Supported bilayers with excess membrane reservoir: a template for reconstituting membrane budding and fission.
Supported bilayers with excess membrane reservoir: a template for reconstituting membrane budding and fission.
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DOI：
10.1016/j.bpj.2010.04.036
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发表时间：
2010-07
影响因子：
3.4
通讯作者：
T. Pucadyil;S. Schmid
中科院分区：
文献类型：
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作者：
T. Pucadyil;S. Schmid
对囊泡运输中膜定位过程的完整机制理解，如膜出芽和裂变，需要用生物化学定义的成分从生物化学定义的底物中重建它们。由囊泡融合形成的支撑双层为这一目的提供了有吸引力的基质。然而，传统的双层支撑层缺乏足够的膜库来重建膜出芽和裂变事件。我们描述了在高离子强度条件下，含负电荷脂质体的脂质体在硅珠上融合形成具有过量膜储层（SUPER）模板的支撑双层。使用基于荧光显微镜的检测来监测支撑双分子层形成的早期和晚期阶段，我们发现离子强度的增加导致脂质体吸附率的增加和随后在支撑双分子层形成过程中的融合。然而，这两种速率不成比例地增加，导致过量储层随着离子强度的增加而积累。超级模板允许无缝应用基于显微镜的分析来分析膜定位过程，以及基于沉淀的分析，以分离从膜上释放的囊泡和非囊泡产物。这里提出的结果强调了这些模板在分析囊泡和非囊泡运输过程中的一般效用。
A complete mechanistic understanding of membrane-localized processes in vesicular transport, such as membrane budding and fission, requires their reconstitution with biochemically-defined components from a biochemically-defined substrate. Supported bilayers formed by vesicle fusion represent an attractive substrate for this purpose. However, conventional supported bilayers lack a sufficient membrane reservoir to recreate membrane budding and fission events. We describe the formation of supported bilayers with excess membrane reservoir (SUPER) templates from the fusion of liposomes containing negatively charged lipids on silica beads under high-ionic-strength conditions. Using a fluorescence microscopy-based assay to monitor early and late stages of supported bilayer formation, we show that an increase in ionic strength leads to an increase in the rates of liposome adsorption and subsequent fusion during formation of supported bilayers. The two rates, however, increase disproportionally, leading to accumulation of excess reservoir with an increase in ionic strength. SUPER templates allow the seamless application of microscopy-based assays to analyze membrane-localized processes together with sedimentation-based assays to isolate vesicular and nonvesicular products released from the membrane. The results presented here emphasize the general utility of these templates for analyzing vesicular and nonvesicular transport processes.