Quantitative analysis of spontaneous mitochondrial depolarizations
Quantitative analysis of spontaneous mitochondrial depolarizations
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DOI：
10.1016/s0006-3495(03)74754-7
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发表时间：
2003-11-01
影响因子：
3.4
通讯作者：
Walsh, JV
中科院分区：
文献类型：
--
作者：
O'Reilly, CM;Fogarty, KE;Walsh, JV
使用荧光探针四甲基罗丹明乙酯 (TMRE) 在分离的线粒体和完整细胞中观察到线粒体膜电位 (DeltaPsi(m)) 的自发瞬时去极化、线粒体闪烁。理论上，细胞质和线粒体中[TMRE]的比率允许用能斯特方程计算DeltaPsi(m)，但由于荧光猝灭和染料与线粒体膜的结合，这在实践中被证明是困难的。我们开发了一种新方法来确定以毫伏去极化为单位的闪烁幅度。使用高速、高灵敏度三维成像来监测 TMRE 荧光，以追踪新鲜分离的平滑肌细胞中的单个线粒体。静息线粒体荧光是静息 DeltaPsi(m) 的指数函数，在线粒体之间存在差异，并且近似正态分布。观察到线粒体荧光的自发变化，表明 DeltaPsi(m) 的去极化和复极化。去极化是可逆的，并且不会导致线粒体永久去极化。闪烁幅度范围为 100 mV，平均值为 17.6 +/- 1.0 mV (n = 360)，分布偏向较小的值。几乎所有线粒体都在闪烁，并且它们彼此独立地闪烁，表明线粒体在此处使用的肌细胞中作为独立单位发挥作用。
Spontaneous transient depolarizations in mitochondrial membrane potential (DeltaPsi(m)), mitochondrial flickers, have been observed in isolated mitochondria and intact cells using the fluorescent probe, tetramethylrhodamine ethyl ester ( TMRE). In theory, the ratio of [TMRE] in cytosol and mitochondrion allows DeltaPsi(m) to be calculated with the Nernst equation, but this has proven difficult in practice due to fluorescence quenching and binding of dye to mitochondrial membranes. We developed a new method to determine the amplitude of flickers in terms of millivolts of depolarization. TMRE fluorescence was monitored using high-speed, high-sensitivity three-dimensional imaging to track individual mitochondria in freshly dissociated smooth muscle cells. Resting mitochondrial fluorescence, an exponential function of resting DeltaPsi(m), varied among mitochondria and was approximately normally distributed. Spontaneous changes in mitochondrial fluorescence, indicating depolarizations and repolarizations in DeltaPsi(m), were observed. The depolarizations were reversible and did not result in permanent depolarization of the mitochondria. The magnitude of the flickers ranged from 100 mV with a mean of 17.6 +/- 1.0 mV (n = 360) and a distribution skewed to smaller values. Nearly all mitochondria flickered, and they did so independently of one another, indicating that mitochondria function as independent units in the myocytes employed here.