In situ localization of mitochondrial DNA replication in intact mammalian cells
In situ localization of mitochondrial DNA replication in intact mammalian cells
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DOI：
10.1083/jcb.135.4.883
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发表时间：
1996-11-01
影响因子：
7.8
通讯作者：
Clayton, DA
中科院分区：
文献类型：
--
作者：
Davis, AF;Clayton, DA
几乎所有已知的线粒体DNA（mtDNA）复制和表达所需的活性都是核编码的基因产物，这使得这两个物理上不同的细胞内区室之间的通信成为必要。近二十年来，关于哺乳动物细胞中mtDNA复制的大量一般和特异性生化信息已经被发现，早期研究实现了胸苷类似物5-溴-2-脱氧尿苷（BrdU）选择性掺入胸苷激酶缺陷（TK[-]）细胞的mtDNA。我们从细胞的角度重新审视了这一方法，以确定mtDNA复制是否存在时空限制。激光扫描共聚焦显微镜使用免疫细胞化学双标记方法来观察BrdU掺入染料标记线粒体的mtDNA，在BrdU处理至少1-2小时后，原位检测BrdU掺入的mtDNA是可行的，这与先前确定完成一轮mtDNA复制所需时间的生化研究一致，有趣的是，BrdU掺入培养的哺乳动物细胞mtDNA的模式始终从核周位置向外辐射，表明mtDNA复制首先发生在核提供的材料附近。然后，新复制的mtDNA似乎迅速分布在整个动态细胞线粒体网络中。
Nearly all of the known activities required for mitochondrial DNA (mtDNA) replication and expression are nuclear-encoded gene products, necessitating communication between these two physically distinct intracellular compartments, A significant amount of both general and specific biochemical information about mtDNA replication in mammalian cells has been known for almost two decades, Early studies achieved selective incorporation of the thymidine analog 5-Bromo-2-deoxy-Uridine (BrdU) into mtDNA of thymidine kinase-deficient (TK[-]) cells. We have revisited this approach from a cellular perspective to determine whether there exist spatiotemporal constraints on mtDNA replication, Laser-scanning confocal microscopy was used to selectively detect mtDNA synthesis in situ in cultured mammalian cells using an immunocytochemical double-labeling approach to visualize the incorporation of BrdU into mtDNA of dye-labeled mitochondria, In situ detection of BrdU-incorporated mtDNA was feasible after a minimum of 1-2 h treatment with BrdU, consistent with previous biochemical studies that determined the time required for completion of a round of mtDNA replication, Interestingly, the pattern of BrdU incorporation into the mtDNA of cultured mammalian cells consistently radiated outward from a perinuclear position, suggesting that mtDNA replication first occurs in the vicinity of nuclear-provided materials. Newly replicated mtDNA then appears to rapidly distribute throughout the dynamic cellular mitochondrial network.