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Abstract：
In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.
摘要：
在神经元细胞类型中，囊泡胞吐作用由SNARE（可溶性NSF附着受体）复合物控制，该复合物由突触蛋白2，SNAP25和syntaxin1组成。这些蛋白质是囊泡引发和融合所必需的。我们生成了一个改进的基于SNAP25的SNARECOmplexReporter（SCORE2），其中包含mCeruelan3和Venus，并在SNAP25敲除的胚胎小鼠嗜铬细胞中过表达。该构建体挽救了囊泡融合,其具有与野生型细胞中的融合无法区分的性质。将使用电化学检测器阵列的单个释放事件的电化学成像与全内反射荧光共振能量转移（TIR-FRET）成像相结合，揭示了在单个融合事件之前65ms的FRET快速增加。实验是在对接的稳态循环条件下进行的，启动,和融合，延迟表明FRET的变化反映了囊泡的紧密对接和启动，然后在〜65ms后进行融合。考虑到不存在野生型SNAP25,SCORE2允许确定融合位点处的分子数量和改变构象的数量。在引发步骤中改变构象的SNAP25分子的数量随囊泡大小和质膜中SNAP25密度的增加而增加，并等于囊泡-质膜接触区中存在的拷贝数。我们估计在wt细胞中，6至7个拷贝的SNAP25在引发步骤期间改变构象。
