Max-Planck-Institute for Dynamics and Self-Organization, Göttingen, Germany.
Max-Planck-Institute for Dynamics and Self-Organization, Göttingen, Germany; Institute of Organic and Biomolecular Chemistry, Georg-August Universität, Göttingen, Germany.
Methods Enzymol. 2024;700:455-483. doi: 10.1016/bs.mie.2024.02.009. Epub 2024 Mar 5.
Over the years, it has become more and more obvious that lipid membranes show a very complex behavior. This behavior arises in part from the large number of different kinds of lipids and proteins and how they dynamically interact with each other. In vitro studies using artificial membrane systems have shed light on the heterogeneity based on lipid-lipid interactions in multicomponent bilayer mixtures. Inspired by the raft hypothesis, the coexistence of liquid-disordered (l) and liquid-ordered (l) phases has drawn much attention. It was shown that ternary lipid mixtures containing low- and high-melting temperature lipids and cholesterol can phase separate into a l phase enriched in the high-melting lipids and cholesterol and a l phase enriched in the low-melting lipids. Depending on the model membrane system under investigation, different domain sizes, shapes, and mobilities have been found. Here, we describe how to generate phase-separated l/l phases in model membrane systems termed pore-spanning membranes (PSMs). These PSMs are prepared on porous silicon substrates with pore sizes in the micrometer regime. A proper functionalization of the top surface of the substrates is required to achieve the spreading of giant unilamellar vesicles (GUVs) to obtain PSMs. Starting with l/l phase-separated GUVs lead to membrane heterogeneities in the PSMs. Depending on the functionalization strategy of the top surface of the silicon substrate, different membrane heterogeneities are observed in the PSMs employing fluorescence microscopy. A quantitative analysis of the heterogeneity as well as the dynamics of the lipid domains is described.
多年来,脂质膜表现出非常复杂的行为这一点变得越来越明显。这种行为部分源于大量不同种类的脂质和蛋白质,以及它们如何与彼此动态相互作用。使用人工膜系统的体外研究揭示了基于多组分双层混合物中脂质-脂质相互作用的异质性。受筏模型假说的启发,液体无序(l)相和液体有序(l)相的共存引起了广泛关注。已经表明,包含低熔点和高熔点脂质以及胆固醇的三元脂质混合物可以相分离成富含高熔点脂质和胆固醇的 l 相和富含低熔点脂质的 l 相。取决于所研究的模型膜系统,已经发现了不同的域大小、形状和迁移率。在这里,我们描述了如何在称为孔贯穿膜(PSM)的模型膜系统中产生相分离的 l/l 相。这些 PSM 是在具有微米级孔径的多孔硅衬底上制备的。需要对衬底的上表面进行适当的功能化,以实现巨大单层囊泡(GUV)的铺展,从而获得 PSM。从 l/l 相分离的 GUV 开始,会导致 PSM 中的膜异质性。根据硅衬底上表面的功能化策略,在使用荧光显微镜观察 PSM 时,会观察到不同的膜异质性。描述了对脂质域的异质性和动力学的定量分析。