Cellular membranes have complex lipid and protein structures that are laterally organized for optimized molecular recognition and signal transduction processes. Knowledge of nanometerscale lateral organization and its function is of great importance in the analysis of receptor-based signaling. In model membranes, we studied in detail the chemical and physical factors which result in lateral organization of lipids and lipid-mediated protein sequestration into signaling domains. In biological

more »

... ranes, we mapped the location and follow the dynamic activity of specific membrane proteins involved in the immunological response of mast cells. These studies were enabled by our development of advanced imaging methods that provided both high spatial resolution and sensitivity to dynamical processes. Our technical approach was to combine the high sensitivity and time resolution of fluorescence imaging with the high lateral resolution of atomic force microscopy (AFM). Simultaneous fluorescence and AFM imaging allows correlation of the distribution and dynamic activity of specific biomolecules via fluorescence labeling with complete topographic information of the membrane. Overall, our unique imaging Simultaneous atomic force microscope (AFM) and submicron confocal fluorescence imaging of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid domain structures in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) is presented. Lipids labeled by fluorescent probes either at the headgroups or tailgroups enable domain contrast in fluorescence imaging on the basis of partitioning between the gel (DPPC) and disordered liquid (DOPC) phases. However, correlation with AFM topographic information reveals that they do not always faithfully report exact gel domain size or shape. Furthermore, we find that the fluorescence contrast decreases significantly with domain size, such that small domains observed with AFM are not observed in fluorescence images despite adequate optical resolution. We attribute these effects in part to broadened partitioning of the probe lipids across the domain boundaries. Binding of fluorescent Alexa 488-conjugated cholera toxin B subunits to GM1 gangliosides in DPPC domains correlates well with AFM topographic information to the limit of optical resolution. However, it also may reveal the presence of dilute GM1 components in the fluid phase that have no topographic contrast. In all cases, the complete correlation of topographic and fluorescence images provides evidence that gel-phase domains occur across both leaflets of the bilayer. ABSTRACT 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 (FceRI), 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 ;50% when cholesterol is extracted with methyl-b-cyclodextrin. Based on composition, the raised domains seen by AFM correspond to the cholesterolenriched 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.