Frankel D J, Pfeiffer J R, Surviladze Z, Johnson A E, Oliver J M, Wilson B S, Burns A R
Biomolecular Materials and Interfaces Department, MS1413 Sandia National Laboratories, Albuquerque, New Mexico 87185, USA.
Biophys J. 2006 Apr 1;90(7):2404-13. doi: 10.1529/biophysj.105.073692. Epub 2006 Jan 13.
Simultaneous atomic force microscopy (AFM) and confocal fluorescence imaging were used to observe in aqueous buffer the three-dimensional landscape of the inner surface of membrane sheets stripped from fixed tumor mast cells. The AFM images reveal prominent, irregularly shaped raised domains that label with fluorescent markers for both resting and activated immunoglobin E receptors (FcepsilonRI), as well as with cholera toxin-aggregated GM1 and clathrin. The latter suggests that coated pits bud from these regions. These features are interspersed with flatter regions of membrane and are frequently surrounded and interconnected by cytoskeletal assemblies. The raised domains shrink in height by approximately 50% when cholesterol is extracted with methyl-beta-cyclodextrin. Based on composition, the raised domains seen by AFM correspond to the cholesterol-enriched dark patches observed in transmission electron microscopy (TEM). These patches were previously identified as sites of signaling and endocytosis based on their localization of activated FcepsilonRI, at least 10 associated signaling molecules, and the presence of clathrin-coated pits. Overall the data suggest that signaling and endocytosis occur in mast cells from raised membrane regions that depend on cholesterol for their integrity and may be organized in specific relationship with the cortical cytoskeleton.
同时使用原子力显微镜（AFM）和共聚焦荧光成像技术，在水性缓冲液中观察从固定的肿瘤肥大细胞剥离的膜片内表面的三维形貌。AFM图像显示出突出的、形状不规则的凸起区域，这些区域可被静息和活化的免疫球蛋白E受体（FcepsilonRI）的荧光标记物标记，也可被霍乱毒素聚集的GM1和网格蛋白标记。后者表明有被小窝从这些区域出芽。这些特征散布在较平坦的膜区域中，并经常被细胞骨架组装体包围和相互连接。当用甲基-β-环糊精提取胆固醇时，凸起区域的高度会收缩约50%。基于组成，AFM观察到的凸起区域对应于透射电子显微镜（TEM）中观察到的富含胆固醇的暗斑。这些斑块先前根据活化的FcepsilonRI的定位、至少10种相关信号分子以及网格蛋白包被小窝的存在，被确定为信号传导和内吞作用的位点。总体而言，数据表明信号传导和内吞作用发生在肥大细胞的凸起膜区域，这些区域的完整性依赖于胆固醇，并且可能与皮质细胞骨架以特定关系组织起来。
