Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.
Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.
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DOI：
10.1371/journal.pone.0042298
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Nagley P
中科院分区：
文献类型：
--
作者：
Ng H;Smith DJ;Nagley P
线粒体介导的细胞凋亡的特征在于线粒体外膜透化后蛋白质从线粒体重新分布到细胞质。我们应用流式细胞术同时定量细胞色素c（cyt c）和Smac/DIABLO（Smac）两种致突变蛋白的再分布。用毛地黄皂苷处理哺乳动物细胞，毛地黄皂苷选择性地使质膜透化。固定后，处理的细胞连续输注一级和二级抗体（后者荧光标记），使独立检测cyt c和Smac。在流式细胞术中，保留线粒体中的cyt c或Smac的洋地黄皂苷处理的细胞产生强荧光信号。细胞色素c或Smac已经穿过线粒体外膜的细胞显示出大大降低的荧光，因为蛋白质从毛地黄皂苷透化的细胞中丢失。定量流式细胞术显示，在用星形孢菌素处理的143 B TK-细胞中，cyt c和Smac不对称地退出线粒体，cyt c重新分布先于Smac。然而，在同样处理的HeLa细胞中，cyt c和Smac同时退出线粒体。在其它诱导凋亡的条件下，例如用MT-21（一种与线粒体腺嘌呤核苷酸转运蛋白结合的凋亡诱导剂）处理的143 B TK-细胞，Smac的再分布先于cyt c的再分布.免疫细胞化学分析和共聚焦显微镜证实了这些蛋白质的再分布的各种模式。我们的结论是，流式细胞术可以有效地同时量化的重新分配的细胞色素c和Smac从线粒体到胞质溶胶。此外，在各种条件下发生细胞色素C和Smac的差异再分配，从而反映了这些蛋白质在外膜透化后退出线粒体的可用性的限制。
Mitochondrially mediated apoptosis is characterized by redistribution of proteins from mitochondria to cytoplasm following permeabilization of the outer mitochondrial membrane. We applied flow cytometry to quantify simultaneously the redistribution of two apoptogenic proteins, cytochrome c (cyt c) and Smac/DIABLO (Smac). Mammalian cells were treated with digitonin that selectively permeabilizes the plasma membrane. Following fixation, treated cells were infused successively with primary and secondary antibodies (the latter fluorescently tagged) enabling independent detection of cyt c and Smac. Digitonin-treated cells that retain cyt c or Smac in mitochondria generate strong fluorescence signals in flow cytometry. Cells in which cyt c or Smac have transited the outer mitochondrial membrane show greatly reduced fluorescence because the proteins are lost from the digitonin-permeabilized cells. Quantitative flow cytometry revealed that in 143B TK- cells treated with staurosporine, cyt c and Smac exit mitochondria asymmetrically, with cyt c redistribution preceding that of Smac. However, in HeLa cells likewise treated, cyt c and Smac exit mitochondria concurrently. Under other conditions of apoptotic induction, for example, 143B TK- cells treated with MT-21 (an apoptotic inducer that binds to the mitochondrial adenine nucleotide transporter), redistribution of Smac precedes that of cyt c. The various patterns of redistribution of these proteins were confirmed by immunocytochemical analysis and confocal microscopy. We conclude that flow cytometry can be employed effectively to quantify simultaneously the redistribution of cyt c and Smac from mitochondria to the cytosol. Moreover, differential redistribution of cyt c and Smac occurs under various conditions, thereby reflecting constraints on availability of these proteins to exit mitochondria after permeabilization of the outer membrane.
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