Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors
Asymmetric Bilayers by Hemifusion: Method and Leaflet Behaviors
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DOI：
10.1016/j.bpj.2019.07.054
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发表时间：
2019-09-17
影响因子：
3.4
通讯作者：
Feigenson, Gerald W.
中科院分区：
文献类型：
--
作者：
Enokit, Thais A.;Feigenson, Gerald W.
我们描述了一种通过半融合制备不对称巨单层囊泡（aGUV）的新方法。半融合的巨型单层囊泡和支持的脂质双层，由钙触发，促进脂质扩散介导的融合外小叶的脂质交换。我们使用不同的荧光染料来监测无支撑的aGUV的内小叶和外小叶。我们证实，几乎所有新交换的脂质在aGUV中发现在这些不对称囊泡的外小叶。此外，我们测试了通过半融合形成的aGUV在手术过程中保存其内容物的稳定性。对于由1，2-二硬脂酰基-sn-甘油-3-磷酸胆碱/1，2-二油酰基-sn-甘油-3-磷酸胆碱/胆固醇= 0.39/0.39/0.22和1，2-二油酰基-sn-甘油-3-磷酸胆碱/胆固醇= 0.8/0.2的支撑脂质双层组成的巨大单层囊泡的半融合制备的aGUV，我们观察到交换的脂质改变了双层性质。要访问的不对称双层的物理和化学性质，我们监测的染料分配系数的各个小叶和广义偏振的荧光探针6-dodecanoyl-2-[N-甲基-N-（羧甲基）氨基]萘，传感器的脂质包装/其周围的秩序。对于高百分比的脂质交换（>70%），染料分配指示诱导无序和诱导有序结构域。诱导域具有不同的脂质包装/秩序相比，对称的液体无序和液体有序的域。
We describe a new method to prepare asymmetric giant unilamellar vesicles (aGUVs) via hemifusion. Hemifusion of giant unilamellar vesicles and a supported lipid bilayer, triggered by calcium, promotes the lipid exchange of the fused outer leaflets mediated by lipid diffusion. We used different fluorescent dyes to monitor the inner and the outer leaflets of the unsupported aGUVs. We confirmed that almost all newly exchanged lipids in the aGUVs are found in the outer leaflet of these asymmetric vesicles. In addition, we test the stability of the aGUVs formed by hemifusion in preserving their contents during the procedure. For aGUVs prepared from the hemifusion of giant unilamellar vesicles composed of 1,2-distearoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.39/0.39/0.22 and a supported lipid bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol = 0.8/0.2, we observed the exchanged lipids to alter the bilayer properties. To access the physical and chemical properties of the asymmetric bilayer, we monitored the dye partition coefficients of individual leaflets and the generalized polarization of the fluorescence probe 6-dodecanoyl-2-[ N-methyl-N-(carboxymethyl)amino] naphthalene, a sensor for the lipid packing/order of its surroundings. For a high percentage of lipid exchange (>70%), the dye partition indicates induced-disordered and induced-ordered domains. The induced domains have distinct lipid packing/order compared to the symmetric liquid-disordered and liquid-ordered domains.