ABSTRACT
Crosslinking of IgE-bound FcepsilonRI triggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that FcepsilonRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-FcepsilonRI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. FcepsilonRI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a trigger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.
Subject(s)
Protein Multimerization , Receptors, IgE/immunology , Signal Transduction , Animals , Antigens/immunology , Cell Line, Tumor , Immunoglobulin E/immunology , Intracellular Signaling Peptides and Proteins/metabolism , Phosphorylation , Protein Subunits/immunology , Protein Subunits/metabolism , Protein Transport , Protein-Tyrosine Kinases/metabolism , Quantum Dots , Rats , Receptors, IgE/metabolism , Syk KinaseABSTRACT
Antigen-mediated activation of mast cells results in Ca2+-dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time-lapse confocal microscopy on RBL-2H3 cells transfected with a green fluorescent protein-Fas ligand fusion protein (GFP-FasL). Green fluorescent protein-labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca2+ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response.
