title: Live pancreatic acinar imaging of exocytosis using syncollin-pHluorin.
abstract: In this report, a novel live acinar exocytosis imaging technique is described. An adenovirus was engineered, encoding for an endogenous zymogen granule (ZG) protein (syncollin) fused to pHluorin, a pH-dependent green fluorescent protein (GFP). Short-term culture of mouse acini infected with this virus permits exogenous adenoviral protein expression while retaining acinar secretory competence and cell polarity. The syncollin-pHluorin fusion protein was shown to be correctly localized to ZGs, and the pH-dependent fluorescence of pHluorin was retained. Coupled with the use of a spinning disk confocal microscope, the syncollin-pHluorin fusion protein exploits the ZG luminal pH changes that occur during exocytosis to visualize exocytic events of live acinar cells in real-time with high spatial resolution in three dimensions. Apical and basolateral exocytic events were observed on stimulation of acinar cells with maximal and supramaximal cholecystokinin concentrations, respectively. Sequential exocytic events were also observed. Coupled with the use of transgenic mice and/or adenovirus-mediated protein expression, this syncollin-pHluorin imaging method offers a superior approach to studying pancreatic acinar exocytosis. This assay can also be applied to acinar disease models to elucidate the mechanisms implicated in pancreatitis.
authors.affiliation: Dept. of Medicine, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada.
authors.forename: Nestor A
authors.initials: NA
authors.lastname: Fernandez
authors.forename: Tao
authors.initials: T
authors.lastname: Liang
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D016764:Cell Polarity; D002766:Cholecystokinin / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008297:Male; D008561:Membrane Fusion / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence / Q000379:methods*; D010179:Pancreas / Q000166:cytology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D022142:Secretory Vesicles / Q000737:chemistry / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; C485187:PHluorin; D011993:Recombinant Fusion Proteins; C107454:Sycn protein, rat; D049452:Green Fluorescent Proteins; D002766:Cholecystokinin