Early steps of supported bilayer formation probed by single vesicle fluorescence assays

被引:216
作者
Johnson, JM
Ha, T
Chu, S
Boxer, SG
机构
[1] Stanford Univ, Dept Chem, Stanford, CA 94305 USA
[2] Stanford Univ, Dept Phys, Stanford, CA 94305 USA
基金
美国国家科学基金会;
关键词
D O I
10.1016/S0006-3495(02)75337-X
中图分类号
Q6 [生物物理学];
学科分类号
071011 [生物物理学];
摘要
We have developed a single vesicle assay to study the mechanisms of supported bilayer formation. Fluorescently labeled, unilamellar vesicles (30-100 nm diameter) were first adsorbed to a quartz surface at low enough surface concentrations to visualize single vesicles. Fusion and rupture events during the bilayer formation, induced by the subsequent addition of unlabeled vesicles, were detected by measuring two-color fluorescence signals simultaneously. Lipid-conjugated dyes monitored the membrane fusion while encapsulated dyes reported on the vesicle rupture. Four dominant pathways were observed, each exhibiting characteristic two-color fluorescence signatures: 1) primary fusion, in-which an unlabeled vesicle fuses with a labeled vesicle on the surface, is signified by the dequenching of the lipid-conjugated dyes followed by rupture and final merging into the bilayer; 2) simultaneous fusion and rupture, in which a labeled vesicle on the surface ruptures simultaneously upon fusion with an unlabeled vesicle; 3) no dequenching, in which loss of fluorescence signal from both dyes occur simultaneously with the final merger into the bilayer; and 4) isolated rupture (pre-ruptured vesicles), in which a labeled vesicle on the surface spontaneously undergoes content loss, a process that occurs with high efficiency in the presence of a high concentration of Texas Red-labeled lipids. Vesicles that have undergone content loss appear to be more fusogenic than intact vesicles.
引用
收藏
页码:3371 / 3379
页数:9
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