Woodbury D J, Hall J E
Department of Physiology and Biophysics, University of California, Irvine 92717.
Biophys J. 1988 Dec;54(6):1053-63. doi: 10.1016/S0006-3495(88)83042-X.
Fluorescence microscopy combined with electrical conductance measurements were used to assess fusion of phospholipid vesicles with a planar bilayer. Large unilamellar vesicles (0.5-3 microns diam.) filled with the fluorescent dye, calcein, were made both with or without porin channels. Vesicle-bilayer fusion was induced by increasing the osmolarity of the solution on the side of the bilayer to which the vesicles were added. Fusion was detected optically by the fluorescent flash due to release of vesicular contents. Although both porin-containing and porin-free vesicles give the same kind of flash upon content release, the conditions necessary to induce release are very different. Only 4% of the porin-free vesicles fuse (release their contents) when subjected to 3 M urea. However, the same conditions induce 53% of the porin-containing vesicles to fuse and most of these fusions occur at a lower osmolarity ([urea] less than 400 mM). Thus channels greatly enhance fusion in this model system. A physical model based on the postulate that fusion is induced by an increase in surface tension, predicts that three conditions are necessary for fusion in this system: (a) an open channel in the vesicle membrane, (b) an osmotic gradient across the bilayer, and (c) the vesicle in contact with the planar membrane. These are the conditions that experimentally produce fusion in the model system.
荧光显微镜结合电导率测量被用于评估磷脂囊泡与平面双层膜的融合。制备了直径为0.5 - 3微米、填充有荧光染料钙黄绿素的大单层囊泡，有孔蛋白通道和无孔蛋白通道的囊泡都有制备。通过增加添加囊泡一侧双层膜溶液的渗透压来诱导囊泡 - 双层膜融合。由于囊泡内容物的释放，通过荧光闪光进行光学检测融合。尽管含孔蛋白和不含孔蛋白的囊泡在内容物释放时都会产生相同类型的闪光，但诱导释放所需的条件却大不相同。当用3M尿素处理时，只有4%的无孔蛋白囊泡融合（释放其内容物）。然而，相同条件下会诱导53%的含孔蛋白囊泡融合，并且这些融合大多发生在较低的渗透压下（尿素浓度小于400mM）。因此，在这个模型系统中通道极大地增强了融合。基于融合是由表面张力增加所诱导这一假设的物理模型预测，在这个系统中融合需要三个条件：（a）囊泡膜上有开放通道，（b）双层膜上存在渗透梯度，以及（c）囊泡与平面膜接触。这些就是在模型系统中通过实验产生融合的条件。
