Topography of signaling molecules as detected by electron microscopy on plasma membrane sheets isolated from non-adherent mast cells

Immunolabeling of isolated plasma membrane (PM) sheets combined with high-resolution electron microscopy is a powerful technique for understanding the topography of PM-bound signaling molecules. However, this technique has been mostly confined to analysis of membrane sheets from adherent cells. Here we present a rapid, simple and versatile method for isolation of PM sheets from non-adherent cells, and show its use for examination of the topography of Fcɛ receptor I (FcɛRI) and transmembrane adaptors, LAT (linker for activation of T cells) and NTAL (non-T cell activation linker), in murine bone marrow-derived mast cells (BMMC). The data were compared with those obtained from widely used but tumor-derived rat basophilic leukemia (RBL) cells. In non-activated cells, FcɛRI was distributed either individually or in small clusters of comparable size in both cell types. In multivalent antigen-activated BMMC as well as RBL cells, FcɛRI was internalized to a similar extent, but, strikingly, internalization in BMMC was not preceded by formation of large (∼ 200 nm) aggregates of FcɛRI, described previously in activated RBL cells. On the other hand, downstream adaptor proteins, LAT and NTAL, were localized in independent domains in both BMMC and RBL cells before and after FcɛRI triggering. The combined data demonstrate unexpected properties of FcɛRI signaling assemblies in BMMC and emphasize the importance of studies of PM sheets isolated from non-tumor cells.