Wnt信號在突觸發(fā)展過程中起著決定性作用。然而,，Wnt被釋放并作用到靶細胞的機制還未闡明,。近日，來自美國馬薩諸塞大學醫(yī)學院的Vivian Budnik等人建立了一個體內系統(tǒng)來闡明外切體釋放機制,，結果發(fā)現(xiàn)了與之有關的三種蛋白Rab11, Syntaxin 1A 和 Myosin5,。相關研究發(fā)表于3月21日的美國《生化周刊》（Journal of Biological Chemistry）上。
外切體復合物,，又簡稱為外切酶體或外切體,，是一種蛋白質復合物，能夠降解各種不同的RNA,。由于復合物表現(xiàn)為核糖核酸外切酶活性,，所以被命名為外切體。外切體復合物只存在于真核細胞和古菌中,；而細菌中則對應有組成和結構更為簡單的“降解體”復合物來發(fā)揮類似的功能,。
Evi/Wls是一種Wg結合蛋白，可以連同Wg并釋放于肌肉神經節(jié)點(NMJ),。在突觸發(fā)展階段,，果蠅Wnt1的分泌需要Evi/Wls的作用?？紤]到Evi是一種跨膜蛋白,，這些研究表明跨突觸通訊的機制可能是以外切體的形式進行。為了研究Evi囊泡釋放的機制,，研究人員使用了dsRNA分析培養(yǎng)的細胞,，來篩選被下調時可以阻止Evi囊泡釋放的基因。結果他們鑒定了兩種蛋白,，即Rab11和Syntaxin 1A (Syx1A),，都與Evi囊泡釋放有關。為了驗證是否在體內NMJ中存在相同的機制,，研究人員改變了Rab11和Syntaxin 1A (Syx1A)在運動神經元中的活性來確定其對Evi囊泡釋放的影響,。結果發(fā)現(xiàn)，Syx1A, Rab11及其效應物Myosin5,，都被需要于Evi囊泡的正常釋放,。此外，超微結構分析這些突觸扣結證明了多囊泡體(MVBs)的存在，細胞器參與了外切體的產生以及釋放,，而且這些多囊泡體包含了Evi,。研究人員還使用了質譜分析法，電子顯微鏡以及生化技術來研究來自培養(yǎng)細胞中外切體的組分,。
此次研究表明：在其它系統(tǒng)中,，分泌的Evi囊泡表現(xiàn)對外切體出顯著性的保護作用,?？偟恼f來，該研究闡明了一些體內Evi囊泡的釋放機制,，并且這是第一次在體內闡明外切體在神經系統(tǒng)中的通訊機制,。（生物谷Deepblue編譯）
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.
