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© The Rockefeller University Press, 0021-9525/2000/5/1131/ $5.00
The Journal of Cell Biology, Volume 149, Number 5, May 29, 2000 1131-1142

Original Article

Observing Fc{epsilon}RI Signaling from the Inside of the Mast Cell Membrane

Bridget S. Wilsona, Janet R. Pfeiffera, and Janet M. Olivera
a Department of Pathology and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, 87131

Correspondence to: Bridget S. Wilson, Department of Pathology, University of New Mexico Health Sciences Center, CRF 205, 2325 Camino de Salud, Albuquerque, NM 87131. Tel:(505) 272-8852 Fax:(505) 272-1435 E-mail:bwilson{at}thor.unm.edu.

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

Key Words: microdomains, Lyn, Syk, rafts


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