Imaging single membrane fusion events mediated by SNARE proteins
Imaging single membrane fusion events mediated by SNARE proteins
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DOI：
10.1073/pnas.0401779101
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发表时间：
2004-05-11
影响因子：
11.1
通讯作者：
Simon, SM
中科院分区：
文献类型：
--
作者：
Fix, M;Melia, TJ;Simon, SM
利用全内反射荧光显微镜，我们开发了一种方法来监测含有囊泡可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARES)的蛋白脂质体与含有同源靶标SNARES的支撑平面双层之间的个别融合事件。通过荧光磷脂从囊泡膜向靶双层的递送和随后的侧向铺展来量化单个囊泡到靶膜的接近、对接和融合。提高二价阳离子(Ca~(2+)和Mg~(2+))的融合几率。单个囊泡的融合起始时间是全长蛋白的30倍，但即使在没有H-ABC结构域的情况下，囊泡融合仍然对钙离子的增加做出反应。我们的观察证实，SNARE核心复合体足以以与细胞中大多数膜融合过程相称的速度融合两个相对的膜双层。这种对单个囊泡融合的实时分析为研究圈套和辅助蛋白如何调节体内的融合过程打开了机制研究的大门。
Using total internal reflection fluorescence microscopy, we have developed an assay to monitor individual fusion events between proteoliposomes containing vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and a supported planar bilayer containing cognate target SNAREs. Approach, docking, and fusion of individual vesicles to the target membrane were quantified by delivery and subsequent lateral spread of fluorescent phospholipids from the vesicle membrane into the target bilayer. Fusion probability was increased by raising divalent cations (Ca2+ and Mg2+). Fusion of individual vesicles initiated in 30-fold compared to the full-length protein, but even in the absence of the H-abc domain, vesicle fusion was still enhanced in response to Ca2+ increase. Our observations establish that the SNARE core complex is sufficient to fuse two opposing membrane bilayers at a speed commensurate with most membrane fusion processes in cells. This real-time analysis of single vesicle fusion opens the door to mechanistic studies of how SNARE and accessory proteins regulate fusion processes in vivo.