Bearer E L, Düzgünes N, Friend D S, Papahadjopoulos D
Biochim Biophys Acta. 1982 Dec 8;693(1):93-8. doi: 10.1016/0005-2736(82)90474-6.
We have examined the early events in Ca2+-induced fusion of large (0.2 microns diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1-2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal HII phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca2+ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.
我们通过快速冷冻断裂电子显微镜检查了钙离子诱导的大尺寸（直径0.2微米）单层心磷脂/磷脂酰胆碱和磷脂酰丝氨酸/磷脂酰乙醇胺囊泡融合的早期事件，无需使用甘油作为冷冻保护剂。刺激1-2秒后冷冻的囊泡悬浮液的冷冻断裂复制品显示，大多数囊泡已经发生了膜融合，哑铃状结构和大囊泡证明了这一点。在没有甘油的情况下，在融合位点未观察到被认为是膜融合中间结构的脂质颗粒或六方HII相。与钙离子长期孵育后，不到5%的心磷脂/磷脂酰胆碱囊泡中可见脂质颗粒，添加甘油会产生更多显示脂质颗粒的囊泡。我们还通过两个独立囊泡群体的水性内容物混合产生的荧光发射时间进程表明，在添加钙离子后几秒钟内就发生了快速融合。因此，这些研究没有证据表明脂质颗粒是膜融合所必需的中间结构。相反，它们表明脂质颗粒是融合发生很长时间后在平衡状态下获得的结构，并且在甘油存在的情况下它们变得特别普遍。
