Single-lipid dynamics in phase-separated supported lipid bilayers
Single-lipid dynamics in phase-separated supported lipid bilayers
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DOI：
10.1101/2020.05.28.121830
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发表时间：
2020-05
期刊：
影响因子：
--
通讯作者：
X. Woodward;C. Kelly
中科院分区：
文献类型：
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作者：
X. Woodward;C. Kelly
相分离是细胞膜上的基本组织机制。脂质相对膜组成、曲率、张力和温度具有复杂的依赖性。单分子扩散测量膜行为的关键特征，并且与有效膜粘度相关。脂质扩散速率在液体无序（Ld）和液体有序（Lo）相之间变化高达10倍，这取决于膜组成、测量技术和周围环境。本文报道了在不同温度、组成和脂相的情况下，脂在相分离的支撑脂双层（SLB）上的扩散。脂质扩散通过单粒子跟踪（SPT）和荧光相关光谱（FCS）通过适用于不同膜系统的定制数据采集和分析协议进行测量。我们证明FCS和SPT分析与单步长度分布和均方位移的脂质与显着的固定扩散之间的协议。传统上，SPT是敏感的扩散聚集，而FCS在很大程度上排除了聚集体的报告数据。协议报告用于识别和挑选的聚集体之前，通过SPT计算扩散速率。通过聚集体剔除，所有扩散测量方法都能提供一致的结果。随着不同的膜组合物和温度，我们证明的重要性，分离共存的脂质相预测的差异扩散之间的Ld和Lo相的连接线长度。摘要脂质扩散随脂质相、温度和聚集体而变化聚集体剔除产生了来自单粒子跟踪和荧光相关光谱的一致测量具有较高胆固醇含量或在低温下具有更多聚集体的膜在低温和低胆固醇含量下共存的脂质相之间发生更多扩散速率的变化
Phase separation is a fundamental organizing mechanism on cellular membranes. Lipid phases have complex dependencies on the membrane composition, curvature, tension, and temperature. Single-molecule diffusion measures a key characteristic of membrane behavior and relates to the effective membrane viscosity. Lipid diffusion rates vary by up to ten-fold between liquid-disordered (Ld) and liquid-ordered (Lo) phases depending on the membrane composition, measurement technique, and the surrounding environment. This manuscript reports the lipid diffusion on phase-separated supported lipid bilayers (SLBs) with varying temperature, composition, and lipid phase. Lipid diffusion is measured by single-particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) via custom data acquisition and analysis protocols that apply to diverse membranes systems. We demonstrate agreement between FCS and SPT analyses with both the single-step length distribution and the mean squared displacement of lipids with significant immobile diffusers. Traditionally, SPT is sensitive to diffuser aggregation, whereas FCS largely excludes aggregates from the reported data. Protocols are reported for identifying and culling the aggregates prior to calculating diffusion rates via SPT. With aggregate culling, all diffusion measurement methods provide consistent results. With varying membrane composition and temperature, we demonstrate the importance of the tie-line length that separates the coexisting lipid phases in predicting the differences in diffusion between the Ld and Lo phases. HIGHLIGHTS Lipid diffusion varies with the lipid phases, temperature, and aggregation Aggregate culling yields consistent measurements from single-particle tracking and fluorescence correlation spectroscopy Membrane with higher cholesterol content or at low temperature have more aggregates A more variation in the diffusion rates occurred between the coexisting lipid phases at low temperatures and low cholesterol content