title: Spatiotemporal resolution of mast cell granule exocytosis reveals correlation with Ca2+ wave initiation.
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 crucial role of Ca(2+) mobilization, but spatio-temporal relationships between stimulated Ca(2+) mobilization and granule exocytosis are incompletely understood. Here we use a novel imaging-based method that uses 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 to substrate attachment. We combine imaging of degranulation and Ca(2+) dynamics to demonstrate a spatial relationship between the sites of Ca(2+) wave initiation in extended cell protrusions and exocytosis under conditions of limited antigen stimulation. In addition, we find that the spatially proximal Ca(2+) signaling and secretory events correlate with participation of TRPC1 channels in Ca(2+) mobilization.
authors.affiliation: Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301,  USA.
authors.forename: Roy
authors.initials: R
authors.lastname: Cohen
authors.forename: Kathryn
authors.initials: K
authors.lastname: Corwith
authors.forename: David
authors.initials: D
authors.lastname: Holowka
authors.forename: Barbara
authors.initials: B
authors.lastname: Baird
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D003584:Cytological Techniques; D003594:Cytoplasmic Granules / Q000378:metabolism; D005089:Exocytosis; D008247:Lysosomes / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology* / Q000378:metabolism*; D051381:Rats; D059008:Time-Lapse Imaging / Q000379:methods*
chemical_list: D002118:Calcium