Lipid mixing and content release in single-vesicle, SNARE-driven fusion assay with 1-5 ms resolution.
Lipid mixing and content release in single-vesicle, SNARE-driven fusion assay with 1-5 ms resolution.
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DOI：
10.1016/j.bpj.2009.02.050
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发表时间：
2009-05
影响因子：
3.4
通讯作者：
Tingting Wang;Elizabeth A. Smith;E. Chapman;J. Weisshaar
中科院分区：
文献类型：
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作者：
Tingting Wang;Elizabeth A. Smith;E. Chapman;J. Weisshaar
单泡、基于荧光、陷阱驱动的融合分析能够以5ms/帧、甚至1ms/帧的时间分辨率同时测量脂质混合和内容物释放。V-SNARE囊泡用不同颜色的脂质和含量标记物标记，与支撑在玻璃上的平面t-SNARE双层对接和融合。钙黄绿素荧光的窄峰(持续5毫秒)，由于内容物的释放和去猝灭，在10毫秒内与小叶内的脂质混合相吻合。与单独使用脂质标记相比，峰值提供了更灵敏的检测生产性半输血事件的方法。因此，许多以前被认为是迅速的、完全融合的快速事件现在被重新归类为生产性半融合。完全融合和半融合的时间常数均为5-10ms。在60%的磷脂酰乙醇胺类脂成分中，生产性和死端半融合占所有融合事件的65%。然而，定量分析表明，钙黄绿素被释放到双层以上的空间(囊泡破裂)，而不是双层和玻璃之间的薄水空间。显然，在小叶内混合的瞬间，囊泡的扁平增加了超过爆破点的内部压力。这可能与体内观察表明，膜溶解往往与膜融合竞争有关。
A single-vesicle, fluorescence-based, SNARE-driven fusion assay enables simultaneous measurement of lipid mixing and content release with 5 ms/frame, or even 1 ms/frame, time resolution. The v-SNARE vesicles, labeled with lipid and content markers of different color, dock and fuse with a planar t-SNARE bilayer supported on glass. A narrow (<5 ms duration), intense spike of calcein fluorescence due to content release and dequenching coincides with inner-leaflet lipid mixing within 10 ms. The spike provides more sensitive detection of productive hemifusion events than do lipid labels alone. Consequently, many fast events previously thought to be prompt, full fusion events are now reclassified as productive hemifusion. Both full fusion and hemifusion occur with a time constant of 5–10 ms. At 60% phosphatidylethanolamine lipid composition, productive and dead-end hemifusion account for 65% of all fusion events. However, quantitative analysis shows that calcein is released into the space above the bilayer (vesicle bursting), rather than the thin aqueous space between the bilayer and glass. Evidently, at the instant of inner-leaflet mixing, flattening of the vesicle increases the internal pressure beyond the bursting point. This may be related to in vivo observations suggesting that membrane lysis often competes with membrane fusion.