Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells.
Quantitative characterization of extracellular vesicle uptake and content delivery within mammalian cells.
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DOI：
10.1038/s41467-021-22126-y
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发表时间：
2021-03-25
影响因子：
16.6
通讯作者：
Lavieu G
中科院分区：
文献类型：
--
作者：
Bonsergent E;Grisard E;Buchrieser J;Schwartz O;Théry C;Lavieu G
细胞外囊泡（EV），包括外来体，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。受体细胞内EV内容物递送的发生尚未明确证明，更不用说量化，并且仍然存在争议。在这里，我们开发了一种基于细胞的测定，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摄取是一个低产率过程（1 h时自发率约为1%）。通过膜和胞质溶胶的分级分离，进一步表征这种有限的EV摄取，揭示了受体细胞中的胞质溶胶释放（约30%的摄取的EV）。这种释放被巴弗洛霉素A1和IFITM蛋白的过表达抑制，这阻止了病毒进入和融合。我们的研究结果表明，EV内容物的释放需要内体酸化，并建议膜融合的参与。细胞外囊泡介导细胞-细胞通讯，然而，它们在受体细胞内递送其内容物的能力尚不清楚。在这里，作者开发了一种定量分析，并表明细胞外囊泡内容物的释放需要内体酸化，并可能涉及膜融合。
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. Extracellular vesicles mediate cell–cell communication, however, their capacity to deliver their content within acceptor cells is unclear. Here, the authors develop a quantitative assay and show that release of extracellular vesicle contents requires endosomal acidification and may involve membrane fusion.
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影响因子：
16
作者：
Lötvall J;Hill AF;Hochberg F;Buzás EI;Di Vizio D;Gardiner C;Gho YS;Kurochkin IV;Mathivanan S;Quesenberry P;Sahoo S;Tahara H;Wauben MH;Witwer KW;Théry C
通讯作者：
Théry C
DOI：
10.1073/pnas.1418401112
发表时间：
2015-03-24
影响因子：
11.1
作者：
Kanada, Masamitsu;Bachmann, Michael H.;Contag, Christopher H.
通讯作者：
Contag, Christopher H.
影响因子：
17.1
作者：
Joshi, Bhagyashree S.;de Beer, Marit A.;Zuhorn, Inge S.
通讯作者：
Zuhorn, Inge S.
影响因子：
50.3
作者：
Becker A;Thakur BK;Weiss JM;Kim HS;Peinado H;Lyden D
通讯作者：
Lyden D
影响因子：
64.5
作者：
Crewe C;Joffin N;Rutkowski JM;Kim M;Zhang F;Towler DA;Gordillo R;Scherer PE
通讯作者：
Scherer PE