Wüstner Daniel, Modzel Maciej, Lund Frederik W, Lomholt Michael A
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark.
Chem Phys Lipids. 2016 Sep;199:106-135. doi: 10.1016/j.chemphyslip.2016.03.003. Epub 2016 Mar 22.
Cholesterol is an important lipid component of the plasma membrane (PM) of mammalian cells, where it is involved in control of many physiological processes, such as endocytosis, cell migration, cell signalling and surface ruffling. In an attempt to explain these functions of cholesterol, several models have been put forward about cholesterol's lateral and transbilayer organization in the PM. In this article, we review imaging techniques developed over the last two decades for assessing the distribution and dynamics of cholesterol in the PM of mammalian cells. Particular focus is on fluorescence techniques to study the lateral and inter-leaflet distribution of suitable cholesterol analogues in the PM of living cells. We describe also several methods for determining lateral cholesterol dynamics in the PM including fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), single particle tracking (SPT) and spot variation FCS coupled to stimulated emission depletion (STED) microscopy. For proper interpretation of such measurements, we provide some background in probe photophysics and diffusion phenomena occurring in cell membranes. In particular, we show the equivalence of the reaction-diffusion approach, as used in FRAP and FCS, and continuous time random walk (CTRW) models, as often invoked in SPT studies. We also discuss mass spectrometry (MS) based imaging of cholesterol in the PM of fixed cells and compare this method with fluorescence imaging of sterols. We conclude that evidence from many experimental techniques converges towards a model of a homogeneous distribution of cholesterol with largely free and unhindered diffusion in both leaflets of the PM.
胆固醇是哺乳动物细胞质膜（PM）的重要脂质成分，它参与许多生理过程的调控，如内吞作用、细胞迁移、细胞信号传导和表面褶皱。为了解释胆固醇的这些功能，人们提出了几种关于胆固醇在质膜中的横向和跨双层组织的模型。在本文中，我们回顾了过去二十年来为评估哺乳动物细胞质膜中胆固醇的分布和动态而开发的成像技术。特别关注用于研究活细胞质膜中合适胆固醇类似物的横向和叶间分布的荧光技术。我们还描述了几种测定质膜中横向胆固醇动态的方法，包括光漂白后荧光恢复（FRAP）、荧光相关光谱（FCS）、单粒子跟踪（SPT）以及与受激发射损耗（STED）显微镜耦合的斑点变化FCS。为了正确解释这些测量结果，我们提供了一些关于探针光物理和细胞膜中发生的扩散现象的背景知识。特别是，我们展示了FRAP和FCS中使用的反应扩散方法与SPT研究中经常引用的连续时间随机游走（CTRW）模型的等效性。我们还讨论了基于质谱（MS）的固定细胞质膜中胆固醇的成像，并将该方法与固醇的荧光成像进行了比较。我们得出结论，来自许多实验技术的证据都趋向于一个胆固醇均匀分布的模型，胆固醇在质膜的两个叶中基本自由且不受阻碍地扩散。
