Institut Curie, PSL Research University, INSERM U932, Paris, France.
Université de Paris, INSERM, CNRS UMR 7057, Paris, France.
Nat Commun. 2021 Mar 25;12(1):1864. doi: 10.1038/s41467-021-22126-y.
Extracellular vesicles (EVs), including exosomes, are thought to mediate intercellular communication through the transfer of cargoes from donor to acceptor cells. Occurrence of EV-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. Here, we developed a cell-based assay in which EVs containing luciferase- or fluorescent-protein tagged cytosolic cargoes are loaded on unlabeled acceptor cells. Results from dose-responses, kinetics, and temperature-block experiments suggest that EV uptake is a low yield process (1% spontaneous rate at 1 h). Further characterization of this limited EV uptake, through fractionation of membranes and cytosol, revealed cytosolic release (30% of the uptaken EVs) in acceptor cells. This release is inhibited by bafilomycin A1 and overexpression of IFITM proteins, which prevent virus entry and fusion. Our results show that EV content release requires endosomal acidification and suggest the involvement of membrane fusion.
细胞外囊泡（EVs），包括外泌体，被认为通过将货物从供体细胞转移到受体细胞来介导细胞间通讯。EV 内容物在受体细胞内的传递尚未得到明确证明，更不用说定量了，这仍然存在争议。在这里，我们开发了一种基于细胞的测定方法，其中含有荧光素酶或荧光蛋白标记的胞质货物的 EV 被加载到未标记的受体细胞上。剂量反应、动力学和温度阻断实验的结果表明，EV 摄取是一个低产过程（在 1 小时时自发率约为 1%）。通过对膜和细胞质进行分级分离，进一步对这种有限的 EV 摄取进行了表征，发现受体细胞中存在细胞质释放（摄取的 EV 的约 30%）。该释放被巴佛洛霉素 A1 和 IFITM 蛋白的过表达抑制，IFITM 蛋白可阻止病毒进入和融合。我们的结果表明，EV 内容物的释放需要内体酸化，并提示膜融合的参与。
