Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes
Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes
复制标题
DOI：
10.1021/acsnano.9b10033
复制
发表时间：
2020-04-28
期刊：
影响因子：
17.1
通讯作者：
Zuhorn, Inge S.
中科院分区：
文献类型：
--
作者：
Joshi, Bhagyashree S.;de Beer, Marit A.;Zuhorn, Inge S.
细胞外囊泡（EV），如外来体，可以通过递送生物分子货物来介导细胞之间的长距离通信。据推测，EV在受体细胞中的多泡体（MVB）处经历回融合以释放其功能性货物。然而，缺乏直接证据。以高分辨率追踪EV的细胞摄取以及获取EV货物释放的直接证据具有挑战性，主要是因为技术限制。在这里，我们开发了一种分析方法，结合国家的最先进的分子工具和相关的光学和电子显微镜，以确定EV货物释放的细胞内网站。通过在EV生产细胞中表达GFP-CD 63（GFP与CD 63的胞质尾部的融合物）将GFP装载到EV内。此外，我们基因工程改造的细胞系，其表达特异性识别EV货物（GFP）的抗GFP荧光抗体。将表达抗GFP荧光抗体的细胞与GFP-CD 63 EV一起孵育导致荧光抗体斑点的形成，表明GFP的胞质暴露。在EV受体细胞中未观察到内体损伤。在GFP/荧光抗体双阳性斑点的基础结构的超微结构分析表明，EV货物释放发生从内体/溶酶体。最后，我们表明，中和内体pH值和胆固醇积累的内体导致堵塞EV货物曝光。总之，我们报告了一部分内化的EV以酸化依赖的方式与内体/溶酶体的限制膜融合，这导致EV货物暴露于细胞胞质溶胶。
Extracellular vesicles (EVs), such as exosomes, can mediate long-distance communication between cells by delivering biomolecular cargo. It is speculated that EVs undergo back-fusion at multivesicular bodies (MVBs) in recipient cells to release their functional cargo. However, direct evidence is lacking. Tracing the cellular uptake of EVs with high resolution as well as acquiring direct evidence for the release of EV cargo is challenging mainly because of technical limitations. Here, we developed an analytical methodology, combining state-of-theart molecular tools and correlative light and electron microscopy, to identify the intracellular site for EV cargo release. GFP was loaded inside EVs through the expression of GFP-CD63, a fusion of GFP to the cytosolic tail of CD63, in EV producer cells. In addition, we genetically engineered a cell line which expresses anti-GFP fluobody that specifically recognizes the EV cargo (GFP). Incubation of anti-GFP fluobody- expressing cells with GFP-CD63 EVs resulted in the formation of fluobody punctae, designating cytosolic exposure of GFP. Endosomal damage was not observed in EV acceptor cells. Ultrastructural analysis of the underlying structures at GFP/ fluobody double-positive punctae demonstrated that EV cargo release occurs from endosomes/lysosomes. Finally, we show that neutralization of endosomal pH and cholesterol accumulation in endosomes leads to blockage of EV cargo exposure. In conclusion, we report that a fraction of internalized EVs fuse with the limiting membrane of endosomes/lysosomes in an acidification-dependent manner, which results in EV cargo exposure to the cell cytosol.