Improved Split-GFP Systems for Visualizing Organelle Contact Sites in Yeast and Human Cells.
Improved Split-GFP Systems for Visualizing Organelle Contact Sites in Yeast and Human Cells.
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DOI：
10.3389/fcell.2020.571388
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发表时间：
2020
影响因子：
5.5
通讯作者：
Tamura Y
中科院分区：
文献类型：
--
作者：
Tashiro S;Kakimoto Y;Shinmyo M;Fujimoto S;Tamura Y
细胞器间接触位点作为介导不同细胞器之间代谢物（包括脂质和离子）交换的功能专门区域而引起了广泛关注。然而，对细胞器间接触位点的研究还处于早期阶段，直接介导细胞器间相互作用的因素以及接触的数量和程度如何调节仍然是个谜。作为回答这些问题的第一步，我们之前开发了 split-GFP 探针，可以可视化和量化酵母和人类培养细胞中的多个细胞器间接触位点。然而，分裂 GFP 探针具有诱导两个不同细胞器膜之间人工连接的缺点，特别是在过度表达时。在本研究中，我们开发了一种表达 split-GFP 探针的方法，其表达保持在低水平，不同酵母细胞之间的差异最小。此外，我们构建了 HeLa 细胞系，其中可以通过添加强力霉素来诱导 split-GFP 探针的表达，以最大程度地减少人为影响。改进的 split-GFP 系统可能是量化细胞器接触位点和筛选参与酵母和哺乳动物细胞细胞器-细胞器束缚的新因子的可靠工具。
Inter-organelle contact sites have attracted a lot of attention as functionally specialized regions that mediate the exchange of metabolites, including lipids and ions, between distinct organelles. However, studies on inter-organelle contact sites are at an early stage and it remains enigmatic what directly mediates the organelle-organelle interactions and how the number and degree of the contacts are regulated. As a first step to answer these questions, we previously developed split-GFP probes that could visualize and quantify multiple inter-organelle contact sites in the yeast and human cultured cells. However, the split-GFP probes possessed a disadvantage of inducing artificial connections between two different organelle membranes, especially when overexpressed. In the present study, we developed a way to express the split-GFP probes whose expressions remained at low levels, with minimal variations between different yeast cells. Besides, we constructed a HeLa cell line in which the expression of the split-GFP probes could be induced by the addition of doxycycline to minimize the artificial effects. The improved split-GFP systems may be faithful tools to quantify organelle contact sites and screen new factors involved in organelle-organelle tethering in yeast and mammalian cells.
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影响因子：
14.8
作者：
Gietz, R. Daniel;Schiestl, Robert H.
通讯作者：
Schiestl, Robert H.
影响因子：
4.6
作者：
Kakimoto Y;Tashiro S;Kojima R;Morozumi Y;Endo T;Tamura Y
通讯作者：
Tamura Y
影响因子：
16
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
3.3
作者：
Pan, XZ;Roberts, P;Goldfarb, DS
通讯作者：
Goldfarb, DS