Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles
Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles
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DOI：
10.1242/jcs.128868
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发表时间：
2013-12-15
影响因子：
4
通讯作者：
Raposo, Graca
中科院分区：
文献类型：
--
作者：
Colombo, Marina;Moita, Catarina;Raposo, Graca
外泌体是内体多泡体（MVB）与质膜融合后分泌的细胞外囊泡（EV）。它们的生物发生机制尚未完全确定，但它们可用于调节外泌体的形成，因此是了解外泌体功能的一个有前途的工具。我们已经进行了RNA干扰筛选靶向23个组件的内体分选复合物所需的运输（ESCRT）机械和相关的蛋白质在MHC II类（MHC II）表达HeLa-CIITA细胞。HRS、STAM 1或TSG 101的沉默减少了EV相关的CD 63和MHC II的分泌，但每个基因不同地改变了分泌的EV的大小和/或蛋白质组成，如通过免疫电子显微镜定量的。相比之下，VPS 4 B的消耗增加了这种分泌，同时不改变EV的特征。对于其他几个ESCRT亚基，不可能从筛选中得出关于它们参与外泌体生物发生的任何结论。有趣的是，阿利克斯的沉默增加了MHC II外泌体的分泌，这是细胞内MHC II蛋白和mRNA水平总体增加的结果。在人树突状细胞（DC）中，阿利克斯耗竭也增加了细胞中的MHC II，但在释放的CD 63阳性EV中没有。这种差异可能是由于更大的异质性的大小，较高的MHC II和较低的CD 63水平从DC回收的囊泡相比，HeLa CIITA。结果揭示了选定的ESCRT组分和辅助蛋白在HeLa-CIITA的外泌体分泌和组成中的作用。他们还强调了肿瘤细胞系和原代DC分泌的囊泡的生物遗传差异。
Exosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis have not yet been fully identified although they could be used to modulate exosome formation and therefore are a promising tool in understanding exosome functions. We have performed an RNA interference screen targeting 23 components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells. Silencing of HRS, STAM1 or TSG101 reduced the secretion of EV-associated CD63 and MHC II but each gene altered differently the size and/or protein composition of secreted EVs, as quantified by immuno-electron microscopy. By contrast, depletion of VPS4B augmented this secretion while not altering the features of EVs. For several other ESCRT subunits, it was not possible to draw any conclusions about their involvement in exosome biogenesis from the screen. Interestingly, silencing of ALIX increased MHC II exosomal secretion, as a result of an overall increase in intracellular MHC II protein and mRNA levels. In human dendritic cells (DCs), ALIX depletion also increased MHC II in the cells, but not in the released CD63-positive EVs. Such differences could be attributed to a greater heterogeneity in size, and higher MHC II and lower CD63 levels in vesicles recovered from DCs as compared with HeLa-CIITA. The results reveal a role for selected ESCRT components and accessory proteins in exosome secretion and composition by HeLa-CIITA. They also highlight biogenetic differences in vesicles secreted by a tumour cell line and primary DCs.