Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843, USA.
J Lipid Res. 2010 May;51(5):1157-72. doi: 10.1194/jlr.M003244. Epub 2009 Dec 11.
While plasma membrane cholesterol-rich microdomains play a role in cholesterol trafficking, little is known about the appearance and dynamics of cholesterol through these domains in living cells. The fluorescent cholesterol analog 6-dansyl-cholestanol (DChol), its biochemical fractionation, and confocal imaging of L-cell fibroblasts contributed the following new insights: i) fluorescence properties of DChol were sensitive to microenvironment polarity and mobility; (ii) DChol taken up by L-cell fibroblasts was distributed similarly as cholesterol and preferentially into cholesterol-rich vs. -poor microdomains resolved by affinity chromatography of purified plasma membranes; iii) DChol reported similar polarity (dielectric constant near 18) but higher mobility near phospholipid polar head group region for cholesterol in purified cholesterol-rich versus -poor microdomains; and iv) real-time confocal imaging, quantitative colocalization analysis, and fluorescence resonance energy transfer with cholesterol-rich and -poor microdomain markers confirmed that DChol preferentially localized in plasma membrane cholesterol-rich microdomains of living cells. Thus, DChol sensed a unique, relatively more mobile microenvironment for cholesterol in plasma membrane cholesterol-rich microdomains, consistent with the known, more rapid exchange dynamics of cholesterol from cholesterol-rich than -poor microdomains.
虽然富含胆固醇的质膜微域在胆固醇转运中发挥作用，但对于胆固醇在活细胞中通过这些微域的出现和动态变化知之甚少。荧光胆固醇类似物 6-二氢-胆甾醇（DChol）、其生化分级分离以及 L 细胞成纤维细胞的共焦成像提供了以下新的见解：i）DChol 的荧光性质对微环境的极性和流动性敏感；ii）L 细胞成纤维细胞摄取的 DChol 与胆固醇分布相似，并且优先进入胆固醇丰富与胆固醇贫乏的微域，这些微域通过纯化质膜的亲和层析分离；iii）DChol 报告了类似的极性（介电常数接近 18），但在纯化的富含胆固醇和贫胆固醇微域中靠近磷脂极性头部基团区域的流动性更高；iv）实时共焦成像、定量共定位分析和与富含胆固醇和贫胆固醇微域标记物的荧光共振能量转移证实，DChol 优先定位于活细胞质膜富含胆固醇的微域中。因此，DChol 感知到胆固醇在富含胆固醇的质膜微域中具有独特的、相对更具流动性的微环境，这与胆固醇从富含胆固醇的微域比贫胆固醇的微域更快的交换动力学一致。
