Department of Chemistry, Stanford University, Stanford, California, USA.
Biophys J. 2013 Jul 16;105(2):409-19. doi: 10.1016/j.bpj.2013.05.056.
Membrane fusion consists of a complex rearrangement of lipids and proteins that results in the merger of two lipid bilayers. We have developed a model system that employs synthetic DNA-lipid conjugates as a surrogate for the membrane proteins involved in the biological fusion reaction. We previously showed that complementary DNA-lipids, inserted into small unilamellar vesicles, can mediate membrane fusion in bulk. Here, we use a model membrane architecture developed in our lab to directly observe single-vesicle fusion events using fluorescence microscopy. In this system, a planar tethered membrane patch serves as the target membrane for incoming vesicles. This allows us to quantify the kinetics and characteristics of individual fusion events from the perspective of the lipids or the DNA-lipids involved in the process. We find that the fusion pathways are heterogeneous, with an arrested hemi-fusion state predominating, and we quantitate the outcome and rate of fusion events to construct a mechanistic model of DNA-mediated vesicle fusion. The waiting times between docking and fusion are distributed exponentially, suggesting that fusion occurs in a single step. Our analysis indicates that when two lipid bilayers are brought into close proximity, fusion occurs spontaneously, with little or no dependence on the number of DNA hybrids formed.
膜融合包括脂质和蛋白质的复杂重排，导致两个脂质双层的融合。我们开发了一种模型系统，该系统使用合成 DNA-脂质缀合物作为参与生物融合反应的膜蛋白的替代物。我们之前表明，插入到小单层囊泡中的互补 DNA-脂质可以介导大量的膜融合。在这里，我们使用我们实验室开发的模型膜结构，使用荧光显微镜直接观察单个囊泡融合事件。在该系统中，平面固定化膜片作为进入囊泡的靶膜。这使我们能够从参与过程的脂质或 DNA-脂质的角度量化单个融合事件的动力学和特征。我们发现融合途径是异构的，以被阻止的半融合状态为主，我们定量融合事件的结果和速率，以构建 DNA 介导的囊泡融合的机制模型。对接和融合之间的等待时间呈指数分布，表明融合是在单个步骤中发生的。我们的分析表明，当两个脂质双层紧密接近时，融合会自发发生，与形成的 DNA 杂交的数量几乎没有关系。
