Tagging and tracking individual networks within a complex mitochondrial web with photoactivatable GFP.
Tagging and tracking individual networks within a complex mitochondrial web with photoactivatable GFP.
复制标题
使用可光激活的 GFP 标记和跟踪复杂线粒体网络中的各个网络。
DOI：
10.1152/ajpcell.00348.2005
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Shirihai,OrianS
中科院分区：
文献类型：
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作者：
Twig,Gilad;Graf,SolomonA;Wikstrom,JakobD;Mohamed,Hibo;Haigh,SarahE;Elorza,Alvaro;Deutsch,Motti;Zurgil,Naomi;Reynolds,Nicole;Shirihai,OrianS
线粒体组装成网络支持燃料代谢和钙运输，并参与细胞对凋亡刺激的反应。线粒体网络被定义为一个连续的矩阵腔，其边界限制了分子的扩散。在拥有密集线粒体种群的活细胞中观察个体网络已被证明具有挑战性。因此，对线粒体网络的电学和形态学特性的研究没有得出一致的结论。在这项研究中，我们使用基质靶向，光激活的绿色荧光蛋白来标记单个线粒体网络。这种方法与线粒体膜电位的实时监测相结合，可以随着时间的推移检查基质管腔连续性、融合和裂变事件。我们发现相邻和交织的线粒体结构通常代表不同网络的集合。我们还发现，单个网络的所有区域都是等电位的，这表明细胞线粒体内膜电位的异质模式代表了离散网络之间的差异。有趣的是，裂变事件经常发生时没有任何明显的形态变化，特别是没有碎裂。这些事件在标准共聚焦显微镜下是看不见的，它们重新定义了线粒体网络的边界，并导致了电分离的子单元。
Assembly of mitochondria into networks supports fuel metabolism and calcium transport and is involved in the cellular response to apoptotic stimuli. A mitochondrial network is defined as a continuous matrix lumen whose boundaries limit molecular diffusion. Observation of individual networks has proven challenging in live cells that possess dense populations of mitochondria. Investigation into the electrical and morphological properties of mitochondrial networks has therefore not yielded consistent conclusions. In this study we used matrix-targeted, photoactivatable green fluorescent protein to tag single mitochondrial networks. This approach, coupled with real-time monitoring of mitochondrial membrane potential, permitted the examination of matrix lumen continuity and fusion and fission events over time. We found that adjacent and intertwined mitochondrial structures often represent a collection of distinct networks. We additionally found that all areas of a single network are invariably equipotential, suggesting that a heterogeneous pattern of membrane potential within a cell's mitochondria represents differences between discrete networks. Interestingly, fission events frequently occurred without any gross morphological changes and particularly without fragmentation. These events, which are invisible under standard confocal microscopy, redefine the mitochondrial network boundaries and result in electrically disconnected daughter units.