Regulation of mitochondrial morphology by membrane potential, and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells
Regulation of mitochondrial morphology by membrane potential, and DRP1-dependent division and FZO1-dependent fusion reaction in mammalian cells
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DOI：
10.1016/s0006-291x(03)00050-0
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发表时间：
2003-02-21
影响因子：
3.1
通讯作者：
Mihara, K
中科院分区：
文献类型：
--
作者：
Ishihara, N;Jofuku, A;Mihara, K
线粒体是动态的细胞器，经常发生裂变、融合或分支。为了分析线粒体融合反应，在hela细胞中分别用绿色或红色荧光蛋白（GFP和RFP）标记线粒体，并用日本血凝病毒（HVJ）融合细胞。然后使用荧光显微镜跟踪荧光报告的混合结果。该系统揭示了哺乳动物细胞中线粒体的频繁融合，其融合依赖于跨越内膜的膜电位。质子载体羰基氰化间氯苯腙（CCCP）导致线粒体断裂，抑制融合反应。去除CCCP可恢复融合活性，从而重组丝状线粒体网络。分析gtp结合蛋白、DRP1和两种FZO1亚型以及gtpase结构域突变体对cccp诱导的线粒体形态变化的影响发现，DRP1和FZO1分别参与了膜出芽和融合。此外，结合RNA干扰（RNAi）的hvj依赖性细胞融合实验表明，两种FZO1亚型都是必不可少的，并且必须顺式作用才能发生线粒体融合反应。(C) 2003 Elsevier Science（美国）版权所有。
Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. To analyze the mitochondrial fusion reaction, mitochondria were separately labeled with green or red fluorescent protein (GFP and RFP, respectively) in HeLa-cells, and the cells were fused using hemagglutinating virus of Japan (HVJ). The resulting mixing of the fluorescent reporters was then followed using fluorescence microscopy. This system revealed that mitochondria fuse frequently in mammalian cells, and the fusion depends on the membrane potential across the inner membrane. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to fragmentation of the mitochondria and inhibited the fusion reaction. Removal of CCCP recovered the fusion activity to reform filamentous mitochondrial networks. Analysis of the effects of GTP-binding proteins, DRP1 and two FZO1 isoforms, and the GTPase-domain mutants on the CCCP-induced mitochondrial morphologic changes revealed that DRP1 and FZO1 are involved in membrane budding and fusion, respectively. Furthermore, a HVJ-dependent cell fusion assay combined with RNA interference (RNAi) demonstrated that both FZO1 isoforms are essential and must be acting in cis for the mitochondrial fusion reaction to occur. (C) 2003 Elsevier Science (USA). All rights reserved.