High resolution mapping of mast cell membranes reveals primary and secondary domains of FcεRI and LAT

In mast cells, cross-linking the high-affinity IgE receptor (Fc epsilon RI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting Fc epsilon RI colocalize loosely with Lyn, whereas cross-linked Fc epsilon RI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of Fc epsilon RI beta is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLC gamma isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (P13-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLC gamma1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLC gamma2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLC gamma2, Gab2, and a portion of p85 colocalize with Fc epsilon RI beta in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLC-gamma1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of P13-kinase, Fc epsilon RI cross-linking increases P13-kinase activity in anti-LAT, anti-Fc epsilon RI beta, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around Fc epsilon RI beta and from secondary domains, including one organized around LAT.