Planar pore-spanning membranes (PSMs) have been shown to be a versatile tool to resolve elementary steps of the neuronal fusion process. However, in previous studies, we monitored only lipid mixing between fusing large unilamellar vesicles and PSMs and did not gather information about the formation of fusion pores. To address this important step of the fusion process, we entrapped sulforhodamine B at self-quenching concentrations into large unilamellar vesicles containing the v-SNARE synaptobrevin 2, which were docked and fused with lipid-labeled PSMs containing the t-SNARE acceptor complex ΔN49 prepared on gold-coated porous silicon substrates. By dual-color spinning disk fluorescence microscopy with a time resolution of ∼20 ms, we could unambiguously distinguish between bursting vesicles, which was only rarely observed (<0.01%), and fusion pore formation. From the time-resolved dual-color fluorescence time traces, we were able to identify different fusion pathways, including remaining three-dimensional postfusion structures with released content and transient openings and closings of the fusion pores. Our results on fusion pore formation and lipid diffusion from the PSM into the fusing vesicle let us conclude that the content release, i.e., fusion pore formation after the merger of the two lipid membranes occurs almost simultaneously.

中文翻译：

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在 SNARE 介导的囊泡与跨孔膜融合过程中观察到的融合孔形成

平面跨孔膜 (PSM) 已被证明是解决神经元融合过程基本步骤的通用工具。然而，在之前的研究中，我们仅监测融合大单层囊泡和 PSM 之间的脂质混合，并没有收集有关融合孔形成的信息。为了解决融合过程的这一重要步骤，我们以自淬灭浓度将硫罗丹明 B 捕获到包含 v-SNARE 突触释放蛋白 2 的大单层囊泡中，这些囊泡与脂质标记的 PSM 对接并融合，其中包含制备的 t-SNARE 受体复合物 ΔN49在镀金多孔硅衬底上。通过时间分辨率约为 20 ms 的双色旋转圆盘荧光显微镜，我们可以明确区分爆裂的囊泡，这很少观察到（<0.01%），和融合孔的形成。从时间分辨的双色荧光时间轨迹中，我们能够识别出不同的融合途径，包括剩余的具有释放内容的三维后融合结构以及融合孔的瞬时开闭。我们关于融合孔形成和脂质从 PSM 扩散到融合囊泡的结果让我们得出结论，内容释放，即两种脂质膜合并后的融合孔形成几乎同时发生。