Single Vesicle Assaying of SNARE-Synaptotagmin-Driven Fusion Reveals Fast and Slow Modes of Both Docking and Fusion and Intrasample Heterogeneity
Single Vesicle Assaying of SNARE-Synaptotagmin-Driven Fusion Reveals Fast and Slow Modes of Both Docking and Fusion and Intrasample Heterogeneity
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DOI：
10.1016/j.bpj.2010.12.3730
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发表时间：
2011-02-16
影响因子：
3.4
通讯作者：
Stamou, Dimitrios G.
中科院分区：
文献类型：
--
作者：
Christensen, Sune M.;Mortensen, Michael W.;Stamou, Dimitrios G.
SNAREs功能化的囊泡和synaptotagmin-1胞浆C2AB结构域之间的脂质混合概括了神经元胞外分泌的基本Ca2+依赖性。然而，在传统的集合脂质混合实验中，不可能区分Ca2+是否加速了囊泡的对接或融合。在这里，我们报告了一种基于荧光显微镜的检测，以监测单个囊泡对的snare介导的对接和融合。原位测量扩散粒子的浓度使我们能够通过最大似然方法量化对接率。该分析表明，C2AB和Ca2+加速囊泡-囊泡时钟超过两个数量级。然而，将测量的对接速率与整体脂质混合动力学进行比较，表明在大多数情况下，双层融合仍然是限速步骤。我们的单囊泡结果显示，只有接近60%的囊泡对接，只有接近6%的对接囊泡融合。单个囊泡的脂质混合速度很快（t（混合）< 1 s），而集合实验显示两个缓慢的混合过程，t（混合）近似于1分钟，t（混合）近似于20分钟。在这个阶段，几种不同的对接和融合途径的存在还不能被合理化，但可能与样品内的异质性有关，可能以脂质和/或蛋白质组成的形式存在。
Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca2+ dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca2+ accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca2+ accelerate vesicle-vesicle clocking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only similar to 60% of the vesicles dock and only similar to 6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t(mix) < 1 s) while an ensemble assay revealed two slow mixing processes with t(mix) similar to 1 min and t(mix) similar to 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition.