Simultaneous atomic force microscopy (AFM) and confocal fluorescence imaging was used to observe in aqueous buffer the 3-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 IgE 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 roughly 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 (1). 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.
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