title: Observing FcepsilonRI signaling from the inside of the mast cell membrane.
abstract: 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.
authors.affiliation: Department of Pathology and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, 87131, USA. bwilson@thor.unm.edu
authors.forename: B S
authors.initials: BS
authors.lastname: Wilson
authors.forename: J R
authors.initials: JR
authors.lastname: Pfeiffer
authors.forename: J M
authors.initials: JM
authors.lastname: Oliver
mesh_terms: D000818:Animals; D002451:Cell Compartmentation / Q000502:physiology; D003034:Coated Pits, Cell-Membrane / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008407:Mast Cells / Q000201:enzymology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000032:analysis / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D000072377:Syk Kinase; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D017455:Receptors, IgE; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606898:Syk protein, rat; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases