Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging
Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging
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DOI：
10.1529/biophysj.105.073692
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发表时间：
2006-04-01
影响因子：
3.4
通讯作者：
Burns, AR
中科院分区：
文献类型：
--
作者：
Frankel, DJ;Pfeiffer, JR;Burns, AR
应用同步原子力显微镜(AFM)和共聚焦荧光成像技术，在水溶液缓冲液中观察固定的肿瘤肥大细胞膜片内表面的三维景观。AFM图像显示了显著的、形状不规则的突起结构域，这些结构域标记有静息和激活的免疫球蛋白E受体(Fc Epsilon RI)的荧光标记，以及霍乱毒素聚集的GM1和网格蛋白。后者表明，被涂的坑从这些地区发芽。这些特征穿插在较平坦的膜区域中，并经常被细胞骨架组件包围和相互连接。当用甲基-β-环糊精提取胆固醇时，升高的结构域高度缩小了50%。根据组成，AFM看到的凸起的结构域对应于在透射电子显微镜(TEM)中观察到的富含胆固醇的深色斑块。这些斑块以前被认为是信号和内吞作用的部位，基于它们对激活的FceRI的定位，至少10个相关的信号分子，以及笼状蛋白包裹的凹坑的存在。总体而言，数据表明，信号和内吞作用发生在肥大细胞中，这些肥大细胞来自于依赖胆固醇完整性的隆起的膜区，可能与皮质细胞骨架有特定的关系。
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 (Fc epsilon RI), 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 similar to 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 FceRI, 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.