Hemifusion of giant unilamellar vesicles with planar hydrophobic surfaces: a fluorescence microscopy study.
Hemifusion of giant unilamellar vesicles with planar hydrophobic surfaces: a fluorescence microscopy study.
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作者：
Zan GH;Tan C;Deserno M;Lanni F;Lösche M
囊泡与界面的粘附和融合常被用于构建生物传感器和药物递送等领域的仿生表面。囊泡融合在细胞生物学中普遍存在，涉及两个分离的膜转化为一个连续的脂质双层。与此不同的是，囊泡膜沉积到疏水表面需要将双分子层转化为单分子层--一种拓扑学上不同的过程，称为半融合。在这里，我们使用疏水终止的自组装单分子膜（SAM）在固体表面上跟踪半融合的荧光标记的巨型单层囊泡（GUV）在单个囊泡水平的视频时间分辨率（1053毫秒）。我们观察到，一个稀释的单层，从外部GUV小叶提取的脂质组成，从囊泡粘附部位的疏水表面向外传播。随后，通过疏水表面附近的囊泡破裂发生双层半融合，然后在致密的单层中扩散脂质。因此，GUV脂质在两个同心区域转移到SAM表面：外部半融合区包含从外部GUV小叶提取的脂质，内部半融合区包含来自内部和外部GUV小叶的脂质，并以1000 µm2 s-1的速率生长（在n = 22个独立实验中，dA/dt = 970 ± 430 µm2 s-1）。这种增长率是定量一致的假设，即单层的传播是完全由疏水和脂质覆盖的SAM表面，这是分散的摩擦单层上的SAM的表面能的差异驱动。脂质转移之间的内部和外部GUV小叶发生通过半融合孔，在膜接触部位附近的过程中形成早期。当囊泡收缩到接触部位时，该孔还允许将水从GUV内部排出。
Vesicle adhesion and fusion to interfaces are frequently used for the construction of biomimetic surfaces in biosensors and drug delivery. Ubiquitous in cell biology, vesicle fusion involves the transformation of two separate membranes into one contiguous lipid bilayer. In distinction, the deposition of vesicle membranes to hydrophobic surfaces requires the transformation of a lipidic bilayer into a monomolecular layer – a topologically distinct process termed hemifusion. Here, we used hydrophobically terminated self-assembled monolayers (SAMs) on solid surfaces to track the hemifusion of fluorescently labeled giant unilamellar vesicles (GUVs) at the single vesicle level with video time resolution (≈53 ms). We observed that a dilute monolayer, consisting of lipid extracted from the outer GUV leaflet, spreads outward across the hydrophobic surface from the vesicle adhesion site. Subsequently, bilayer hemifusion occurs by vesicle rupture near the hydrophobic surface, followed by spreading of lipid in a dense monolayer. GUV lipids thus transfer to the SAM surface in two concentric zones: an outer hemifusion zone comprises lipids drawn from the outer GUV leaflet and an inner hemifusion zone comprises lipids from both the inner and outer GUV leaflets and grows at a rate of ≈1000 µm2 s−1 (dA/dt = 970 ± 430 µm2 s−1 in n = 22 independent experiments). This growth rate is quantitatively consistent with the assumption that the spreading of the monolayer is entirely driven by the difference in surface energies of the hydrophobic and the lipid-covered SAM surfaces, which is dissipated by friction of the spreading monolayer on the SAM. Lipid transfer between the inner and outer GUV leaflets occurs via a hemifusion pore that forms early in the process near the membrane contact site. This pore also permits expulsion of water from the GUV interior as the vesicle contracts onto the contact site.