Active Wnt proteins are secreted on exosomes

Julia Christina Gross, Varun Chaudhary, Kerstin Bartscherer and Michael Boutros 

Wnt信号在发育过程中和许多疾病中起着重要作用。作为形态素，疏水性Wnt蛋白在距离上发挥其功能，诱导细胞形态建模和分化决定。最近的研究已经确定了几个促进Wnt蛋白分泌所需的因素；然而，Wnts如何在细胞外空间中传播仍然是一个尚未解决的问题。在这里，我们展示了在果蝇发育过程中和人类细胞中，Wnts通过外泌体分泌。我们证明外泌体携带Wnts表面来诱导靶细胞中的Wnt信号活性。与货物受体Evi/WIs一起，Wnts通过内体区域转运到外泌体，这个过程需要R-SNARE Ykt6。我们的研究证明了Wnt蛋白的细胞外囊泡转运在功能上具有进化保守性。

Figure 1 Wnt proteins are secreted on exosomes. (a) Differential centrifugation protocol for the isolation of exosomes from cell culture supernatants. (b,c) Representative transmission electron microscope image of the 100,000g pellet fraction from supernatant of HEK293 cells (b) and of Drosophila Kc167 cells contrast stained with 3% uranyl acetate (c). Scale bars, 250 nm. (d) Immunoblot of cell extract (CX), supernatant (SN) and 100,000g pellet (P100) of mouse Wnt3A–L cell supernatant. (e) P100 of mouse Wnt3A–L cell supernatant was loaded on top of a step sucrose gradient (1.08–1.28 g ml−1 ; that is, 0.8–2 M) and ten fractions collected and analysed for Wnt and exosomal proteins. (f,g) Immunoblot analysis of cell extract, P100 and supernatant before and after ultracentrifugation of Drosophila Kc167 cells. Full images of blots (d–g) are presented in Supplementary Fig. S3c. (h) Immunogold labelling of Kc167 P100 with anti-Wg antibodies. Scale bar, 100 nm. A representative example of three replicates is shown.

图1 Wnt蛋白质通过外泌体分泌。(a) 细胞培养上清液中外泌体的差速离心分离。(b,c) HEK293细胞上清液中透射电镜图像(b)，以及用3%乙酸铀酸盐对果蝇Kc167细胞进行对比染色的图像(c)。比例尺，250纳米。(d) 对小鼠Wnt3A-L细胞上清液的细胞提取物（CX）、上清液（SN）和100,000g沉淀（P100）进行免疫印迹分析。(e) 将小鼠Wnt3A-L细胞上清液的P100加载在阶梯蔗糖梯度（1.08–1.28 g ml−1；即0.8–2 M）的顶部，收集十个分数并分析Wnt和外泌体蛋白。(f,g) 对果蝇Kc167细胞进行超速离心前后的细胞提取物、P100和上清液的免疫印迹分析。免疫印迹的完整图像（d–g）见补充图S3c。(h) 用抗-Wg抗体对Kc167 P100进行免疫金标记。比例尺，100纳米。显示了三个重复实验的代表性示例。

Electron microscopy.

Purified exosomes were left to settle on carbon-coated grids. After staining with 3% uranyl acetate, grids were air-dried and visualized using a transmission electron microscope (Zeiss EM900). For immunogold labelling, pelleted exosomes were plated on grids, blocked and stained in anti-Wg antibody (4D4, 1:5), then incubated in secondary anti-mouse antibody and labelled with protein A–gold particles (10 nm). Each staining step was followed by five PBS washes and finally ten washes in H2O before contrast staining with 3% uranyl acetate. Cryosectioning was carried out as described previously72.

电子显微镜。

纯化的外泌体留在碳涂层网格上。经过3%乙酸铀酸盐染色后被风干并使用透射电子显微镜（Zeiss EM900）进行观察，免疫电镜，沉淀的外泌体被铺在网格上，被阻断并用抗-Wg抗体（4D4，1:5）染色，然后孵育在次级抗小鼠抗体中，并用蛋白A-金颗粒（10nm）标记。每个染色步骤后进行五次PBS洗涤，最后进行十次H2O洗涤，在使用3%乙酸铀酸盐对比染色前。切片过程按照之前的描述进行。