Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS
Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS
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DOI：
10.1073/pnas.0906677106
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发表时间：
2009-11-03
影响因子：
11.1
通讯作者：
Walla, Peter J.
中科院分区：
文献类型：
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作者：
Cypionka, Anna;Stein, Alexander;Walla, Peter J.
神经元胞吐由SNARE蛋白小突触蛋白2/VAMP、突触融合蛋白1A和SNAP-25 A介导。虽然已经确定这些蛋白质在人工膜中重构后介导膜融合，但迄今为止难以监测反应的中间阶段。使用共聚焦双光子装置，我们应用荧光互相关光谱（FCCS）和荧光寿命分析来区分对接和融合的脂质体。我们表明，脂质体群体，无论是非相互作用，或正在进行对接和融合，以及多种相互作用，可以定量区分，而不需要固定的脂质双层。当含有稳定的突触融合蛋白1A/SNAP-25 A复合物的脂质体与含有小突触蛋白2的脂质体混合时，我们观察到快速对接先于融合。因此，对接的中间体在反应的初始阶段积累。此外，观察到多个对接状态的快速形成，平均四个脂质体彼此相互作用。当比较不同尺寸的脂质体时，只有脂质混合的速率取决于脂质体尺寸，而不是对接的速率。我们的研究结果表明，在适当的条件下，对接状态，介导的trans-SNARE相互作用，可以被隔离，构成一个中间体的融合途径。
Neuronal exocytosis is mediated by the SNARE proteins synaptobrevin 2/VAMP, syntaxin 1A, and SNAP-25A. While it is well-established that these proteins mediate membrane fusion after reconstitution in artificial membranes, it has so far been difficult to monitor intermediate stages of the reaction. Using a confocal two-photon setup, we applied fluorescence cross-correlation spectroscopy (FCCS) and fluorescence lifetime analysis to discriminate between docking and fusion of liposomes. We show that liposome populations that are either non-interacting, or are undergoing docking and fusion, as well as multiple interactions can be quantitatively discriminated without the need for immobilizing the lipid bilayers. When liposomes containing a stabilized syntaxin 1A/SNAP-25A complex were mixed with liposomes containing synaptobrevin 2, we observed that rapid docking precedes fusion. Accordingly, docked intermediates accumulated in the initial phase of the reaction. Furthermore, rapid formation of multiple docked states was observed with on average four liposomes interacting with each other. When liposomes of different sizes were compared, only the rate of lipid mixing depended on the liposome size but not the rate of docking. Our results show that under appropriate conditions a docked state, mediated by trans-SNARE interactions, can be isolated that constitutes an intermediate in the fusion pathway.