Fernández-Golbano Isabel María, García Patricia, Rebollo Elena, Geli María Isabel, Encinar Del Dedo Javier
Institute for Molecular Biology of Barcelona, CSIC, Barcelona, Spain.
Institute of Functional Biology and Genomics, CSIC, University of Salamanca, Salamanca, Spain.
Methods Mol Biol. 2025;2888:35-52. doi: 10.1007/978-1-0716-4318-1_4.
Cholesterol is a fundamental component of cellular membranes, and its organization, distribution, and recycling are tightly regulated. Cholesterol can form, together with other lipids and proteins, membrane nanodomains, which play important roles in membrane trafficking, the spatiotemporal organization of signal transduction, or the modulation of plasma membrane transporters, among others. Not surprisingly then, the misregulation of cholesterol biosynthetic and transport pathways has been related to numerous diseases, including neurodegenerative and metabolic disorders. Here, we focus on the cholesterol-binding domain 4 (D4) of perfringolysin O (PFO, theta toxin) and its use as a probe to define the dynamics and subcellular localization of yeast sterols using time-lapse live-cell fluorescence microscopy. In combination with drugs that acutely interfere with sterol synthesis, such as terbinafine, the probe can also be used to monitor in real-time the extraction of sterols from specialized endoplasmic reticulum subdomains named ERSES (endoplasmic reticulum sterol exit sites) by the OSBP-related protein Osh2.
胆固醇是细胞膜的基本组成成分，其组织、分布和循环受到严格调控。胆固醇可与其他脂质和蛋白质一起形成膜纳米结构域，这些结构域在膜运输、信号转导的时空组织或质膜转运体的调节等方面发挥重要作用。因此，毫不奇怪，胆固醇生物合成和运输途径的失调与多种疾病有关，包括神经退行性疾病和代谢紊乱。在这里，我们聚焦于产气荚膜梭菌溶素O（PFO，θ毒素）的胆固醇结合结构域4（D4），以及利用延时活细胞荧光显微镜将其用作探针来确定酵母甾醇的动态变化和亚细胞定位。与能急性干扰甾醇合成的药物（如特比萘芬）联合使用时，该探针还可用于实时监测甾醇从名为ERSES（内质网甾醇出口位点）的特殊内质网亚结构域被OSBP相关蛋白Osh2提取的过程。
