Mehta Deeksha, Crumley Elizabeth K, Lou Jinchao, Dzikovski Boris, Best Michael D, Waxham M Neal, Heberle Frederick A
Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37916, United States.
ACERT, National Biomedical Center for Advanced ESR Technology, Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States.
J Phys Chem B. 2025 Jan 16;129(2):671-683. doi: 10.1021/acs.jpcb.4c06318. Epub 2025 Jan 7.
Eukaryotic plasma membranes exhibit nanoscale lateral lipid heterogeneity, a feature that is thought to be central to their function. Studying these heterogeneities is challenging since few biophysical methods are capable of detecting domains at submicron length scales. We recently showed that cryogenic electron microscopy (cryo-EM) can directly image nanoscale liquid-liquid phase separation in extruded liposomes due to its ability to resolve the intrinsic thickness and electron density differences of ordered and disordered phases. However, the intensity contrast between these phases is poor compared with conventional fluorescence microscopy and is thus both a limiting factor and a focal point for optimization. Because the fundamental source of intensity contrast is the spatial variation in electron density within the bilayer, lipid modifications aimed at selectively increasing the electron density of one phase might enhance the ability to resolve coexisting phases. To this end, we investigated model membrane mixtures of DPPC/DOPC/cholesterol in which one hydrogen of cholesterol's C19 methyl group was replaced by an electron-rich halogen atom (either bromine or iodine). We characterized the phase behavior as a function of composition and temperature using fluorescence microscopy, Förster resonance energy transfer, and cryo-EM. Our data suggest that halogenated cholesterol variants distribute approximately evenly between liquid-ordered and liquid-disordered phases and are thus ineffective at enhancing the intensity difference between them. Furthermore, replacing more than half of the native cholesterol with halogenated cholesterol variants dramatically reduces the size of the membrane domains. Our results reinforce how small changes in the sterol structure can have a large impact on the lateral organization of membrane lipids.
真核细胞质膜呈现出纳米尺度的横向脂质异质性，这一特征被认为对其功能至关重要。研究这些异质性具有挑战性，因为很少有生物物理方法能够在亚微米长度尺度上检测结构域。我们最近表明，低温电子显微镜（cryo-EM）能够直接对挤出脂质体中的纳米尺度液-液相分离进行成像，这是由于它能够分辨有序相和无序相的固有厚度和电子密度差异。然而，与传统荧光显微镜相比，这些相之间的强度对比度较差，因此这既是一个限制因素，也是优化的重点。由于强度对比度的根本来源是双层膜内电子密度的空间变化，旨在选择性增加某一相电子密度的脂质修饰可能会增强分辨共存相的能力。为此，我们研究了DPPC/DOPC/胆固醇的模型膜混合物，其中胆固醇C19甲基上的一个氢被富电子的卤原子（溴或碘）取代。我们使用荧光显微镜、Förster共振能量转移和低温电子显微镜表征了作为组成和温度函数的相行为。我们的数据表明，卤化胆固醇变体在液-有序相和液-无序相之间大致均匀分布，因此在增强它们之间的强度差异方面无效。此外，用卤化胆固醇变体取代超过一半的天然胆固醇会显著减小膜结构域的大小。我们的结果强化了甾醇结构的微小变化如何对膜脂质的横向组织产生重大影响。
