Reporter gene assay for membrane fusion of extracellular vesicles
Reporter gene assay for membrane fusion of extracellular vesicles
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细胞外囊泡膜融合的报告基因测定
DOI：
10.1101/2021.02.16.431359
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Kuroda Shun’ichi
中科院分区：
文献类型：
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作者：
Somiya Masaharu;Kuroda Shun’ichi
活细胞分泌的胞外小泡(EVS)有望将包括RNA和蛋白质在内的生物货物分子运送到受体细胞的细胞质中。人们越来越需要了解电动汽车细胞间货物运输的机制。然而，缺乏可行的生物测定方法阻碍了我们对EV摄取、膜融合和向受体细胞运送货物的生物学过程的理解。在这里，我们描述了一种报告基因检测，它可以测量EVS在货物递送到受体细胞过程中的膜融合效率。当含有四环素反式激活剂(TTA)融合蛋白的EV被受体细胞内化并与细胞膜融合时，TTA结构域暴露在细胞质中，并被烟草蚀刻病毒蛋白酶切割，从而诱导四环素反应元件(TrE)介导的报告基因在受体细胞中表达。该试验(称为EV介导的Tetraspanin-tTA递送试验，ETTD试验)使我们能够评估EV在受体细胞中的胞浆货物递送效率。在水泡性口炎病毒衍生的膜融合蛋白的帮助下，ETTD检测可以检测到电动汽车货物递送效率的显著提高。此外，ETTD检测可以评估潜在的货物递送促进剂/抑制剂的效果。因此，ETTD检测可能有助于更好地了解EVS胞质货物运输的潜在机制。
Extracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells. Here, we describe a reporter gene assay that can measure the membrane fusion efficiency of EVs during cargo delivery to recipient cells. When EVs containing tetracycline transactivator (tTA)‐fused tetraspanins are internalized by recipient cells and fuse with cell membranes, the tTA domain is exposed to the cytoplasm and cleaved by tobacco etch virus protease to induce tetracycline responsive element (TRE)‐mediated reporter gene expression in recipient cells. This assay (designated asEV‐mediatedtetraspanin‐tTAdelivery assay, ETTD assay), enabled us to assess the cytoplasmic cargo delivery efficiency of EVs in recipient cells. With the help of a vesicular stomatitis virus‐derived membrane fusion protein, the ETTD assay could detect significant enhancement of cargo delivery efficiency of EVs. Furthermore, the ETTD assay could evaluate the effect of potential cargo delivery enhancers/inhibitors. Thus, the ETTD assay may contribute to a better understanding of the underlying mechanism of the cytoplasmic cargo delivery by EVs.