JBC：首次发现Evi外切体在突触扣结释放的机制

Wnt信号在突触发展过程中起着决定性作用。然而，Wnt被释放并作用到靶细胞的机制还未阐明。近日，来自美国马萨诸塞大学医学院的Vivian Budnik等人建立了一个体内系统来阐明外切体释放机制，结果发现了与之有关的三种蛋白Rab11, Syntaxin 1A 和 Myosin5。相关研究发表于3月21日的美国《生化周刊》（Journal of Biological Chemistry）上。

外切体复合物，又简称为外切酶体或外切体，是一种蛋白质复合物，能够降解各种不同的RNA。由于复合物表现为核糖核酸外切酶活性，所以被命名为外切体。外切体复合物只存在于真核细胞和古菌中；而细菌中则对应有组成和结构更为简单的“降解体”复合物来发挥类似的功能。

Evi/Wls是一种Wg结合蛋白，可以连同Wg并释放于肌肉神经节点(NMJ)。在突触发展阶段，果蝇Wnt1的分泌需要Evi/Wls的作用。考虑到Evi是一种跨膜蛋白，这些研究表明跨突触通讯的机制可能是以外切体的形式进行。为了研究Evi囊泡释放的机制，研究人员使用了dsRNA分析培养的细胞，来筛选被下调时可以阻止Evi囊泡释放的基因。结果他们鉴定了两种蛋白，即Rab11和Syntaxin 1A (Syx1A)，都与Evi囊泡释放有关。为了验证是否在体内NMJ中存在相同的机制，研究人员改变了Rab11和Syntaxin 1A (Syx1A)在运动神经元中的活性来确定其对Evi囊泡释放的影响。结果发现，Syx1A, Rab11及其效应物Myosin5，都被需要于Evi囊泡的正常释放。此外，超微结构分析这些突触扣结证明了多囊泡体(MVBs)的存在，细胞器参与了外切体的产生以及释放，而且这些多囊泡体包含了Evi。研究人员还使用了质谱分析法，电子显微镜以及生化技术来研究来自培养细胞中外切体的组分。

此次研究表明：在其它系统中，分泌的Evi囊泡表现对外切体出显著性的保护作用。总的说来，该研究阐明了一些体内Evi囊泡的释放机制，并且这是第一次在体内阐明外切体在神经系统中的通讯机制。（生物谷Deepblue编译）

doi: 10.1074/jbc.M112.342667
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Mechanism of Evi-exosome release at synaptic boutons

Kate Koles, John Nunnari, Ceren Korkut, Romina Barria, Cassandra Brewer, Yihang Li, John Leszyk, Bing Zhang and Vivian Budnik.

Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells is unresolved. During synaptic development the secretion of Drosophila Wnt1, Wingless (Wg), requires the function of Evi/Wls, a Wg-binding protein that is secreted along with Wg at the neuromuscular junction (NMJ). 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 downregulated prevent the release of Evi-vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi-vesicle release. To determine if the same mechanisms were used in vivo at the NMJ, we altered the activity of Rab11 and Syx1A in motorneurons 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 (MVBs), organelles involved in the production and release of exosomes, and these MVBs 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.