Mechanism of Evenness Interrupted (Evi)-Exosome Release at Synaptic Boutons
Mechanism of Evenness Interrupted (Evi)-Exosome Release at Synaptic Boutons
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DOI：
10.1074/jbc.m112.342667
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发表时间：
2012-05-11
影响因子：
4.8
通讯作者：
Budnik, Vivian
中科院分区：
文献类型：
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作者：
Koles, Kate;Nunnari, John;Budnik, Vivian
Wnt信号在突触发育和可塑性中起着关键作用。然而，Wnt释放并到达靶细胞的机制尚未得到解决。在突触发育过程中，果蝇Wnt 1的分泌需要Evi/Wls的功能，Evi/Wls是一种Wingless结合蛋白，沿着Wingless在神经肌肉接头处分泌。考虑到Evi是一种跨膜蛋白，这些研究表明存在一种新的跨突触通讯的囊泡机制，可能是外泌体的形式。为了建立Evi囊泡释放的机制，我们在培养的细胞中使用dsRNA测定来筛选当下调时阻止Evi囊泡释放的基因。我们确定了两种蛋白质，Rab 11和Syntaxin 1A（Syx 1A），这是Evi囊泡释放所需的。为了确定是否在神经肌肉接头中使用相同的机制，我们改变了运动神经元中Rab 11和Syx 1A的活性，并确定了对Evi释放的影响。我们发现Syx 1A，Rab 11及其效应物Myosin 5是Evi囊泡释放所必需的。此外，突触终扣的超微结构分析表明存在多泡体，参与外泌体的产生和释放的细胞器，这些多泡体含有Evi。我们还使用质谱，电子显微镜和生化技术来表征来自培养细胞的外泌体部分。我们的研究表明，分泌的Evi囊泡在其他系统中与外泌体显示出显着的保守性。总之，我们的观察揭示了Evi囊泡释放所需的一些体内机制。
Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.