Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.
Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.
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DOI：
10.1038/ncomms8029
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发表时间：
2015-05-13
影响因子：
16.6
通讯作者：
Breakefield, Xandra O.
中科院分区：
文献类型：
--
作者：
Lai, Charles P.;Kim, Edward Y.;Badr, Christian E.;Weissleder, Ralph;Mempel, Thorsten R.;Tannous, Bakhos A.;Breakefield, Xandra O.
细胞外囊泡（EV）递送和货物RNA翻译的准确时空评估需要特异性和稳健的活细胞成像技术。在这里，我们设计了光学报告分子来标记多个EV群体，用于可视化和跟踪培养和体内细胞群体之间的肿瘤EV释放，摄取和交换。将增强型绿色荧光蛋白（EGFP）和串联二聚体番茄（tdTomato）在NH 2-末端与棕榈酰化信号（PalmGFP，PalmtdTomato）融合用于EV膜标记。为了监测EV-RNA货物，用MS 2 RNA结合序列标记编码PalmtdTomato的转录物，并通过与EGFP融合的噬菌体MS 2外壳蛋白的共表达来检测。通过多重荧光和生物发光EV膜报告，我们揭示了EV摄取和EV递送的货物mRNA在癌细胞中的翻译的快速动力学，其发生在细胞之间水平转移后1小时内。这些研究证实，EV介导的通信是动态的和多向的细胞之间的功能mRNA的交付。 细胞外囊泡（EV）通过细胞物质的交换而充当细胞间通信的管道，而没有直接的细胞与细胞接触。在这里，作者开发了一种多重报告系统，可以监测不同细胞群之间的EV交换，货物递送和蛋白质翻译。
Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA. Extracellular vesicles (EVs) act as a conduit for intercellular communication through the exchange of cellular materials without direct cell-to-cell contacts. Here the authors develop a multiplexed reporter system that allows monitoring of EV exchange, cargo delivery and protein translation between different cell populations.
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影响因子：
46.9
作者：
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通讯作者：
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影响因子：
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