Faculty of Science, Yamagata University, 1-4-12 Kojirakawa-machi, Yamagata, Yamagata, 990-8560, Japan.
Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo-motoyama, Kita-ku, Kyoto, 603-8555, Japan.
Sci Rep. 2018 Apr 18;8(1):6175. doi: 10.1038/s41598-018-24466-0.
Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.
真核细胞器的功能完整性依赖于不同细胞器之间的直接物理接触。然而，由于缺乏在活细胞中分析细胞器相互作用的手段，因此对细胞器连接因子的实体并不了解。在这里，我们评估了用于在体内可视化细胞器接触位点的分裂 GFP 系统，并展示了其优缺点。我们观察到来自酵母细胞中内质网、线粒体、过氧化物酶体、液泡和脂滴之间任何两对细胞器的靶向的分裂 GFP 片段的点状 GFP 信号，这表明尽管很难排除这些细胞器接触位点是由分裂 GFP 探针的不可逆结合人工形成的可能性，但这些细胞器同时与多个细胞器形成接触位点。重要的是，内质网和线粒体的重叠区域中的分裂 GFP 信号主要与 ERMES 共定位，后者是一种真实的内质网-线粒体连接结构，这表明分裂 GFP 的组装取决于预先存在的细胞器之间的接触位点。我们还证实，该分裂 GFP 系统可用于检测 HeLa 细胞中的 ER-线粒体接触位点。因此，我们提出分裂 GFP 系统是一种在活酵母和哺乳动物细胞中观察和分析细胞器接触位点的潜在工具。
