Productive hemifusion intermediates in fast vesicle fusion driven by neuronal SNAREs
Productive hemifusion intermediates in fast vesicle fusion driven by neuronal SNAREs
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DOI：
10.1529/biophysj.107.107896
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发表时间：
2008-02-15
影响因子：
3.4
通讯作者：
Weisshaar, James C.
中科院分区：
文献类型：
--
作者：
Liu, Tingting;Wang, Tingting;Weisshaar, James C.
体外融合试验使用标记脂质的荧光显微镜来监测含有t-SNARE的平面脂质双层上的单个v-SNARE囊泡对接和融合事件。对于包含磷脂酰胆碱（POPC，84- 85摩尔%）和磷脂酰丝氨酸（DOPS，15摩尔%）的囊泡和双层，先前的工作证明了迅速的完全融合（tau（fus）= 25 ms）。用包含在t-SNARE双层中的0或20%DOPE取代v-SNARE囊泡中的20-60%磷脂酰乙醇胺（DOPE）引起半融合事件。标记的脂质扩散到平面双分子层作为两个时间上不同的波，大概是半融合的外部莱亚。et后接内莱亚。核聚变。与DOPE的融合动力学是显著不均匀的。一些囊泡/对接位点对表现出迅速的完全融合，而另一些表现出半融合。半融合事件大约是半生产性的（导致随后的核心融合在20秒内）和半死胡同。与无蛋白囊泡-囊泡融合研究的预期定性雅阁，半融合速率k（hemi）比核心融合速率k（core）快15-20倍，并且半融合事件的分数随着DOPE百分比的增加而增加。这表明无蛋白和神经元SNARE驱动的融合的潜在分子途径相似。从v-SNARE囊泡中去除磷脂酰丝氨酸对对接或融合没有影响。
An in vitro fusion assay uses fluorescence microscopy of labeled lipids to monitor single v-SNARE vesicle docking and fusion events on a planar lipid bilayer containing t-SNAREs. For vesicles and bilayer comprising phosphatidylcholine (POPC, 84-85% by mol) and phosphatidylserine (DOPS, 15% by mol), previous work demonstrated prompt, full fusion (tau(fus) = 25 ms). Substitution of 20-60% phosphatidylethanolamine (DOPE) for phosphatidylcholine in the v-SNARE vesicle with either 0 or 20% DOPE included in the t-SNARE bilayer gives rise to hemifusion events. Labeled lipids diffuse into the planar bilayer as two temporally distinct waves, presumably hemifusion of the outer lea. et followed by inner lea. et (core) fusion. The fusion kinetics with DOPE is markedly heterogeneous. Some vesicle/docking site pairs exhibit prompt, full fusion while others exhibit hemifusion. Hemifusion events are roughly half productive (leading to subsequent core fusion within 20 s) and half dead-end. In qualitative accord with expectations from studies of protein-free vesicle-vesicle fusion, the hemifusion rate k(hemi) is 15-20 times faster than the core fusion rate k(core), and the fraction of hemifusion events increases with increasing percentage of DOPE. This suggests similar underlying molecular pathways for protein-free and neuronal SNARE-driven fusion. Removal of phosphatidylserine from the v-SNARE vesicle has no effect on docking or fusion.