Observing FcεRI signaling from the inside of the mast cell membrane

We have determined the membrane topograph!: of the high-affinity IgE receptor, Fc is an element of RI, and its associated tyrosine kinases, Lyn and Syk, by immunogold labeling and transmission electron microscopic (TEM) analysis of membrane sheets prepared from RBL-2H3 mast cells, The method of Sanan and Anderson (Sanan, D.A,. and R,G,W Anderson, 1991.J. Hisrochem. Cytochem. 39:1017-1024) was modified to generate membrane sheets from the dorsal surface of RBL-2H3 cells, Signaling molecules were localized on the cytoplasmic face of these native membranes by immunogold labeling and high-resolution TEM analysis. In unstimulated cells, the majority of gold particles marking both Fc is an element of RI and Lyn are distributed as small clusters (2-9 gold particles) that do not associate with clathrin-coated membrane, Approximately 25% of Fc is an element of RI clusters contain Lyn, In contrast, there is essentially no Fc is an element of RI-Syk colo- calization in resting cells. 2 min after Fc is an element of RI cross-linking, similar to 10% of Lyn colocalizes with small and mediumsized Fc is an element of RI clusters (up to 20 gold particles), whereas similar to 16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20-100 gold particles) that form on characteristically osmiophilic membrane patches. While Lyn is excluded, Syk is dramatically recruited into these larger aggregates. The clathrin-coated pits that internalize cross-linked receptors bud from membrane adjacent to the Syk-containing receptor complexes. The sequential association of Fc is an element of RI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of Fc is an element of RI signaling.