Fabrication and electromechanical characterization of freestanding asymmetric membranes
Fabrication and electromechanical characterization of freestanding asymmetric membranes
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DOI：
10.1016/j.bpj.2021.02.036
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发表时间：
2021-05-04
影响因子：
3.4
通讯作者：
Beltramo, Peter J.
中科院分区：
文献类型：
--
作者：
Liu, Paige;Zabala-Ferrera, Oscar;Beltramo, Peter J.
所有生物细胞膜都保持约100 mV的跨膜电位，部分原因是带电磷脂在膜上的不对称分布。这种不对称性对细胞健康和生理过程如细胞内信号传导、受体介导的内吞作用和膜蛋白功能至关重要。实验性人工膜系统以可控制的方式将基本的细胞膜结构（例如磷脂双层）结合在一起，其中可以分离和检查特定的性质和过程。在这里，我们描述了一种方法来制造和表征平面的，独立的，不对称的膜，并用它来检查头基电荷对膜刚度的影响。该方法依赖于用于通过将水相脂质囊泡吸附到油-水界面并随后使油变薄以形成双层来形成独立膜的薄膜平衡。我们通过分析对称膜的厚度和可压缩性来验证这种水包脂质方法，所述对称膜具有不同的两性离子1，2-二油酰基-sn-甘油-3-磷酸胆碱（DOPC）和阴离子1，2-二油酰基-sn-甘油-3-磷酸-（10 -rac-甘油）钠盐（DOPG）含量，与以前的油包脂质方法相比。我们发现，随着DOPG浓度的增加，膜变得更厚，更硬。不对称膜是通过控制油的两侧的水性储层中的脂质囊泡组成来制造的。膜组成的不对称性定性证明使用荧光猝灭测定和定量表征通过电压依赖性电容测量。在一侧具有DOPC并且在另一侧具有DOPC-DOPG混合物的稳定的不对称膜以15至80 mV的跨膜电位产生。引入膜电荷不对称性与具有相同的总体磷脂组成的对称膜相比降低了厚度和刚度。这些初步的成功证明了一个可行的途径，定量表征不对称的双层，可以扩展到适应更复杂的膜和膜过程的未来。
All biological cell membranes maintain an electric transmembrane potential of around 100 mV, due in part to an asymmetric distribution of charged phospholipids across the membrane. This asymmetry is crucial to cell health and physiological processes such as intracell signaling, receptor-mediated endocytosis, and membrane protein function. Experimental artificial membrane systems incorporate essential cell membrane structures, such as the phospholipid bilayer, in a controllable manner in which specific properties and processes can be isolated and examined. Here, we describe an approach to fabricate and characterize planar, freestanding, asymmetric membranes and use it to examine the effect of headgroup charge on membrane stiffness. The approach relies on a thin film balance used to form a freestanding membrane by adsorbing aqueous phase lipid vesicles to an oil-water interface and subsequently thinning the oil to form a bilayer. We validate this lipid-in-aqueous approach by analyzing the thickness and compressibility of symmetric membranes with varying zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and anionic 1,2-dioleoyl-sn-glycero-3-phospho-(10 -rac-glycerol) sodium salt (DOPG) content as compared with previous lipid-in-oil methods. We find that as the concentration of DOPG increases, membranes become thicker and stiffer. Asymmetric membranes are fabricated by controlling the lipid vesicle composition in the aqueous reservoirs on either side of the oil. Membrane compositional asymmetry is qualitatively demonstrated using a fluorescence quenching assay and quantitatively characterized through voltage-dependent capacitance measurements. Stable asymmetric membranes with DOPC on one side and DOPC-DOPG mixtures on the other were created with transmembrane potentials ranging from 15 to 80 mV. Introducing membrane charge asymmetry decreases both the thickness and stiffness in comparison with symmetric membranes with the same overall phospholipid composition. These initial successes demonstrate a viable pathway to quantitatively characterize asymmetric bilayers that can be extended to accommodate more complex membranes and membrane processes in the future.