Author:
Cohen Roy,Corwith Kathryn,Holowka David,Baird Barbara
Abstract
Mast cell activation initiated by antigen-mediated crosslinking of IgE receptors results in stimulated exocytosis of secretory lysosomes in the process known as degranulation. Much has been learned about the molecular mechanisms important for this process, including the critical role of Ca2+ mobilization, but spatio-temporal relationships between stimulated Ca2+ mobilization and granule exocytosis are incompletely understood. Here we use a novel imaging-based method that utilizes fluorescein isothiocyanate (FITC)-dextran as a reporter for granule exocytosis in RBL mast cells and takes advantage of the pH sensitivity of FITC. We demonstrate the selectivity of FITC-dextran, accumulated by fluid phase uptake, as a marker for secretory lysosomes, and we characterize its capacity to delineate different exocytotic events, including full fusion, kiss-and-run transient fusion, and compound exocytosis. Using this method, we find strong dependence of degranulation kinetics on the duration of cell-substrate attachment. We combine imaging of degranulation and Ca2+ dynamics to demonstrate a spatial relationship between the sites of Ca2+ wave initiation in extended cell protrusions and exocytosis under conditions of limited antigen stimulation. In addition, we find that the spatially proximal Ca2+ signaling and secretory events correlate with participation of TRPC1 channels in Ca2+ mobilization.
Publisher
The Company of Biologists
Cited by
42 articles.
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