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Center for Nonlinear Studies |
The high affinity IgE receptor, FcεRI, is the principal multi-subunit immunoreceptor on the surface of human mast cells and basophils. Crosslinking of IgE-FcεRI complexes by multivalent allergen initiates complex signaling pathways leading to the release of mediators of allergic inflammation. Here, we generated two novel quantum dot (QD)-based probes to follow the dynamic events at the cell membrane that initiate FcεRI signaling: monovalent QD-IgE (binds FcεRI without crosslinking) and polyvalent DNP-QD (mimics allergen by crosslinking DNP-specific IgE). Using these probes and dual-color single QD tracking, we characterized the lateral motion of individual FcεRI in resting and activated states. We employed wide field, confocal and TIRF microscopy for multi-color imaging. In the resting condition, we observed co-confinement of multiple FcεRI in the same region (up to 2 µm in size). By simultaneously imaging QD-IgE-FcεRI and GFP-tagged actin, we directly observed the “corralling” of receptor motion by the actin cytoskeleton. Combining single QD tracking and electron microscopy allowed us to correlate FcεRI diffusional dynamics with topography and demonstrate that small, dynamic clusters reorganize into large, stable signaling domains within seconds of the addition of multivalent antigen.
Host: William Hlavacek