Single Molecule Studies of FcεRI Dynamics Link Mobile Receptors with Signaling

The high affinity IgE receptor, FceRI, is the principal multi-subunit immunoreceptor on the surface of mast cells and basophils. These receptors bind circulating IgE with high affinity and are activated when multivalent allergen crosslinks IgE-bound receptors. Crosslinking initiates a complex signaling pathway that ultimately leads to degranulation and release of key mediators of allergic inflammation. To study the dynamic events that induce FceRI signaling, we generated two novel quantum dot (QD) probes for single particle tracking: monovalent QD-IgE that binds FceRI without crosslinking and multivalent DNP-QD that mimics allergen by crosslinking DNP-specific IgE. Previously, we provided direct evidence that actin filaments “corral” resting receptor motion and are involved in receptor immobilization (Andrews et al, Nature Cell Biology 10:955). Simultaneous imaging of single QD-IgE-FceRI complexes and GFP-tagged actin revealed that membrane-proximal actin bundles form a dynamic labyrinth that restricts receptor diffusion. Real-time imaging revealed that receptors become immobilized within seconds of crosslinking by high doses (1 μg/ml) of multivalent antigen and that immobilization is delayed when actin is disrupted. We have now investigated further the relationship between receptor immobilization and activation. We find that the kinetics of immobilization are dose dependent and receptors remain mobile at antigen doses corresponding to maximal degranulation (0.001-0.01 μg/ml). Using a novel hyperspectral microscope with ∼1 nm spectral resolution, we tracked up to five spectrally distinct QDs simultaneously. Multi-color tracking of QD-IgE-FceRI complexes revealed that small crosslinked clusters (2-4 receptors) remain mobile under activation conditions. In addition, DNP-QDs were shown to induce degranulation, yet DNP-QD-crosslinked receptors continue to diffuse. These results indicate that receptor immobilization is a feature of highly aggregated receptors, depends upon an intact actin cytoskeleton, and is more likely involved in signal termination than activation.