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 which occur during exocytosis to visualize exocytic events of live acinar cells in real-time with high spatial resolution in 3D. Apical and basolateral exocytic events were observed upon stimulation of acinar cells with maximal and supramaximal cholecystokinin (CCK) concentrations, respectively. Sequential exocytic events were also observed. Coupled with the use of transgenic mice and/or adenoviral-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.
- exocrine pancreas
- sequential exocytosis
- basolateral exocytosis
- spinning disk microscopy
- Copyright © 2010, American Journal of Physiology - Cell Physiology