Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells
Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells
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DOI：
10.1194/jlr.m003244
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发表时间：
2010-05-01
影响因子：
6.5
通讯作者：
Schroeder, Friedhelm
中科院分区：
文献类型：
--
作者：
Huang, Huan;McIntosh, Avery L.;Schroeder, Friedhelm
虽然质膜富含胆固醇的微区在胆固醇运输中发挥作用，但对胆固醇在活细胞中通过这些微区的外观和动力学知之甚少。荧光胆固醇类似物6-丹磺酰胆甾烷醇（DChol）、其生化分级和L-细胞成纤维细胞的共聚焦成像贡献了以下新的见解：i）DChol的荧光性质对微环境极性和迁移率敏感;（ii）由L-细胞成纤维细胞摄取的DChol与胆固醇类似地分布，并且优先进入富含胆固醇的细胞，而不是-通过纯化的质膜的亲和色谱分离的不良微区; 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的微区相对于缺乏胆固醇的微区中，胆固醇在磷脂极性头部基团区域附近的迁移率更高;和iv）实时共聚焦成像、定量共定位分析和用富胆固醇和贫胆固醇微区标记物的荧光共振能量转移证实DChol优先定位于活细胞的质膜富胆固醇微区中。因此，DChol在质膜富含胆固醇的微区中感测到胆固醇的独特的、相对更移动的微环境，这与已知的胆固醇从富含胆固醇的微区比从缺乏胆固醇的微区更快速的交换动力学一致。黄，H.，A. L.麦金托什，B。P. Atshaves，Y. Ohno-Iwashita，A. B。Kier和F.施罗德丹磺酰-胆甾烷醇作为活细胞膜中胆固醇行为探针的用途。J. Lipid Res. 2010. 51：1157-1172。
While plasma membrane cholesterol-rich micro-domains 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.-Huang, H., A. L. McIntosh, B. P. Atshaves, Y. Ohno-Iwashita, A. B. Kier, and F. Schroeder. Use of dansyl-cholestanol as a probe of cholesterol behavior in membranes of living cells. J. Lipid Res. 2010. 51: 1157-1172.