细胞外囊泡的内吞作用及其从内体释放的货物。,ACS Nano

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细胞外囊泡的内吞作用及其从内体释放的货物。
ACS Nano ( IF 15.8 ) Pub Date : 2020-04-16 , DOI: 10.1021/acsnano.9b10033
Bhagyashree S Joshi ₁ , Marit A de Beer ₂ , Ben N G Giepmans ₂ , Inge S Zuhorn ₁

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胞外小泡（EVs），例如外泌体，可以通过传递生物分子货物来介导细胞之间的长距离通讯。据推测，电动汽车会在受体细胞中的多泡体（MVB）处进行反向融合，以释放其功能性货物。但是，缺乏直接的证据。追踪高分辨率的电动汽车的细胞吸收以及获得释放电动汽车货物的直接证据是一项挑战，主要是因为技术限制。在这里，我们开发了一种分析方法，结合了最先进的分子工具和相关的光电子显微镜，以鉴定释放电动汽车货物的细胞内位置。通过在EV生产细胞中表达GFP-CD63（GFP与CD63的胞质尾巴的融合），将GFP加载到EV内。此外，我们通过基因工程改造了一种表达抗GFP荧光体的细胞系，该荧光体可以特异性识别EV货物（GFP）。用GFP-CD63 EV将表达GFP的抗GFP抗体的细胞温育导致形成荧光小点，表明GFP的胞质暴露。在EV受体细胞中未观察到内体损伤。对GFP /液体双阳性点的基本结构的超微结构分析表明，EV货物的释放是从内体/溶酶体发生的。最后，我们表明中和内体的内体pH值和胆固醇蓄积会导致EV货物暴露受阻。总之，我们报道了一部分内在化的电动汽车以依赖于酸化的方式与内体/溶酶体的限制膜融合，从而导致电动汽车的货物暴露于细胞质中。

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Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes.

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-the-art 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.

更新日期：2020-04-13

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