Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion.
Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion.
复制标题
速冻可阻止磷脂囊泡的融合。
DOI：
10.1016/0005-2736(82)90474-6
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Papahadjopoulos,D
中科院分区：
文献类型：
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作者：
Bearer,EL;Düzgünes,N;Friend,DS;Papahadjopoulos,D
我们通过速冻冷冻断裂电镜检查了Ca2+诱导的大（0.2 μm直径）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，消除了使用甘油作为冷冻保护剂的必要性。刺激后1 ~ 2 s的囊泡悬浮液冻裂复制品显示，大部分囊泡已经发生了膜融合，表现为哑铃状结构和大囊泡。在没有甘油的情况下，脂质颗粒或六方HIIphase（被认为是膜融合的中间结构）在融合位点未被观察到。与Ca2+长期孵育后，不到5%的心磷脂/磷脂酰胆碱囊泡中有明显的脂质颗粒，而添加甘油会产生更多显示这些颗粒的囊泡。我们还表明，快速融合发生在Ca2+加入后的几秒钟内，通过荧光发射的时间过程，由两个单独的囊泡群体的水内容物的混合产生。因此，这些研究没有证据表明脂质颗粒是膜融合的必要中间体。相反，它们表明脂质颗粒是在融合发生后很久才达到平衡状态的结构，在甘油存在时它们变得特别普遍。
We have examined the early events in Ca2+-induced fusion of large (0.2 μm 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 HIIphase, 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.