SUMMARY Crosslinking of IgE-bound FcεRI triggers mast cell degranulation. Previous FRAP and phosphorescent anisotropy studies suggested that FcεRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods are used to redefine relationships between receptor mobility and signaling. QD-IgE-FcεRI aggregates of at least three receptors remain highly mobile over extended times at low concentrations of antigen that induce Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remains mobile at low doses that support secretion. FcεRI immobilization is marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocks secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates, is a trigger for receptor internalization, and is not required for tyrosine kinase activation leading to secretion.

Antigen-mediated activation of mast cells results in Ca 2+ -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 Ca 2+ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (FcER1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, FcER1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of