Functional nanoscale coupling of Lyn kinase with IgE-FcεRI is restricted by the actin cytoskeleton in early antigen-stimulated signaling

The allergic response is initiated on the plasma membrane of mast cells by phosphorylation of the receptor for immunoglobulin E (IgE), Fc epsilon RI, by Lyn kinase after IgE-Fc epsilon RI complexes are cross-linked by multivalent antigen. Signal transduction requires reorganization of receptors and membrane signaling proteins, but this spatial regulation is not well defined. We used fluorescence localization microscopy (FLM) and pair-correlation analysis to measure the codistribution of IgE-Fc epsilon RI and Lyn on the plasma membrane of fixed cells with 20- to 25-nm resolution. We directly visualized Lyn recruitment to IgE-Fc epsilon RI within 1 min of antigen stimulation. Parallel FLM experiments captured stimulation-induced Fc epsilon RI phosphorylation and colocalization of a saturated lipid-anchor probe derived from Lyn's membrane anchorage. We used cytochalasin and latrunculin to investigate participation of the actin cytoskeleton in regulating functional interactions of Fc epsilon RI. Inhibition of actin polymerization by these agents enhanced colocalization of IgE-Fc epsilon RI with Lyn and its saturated lipid anchor at early stimulation times, accompanied by augmented phosphorylation within Fc epsilon RI clusters. Ising model simulations provide a simplified model consistent with our results. These findings extend previous evidence that IgE-Fc epsilon RI signaling is initiated by colocalization with Lyn in ordered lipid regions and that the actin cytoskeleton regulates this functional interaction by influencing the organization of membrane lipids.