de-energization restored green fluorescence and abolished red fluorescence. Conversely, when MTG was added to TMRM-labeled cells, red fluorescence excited by blue light was enhanced, an effect again reversed by de-energization. These observations of reversible quenching of donor fluorescence and augmentation of acceptor fluorescence signify fluorescence resonance energy transfer (FRET). In undisturbed hepatocytes, spontaneous depolarization of a sub,fraction of mitochondria was an ongoing phenomenon. In conclusion, confocal FRET discriminates individual depolarized mitochondria against a background of hundreds of polarized mitochondria. (C) 2004 Elsevier Inc. All rights reserved.