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Imaging of exocytosis and endocytosis in exocrine cells Enzyme secretion in acinar cells of exocrine pancreas is dependent on compound exocytosis of secretory vesicles (Nemoto et al., 2001). After fusion with the plasma membrane the lifetime of secretory vesicles is in the order of minutes and over this time the vesicles remain docked to the plasma membrane and the fusion pore can remain open (Thorn et al., 2004). However, the dynamics of fusion pore is not fully determined and it is still not clear whether the current models of exocytosis in neurons and endocrine cells (“kiss and collapse”, “kiss and run”) are applicable to acinar pancreatic cells. In the present study we looked at post fusion events in pancreatic acinar cells. We reveal secretory vesicle staining in pre and post fusion periods and fusion pore dynamics using lysine-fixable dyes. Lobules and fragments of exocrine pancreas of male albino CD-1 mouse (weight 18-20g) were prepared by collagenase digestion method. Cells attached to Poly-l-lysine-coated glass coverslips were stimulated with 10 µM acetylcholine for 1 min. Then in control experiments cells were loaded with Lysine-fixable Texas Red and Fluorescein dextran dyes (2mg/ml, Molecular Probes) for 5 min and fixed in 4% PFA. In experiments designed to study post fusion events cells were stimulated with acetylcholine at presence Texas Red dextran dye in the extracellular solution. Then we applied atropine (100 µM) and at time points 1, 5, 10, 20 and 30 min, after we applied Fluorescein dextran dye for 5 min and fixed cells in 4% PFA. Images were obtained on a Zeiss Axiovert LSM510 confocal microscope, 63X oil 1.4 NA objective. Confocal image sections (z ~0.1µm) were processed using Metamorph (Universal Imaging Corporation). We found that in control experiments lysine-fixable Texas Red and Fluorescein dextran dyes produced an identical pattern of staining in cells stimulated with acetylcholine. The mean diameter of the fluorescent spots, determined as the peak width at half-maximal fluorescence, is 0.82 +/-0.03 µm (mean +/- SEM, n=33) and is consistent with diameter of zymogen granules (Nemoto et al., 2001). In the post fusion experiments at earlier points (1 min) Texas Red and Fluorescein dextran dyes staining patterns were identical. However, at later points (5 and 10 min) some granules were filled with Texas Red dextran dye only, indicating closure of the fusion pore. This approach, therefore, allows us to visualize for the first time the fusion pore dynamics and shows that in acinar cells the fusion pore can close. Further experiments will aim to describe the fusion pore dynamics in more details. Nemoto et al. (2001) Nat Cell Biol 75:153-162. |
