Kuhlmann Jan W, Junius Meike, Diederichsen Ulf, Steinem Claudia
Institute of Organic and Biomolecular Chemistry, University of Göttingen, Göttingen, Germany.
Institute of Organic and Biomolecular Chemistry, University of Göttingen, Göttingen, Germany.
Biophys J. 2017 Jun 6;112(11):2348-2356. doi: 10.1016/j.bpj.2017.04.032.
In vitro single-vesicle fusion assays are important tools to analyze the details of SNARE-mediated fusion processes. In this study, we employed planar pore-spanning membranes (PSMs) prepared on porous silicon substrates with large pore diameters of 5 μm, allowing us to compare the process of vesicle docking and fusion on the supported parts of the PSMs (s-PSMs) with that on the freestanding membrane parts (f-PSM) under the exact same experimental conditions. The PSMs harbor the t-SNARE ΔN49-complex to investigate the dynamics and fusogenicity of single large unilamellar vesicles doped with the v-SNARE synaptobrevin 2 by means of spinning-disc confocal microscopy with a time resolution of 10 ms. Our results demonstrate that vesicles docked to the s-PSM were fully immobile, whereas those docked to the f-PSM were mobile with a mean diffusion coefficient of 0.42 μm/s. Despite the different dynamics of the vesicles on the two membrane types, similar fusion kinetics were observed, giving rise to a common fusion mechanism. Further investigations of individual lipid mixing events on the s-PSMs revealed semi-stable post-fusion structures.
体外单囊泡融合测定是分析SNARE介导的融合过程细节的重要工具。在本研究中，我们使用了在孔径为5μm的多孔硅基板上制备的平面跨孔膜（PSM），这使我们能够在完全相同的实验条件下，比较囊泡在PSM的支撑部分（s-PSM）和独立膜部分（f-PSM）上的对接和融合过程。PSM含有t-SNARE ΔN49复合物，通过时间分辨率为10ms的转盘共聚焦显微镜，研究掺杂有v-SNARE突触小泡蛋白2的单个大单层囊泡的动力学和融合能力。我们的结果表明，停靠在s-PSM上的囊泡完全不移动，而停靠在f-PSM上的囊泡是可移动的，平均扩散系数为0.42μm/s。尽管两种膜类型上的囊泡动力学不同，但观察到了相似的融合动力学，从而产生了共同的融合机制。对s-PSM上单个脂质混合事件的进一步研究揭示了半稳定的融合后结构。
