Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee; Shull Wollan Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee.
Department of Biosciences, Rice University, Houston, Texas; Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas.
Biophys J. 2019 Oct 15;117(8):1381-1386. doi: 10.1016/j.bpj.2019.09.006. Epub 2019 Sep 16.
Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Submicroscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would impact interpretations from many model membrane experiments. Using three probe-free methods-small angle X-ray and neutron scattering and cryogenic electron microscopy-we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100-nm diameter pores. In such preparations, only ∼35% of lipids are externally accessible and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein the formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity.
挤出通过多孔过滤器是一种广泛使用的方法来制备仿生模型膜。在这种方法中，最重要的是高效地生产单层（单分子层）囊泡，消除层间相互作用的影响，并严格定义双层表面积，使其可用于外部试剂，如蛋白质。使用挤出法制备的亚微观囊泡被广泛认为是单分子层的，而与该假设的大偏差将影响许多模型膜实验的解释。我们使用三种无探针方法-小角 X 射线和中子散射以及低温电子显微镜-报告了明确的证据，证明由中性磷脂脂质组成的挤出囊泡中存在广泛的多层结构，包括中性脂质在生理缓冲液中分散并通过 100nm 直径的孔挤出的常见情况。在这种制剂中，只有约 35%的脂质是外部可及的，并且这个分数高度依赖于制备条件。带电荷的脂质促进单分子层形成，而降低溶剂离子强度也会促进单分子层形成，这表明静电相互作用在确定挤出囊泡的层状结构方面非常重要。较小的挤出孔尺寸也能有效地增加单分子层囊泡的比例，这表明膜弯曲起作用。综上所述，这些观察结果为挤出的机制模型提供了依据，其中单分子层囊泡的形成涉及双层弯曲和粘附能之间的竞争。这里提出的发现对模型膜研究的设计和解释具有广泛的影响，特别是依赖于单分子层假设的总体平均观察。
