Photoactivatable green fluorescent protein-based visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living cells.
Photoactivatable green fluorescent protein-based visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living cells.
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DOI：
10.1016/b978-0-12-801415-8.00004-7
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发表时间：
2014
影响因子：
--
通讯作者：
Roelofs BA
中科院分区：
文献类型：
--
作者：
Karbowski M;Cleland MM;Roelofs BA
显微镜技术的进步和线粒体特异性成像分子工具的发展阐明了这些重要细胞器的动态重排。这些重排主要是两个相反过程的结果：线粒体融合和线粒体分裂。与此一致的是，除了线粒体的运动性之外，这两个过程也是决定线粒体网络整体连续性程度以及细胞内线粒体平均大小的主要因素。在这一章中，我们详细介绍了使用先进的共聚焦显微镜和线粒体基质靶向的光激活绿色荧光蛋白(mito-PAGFP)来研究线粒体的动力学。我们专注于对活的哺乳动物细胞中线粒体融合和线粒体网络复杂性的直接可视化和量化。这些分析有助于线粒体生物学领域最近的重要发现，包括线粒体融合在激活线粒体凋亡步骤中的作用，Bcl2家族蛋白在线粒体形态发生中的参与以及应激诱导的线粒体高融合。我们提出了一些基本方向，这些方向应该有助于设计基于MITO-PAGFP的实验。此外，由于基于mito-PAGFP的线粒体融合分析依赖于时移成像，因此还讨论了时移显微镜和细胞制备的关键参数。
Technological improvements in microscopy and the development of mitochondria-specific imaging molecular tools have illuminated the dynamic rearrangements of these essential organelles. These rearrangements are mainly the result of two opposing processes: mitochondrial fusion and mitochondrial fission. Consistent with this, in addition to mitochondrial motility, these two processes are major factors determining the overall degree of continuity of the mitochondrial network, as well as the average size of mitochondria within the cell. In this chapter, we detail the use of advanced confocal microscopy and mitochondrial matrix-targeted photoactivatable green fluorescent protein (mito-PAGFP) for the investigation of mitochondrial dynamics. We focus on direct visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living mammalian cells. These assays were instrumental in important recent discoveries within the field of mitochondrial biology, including the role of mitochondrial fusion in the activation of mitochondrial steps in apoptosis, participation of Bcl-2 family proteins in mitochondrial morphogenesis and stress induced mitochondrial hyperfusion. We present some basic directions that should be helpful in designing mito-PAGFP-based experiments. Furthermore, since analyses of mitochondrial fusion using mito-PAGFP-based assay rely on time-lapse imaging, critical parameters of time-lapse microscopy and cell preparation are also discussed.