Kataoka-Hamai Chiho, Yamazaki Tomohiko
International Center for Materials Nanoarchitectonics, National Institute for Materials Science , 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan.
Langmuir. 2015 Feb 3;31(4):1312-9. doi: 10.1021/la5042822. Epub 2015 Jan 22.
Supported lipid bilayers (SLBs) are often formed by spontaneous vesicle rupture and fusion on a solid surface. A well-characterized rupture mechanism for isolated vesicles is pore nucleation and expansion in the solution-exposed nonadsorbed area. In contrast, pore formation in the adsorbed bilayer region has not been investigated to date. In this work, we studied the detailed mechanisms of asymmetric rupture of giant unilamellar vesicles (GUVs) adsorbed on glass using fluorescence microscopy. Asymmetric rupture is the pathway where a rupture pore forms in a GUV near the edge of the glass-bilayer interface with high curvature and then expansion of the pore yields a planar bilayer patch. We show that asymmetric rupture occasionally resulted in SLB patches bearing a defect pore. The defect formation probability depended on lipid composition, salt concentration, and pH. Approximately 40% of negatively charged GUVs under physiological conditions formed pore-containing SLB patches, while negatively charged GUVs at low salt concentration or pH 4.0 and positively charged GUVs exhibited a low probability of defect inclusion. The edge of the defect pore was either in contact with (on-edge) or away from (off-edge) the edge of the planar bilayer. On-edge pores were predominantly formed over off-edge defects. Pores initially formed in the glass-adsorbed region before rupture, most frequently in close contact with the edge of the adsorbed region. When a pore formed near the edge of the adsorbed area or when the edge of a pore reached that of the adsorbed area by pore expansion, asymmetric rupture was induced from the defect site. These induced rupture mechanisms yielded SLB patches with an on-edge pore. In contrast, off-edge pores were produced when defect pore generation and subsequent vesicle rupture were uncoupled. The current results demonstrate that pore formation in the surface-adsorbed region of GUVs is not a negligible event.
支撑脂质双层（SLB）通常通过囊泡在固体表面的自发破裂和融合形成。孤立囊泡的一种特征明确的破裂机制是在溶液暴露的非吸附区域形成孔核并扩展。相比之下，吸附双层区域中的孔形成迄今尚未得到研究。在这项工作中，我们使用荧光显微镜研究了吸附在玻璃上的巨型单层囊泡（GUV）不对称破裂的详细机制。不对称破裂是指在GUV中靠近玻璃 - 双层界面高曲率边缘处形成破裂孔，然后孔扩展产生平面双层斑块的途径。我们表明，不对称破裂偶尔会导致带有缺陷孔的SLB斑块。缺陷形成概率取决于脂质组成、盐浓度和pH值。在生理条件下，约40%带负电荷的GUV形成了含孔的SLB斑块，而低盐浓度或pH值为4.0时带负电荷的GUV以及带正电荷的GUV出现缺陷包含的概率较低。缺陷孔的边缘要么与平面双层的边缘接触（边缘上），要么远离（边缘外）。边缘上的孔比边缘外的缺陷更易形成。破裂前，孔最初在玻璃吸附区域形成，最常与吸附区域的边缘紧密接触。当在吸附区域边缘附近形成孔或孔通过扩展其边缘到达吸附区域边缘时，会从缺陷部位引发不对称破裂。这些引发的破裂机制产生带有边缘上孔的SLB斑块。相比之下，当缺陷孔的产生与随后的囊泡破裂解耦时，会产生边缘外的孔。目前的结果表明，GUV表面吸附区域中的孔形成并非可忽略不计的事件。
