Department of Cell Biology & Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
PLoS One. 2019 Jul 24;14(7):e0220007. doi: 10.1371/journal.pone.0220007. eCollection 2019.
Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.
细胞外囊泡（EVs）被认为在细胞间通讯中很重要，并因其作为疾病潜在生物标志物的潜力而引起了极大的兴趣。然而，能够阐明其释放机制的定量方法却很少。在这里，我们描述了一种基于细胞的测定方法，用于使用与小的、ATP 非依赖性报告酶 Nanoluciferase 融合的富含 EV 的四跨膜蛋白 CD63 来监测 EV 释放。通过比较培养基中与细胞内剩余的荧光素酶活性，监测稳定表达细胞系中含 CD63 的 EV 释放。HEK293、U2OS、U87 和 SKMel28 细胞在 5 小时内以 EV 的形式释放了总细胞 CD63 的 0.3%-0.6%，具体取决于细胞系。为了鉴定对 CD63 含 EV 分泌很重要的细胞机制，我们在 HEK293 细胞中进行了生物活性化合物的筛选。虽然大多数化合物对 EV 释放没有显著影响，但用已知抑制 V-ATP 酶的 plecomacrolides 巴弗洛霉素或康纳霉素处理细胞会显著增加 EV 释放。有趣的是，使用弱碱使内体腔碱化没有效果，这表明 V-ATP 酶抑制剂通过 pH 独立增强了 EV 释放。在小样本中定量 EV 的能力将使未来能够详细研究释放动力学，以及进一步进行化学和遗传筛选以定义参与 EV 分泌的途径。
