Abstract
During exocytosis in the pancreatic acinar cell, zymogen granules fuse directly with the apical plasma membrane and also with granules that have themselves fused with the plasma membrane. Together, these primary and secondary fusion events constitute the process of compound exocytosis. It has been suggested that the sequential nature of primary and secondary fusion is a consequence of the requirement for plasma membrane soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors, such as syntaxin 2, to enter the membrane of the primary fused granule. We have tested this possibility by determining the location of syntaxin 2 in unstimulated and stimulated pancreatic acini. Syntaxin 2 was imaged by confocal immunofluorescence microscopy. Fused granules were detected both through their filling with the aqueous dye lysine-fixable Texas Red-dextran and through the decoration of their cytoplasmic surfaces with filamentous actin. In unstimulated cells, syntaxin 2 was exclusively present on the apical plasma membrane. In contrast, after stimulation, syntaxin 2 had moved into the membranes of fused granules, as judged by its location around dye-filled structures of 1-mum diameter that were coated with filamentous actin. At long times of stimulation (5 min), the majority (85%) of dye-filled granules were also positive for syntaxin 2. In contrast, at shorter times (1 min), more dye-filled granules (29%) were syntaxin 2-negative. We conclude that syntaxin 2 enters the membrane of a fused zymogen granule after the opening of the fusion pore, and we suggest that this movement might permit the onset of secondary fusion.
Highlights
It is accepted that membrane fusion events occurring along the secretory pathway are mediated by trans-membrane complexes of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins [6]
Because the pancreatic acinar cell is known to contain syntaxin isoforms 2, 3, 4, 7, and 8 (8 –10), it was important to establish that the antibody we intended to use to detect syntaxin 2 was specific for that isoform
Syntaxin 2 is observed in regions around zymogen granules that have undergone exocytosis, as marked with the aqueous dye LF Texas Red-dextran
Summary
Reagents—A rabbit polyclonal antiserum raised against recombinant rat syntaxin 2 was provided by Dr R. The cells were washed twice with 2 ml of phosphate-buffered saline (PBS; 10 mM sodium phosphate, pH 7.4, and 150 mM NaCl) for 10 min each time and fixed in 4% (w/v) paraformaldehyde in sucrose/ phosphate buffer (210 mM sucrose and 30 mM sodium phosphate, pH 6.8), for 30 min. The cells were washed twice for 5 min in PBS, twice for 5 min in high-salt PBS (20 mM sodium phosphate, pH 7.4, and 500 mM NaCl), and incubated for 15 min in permeabilizing buffer (PBS containing 0.1% (w/v) Triton X-100). Cells were washed three times for 5 min in permeabilizing buffer containing 5% (w/v) goat serum (blocking buffer) and incubated with primary antibody overnight at 4 °C on Parafilm. The cells were washed twice with high-salt PBS for 5 min and once for 5 min in blocker, before incubation with secondary antibody for 90 min. The statistical significance of differences between means was assessed using Student’s t test for unpaired data
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