Observing Fcεri Signaling from the Inside of the Mast Cell Membrane

We have determined the membrane topography of the high-affinity IgE receptor, FcstraightepsilonRI, 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. Histochem. 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 FcepsilonRI and Lyn are distributed as small clusters (2-9 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of FcepsilonRI clusters contain Lyn. In contrast, there is essentially no FcepsilonRI-Syk colocalization in resting cells. 2 min after FcepsilonRI cross-linking, approximately 10% of Lyn colocalizes with small and medium-sized FcepsilonRI clusters (up to 20 gold particles), whereas approximately 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 FcstraightepsilonRI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of FcstraightepsilonRI signaling.