The Plasma Membrane Q-SNARE Syntaxin 2 Enters the Zymogen Granule Membrane during Exocytosis in the Pancreatic Acinar Cell

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-m 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.Regulated exocytosis in the pancreatic acinar cell, in response to a rise in intracellular Ca 2ϩ concentration (1), involves the fusion of the membranes of zymogen granules both with the apical plasma membrane and with each other (2-5). This latter process, known as sequential or compound exocytosis, occurs in only a few cell types and, in the case of the acinar cell, is likely to be an adaptation to increase the efficiency of digestive enzyme secretion at the spatially restricted apical pole of the cell.It is now 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). The SNARE 1 complex consists of four intertwined amphipathic ␣-helices (7). The SNAREs of one membrane contribute three helices, each of which contains a strategically located glutamine residue; hence, such SNAREs have been termed Q-SNAREs (e.g. syntaxin and 25-kDa synaptosome-associated protein (SNAP-25)). The fourth helix is provided by the SNARE present on the apposing membrane and contains a similarly placed arginine residue, hence the name R-SNARE (e.g. synaptobrevin). Exocytotic membrane fusion in the acinar cell is known to be SNARE-dependent (8 -10). Syntaxin 2, the Q-SNARE present on the apical plasma membrane (8, 9), is required for granule-plasma membrane fusio...

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“…enabling other granules to coalesce with it" (4). Nearly 40 years later this idea was supported by evidence that, after fusion, SNAREs may move into the primary granule membrane (9,10,11). These studies have limited functional information, but the implied model is that the fused granule could take on the SNARE apparatus of the plasma membrane (1).…”

“…To explore this possibility we performed immunoprecipitation experiments using antibodies against either syntaxin 2 or syntaxin 3. We previously reported syntaxin 2 to be the putative Q-SNARE on the apical PM (8,9,25,26), whereas syntaxin 3 is a Q-SNARE most abundant on zymogen granules (8,26,27); hence, syntaxin 2 and 3 would be the likely Q-SNAREs for primary and secondary exocytosis, respectively. Because of the requirement for larger amounts of tissue, we used rat pancreas in these experiments.…”

“…Three lines of evidence support this idea. First, there is evidence that syntaxin 2 moves into the primary granule membrane in acinar cells (9). Second, evidence in pancreatic ␤ cells that shows that another SNARE, SNAP25, also moves selectively into primary granules as a prelude to secondary fusion (10).…”

Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy

“…Second, we have shown that F-actin prevents the fusion between the SCGs accumulated in the apical area and those fused with the APM. In secretory systems in which the collapse of the SCGs is a slow process, the fused granules may have acquired components of the APM, which makes them more susceptible to fusion with other SCGs (44). To prevent these fusion events, F-actin could be recruited onto the SCGs, performing a "barrier" function similar to what has been proposed for the F-actin at the APM (18,23).…”

Serous Cells in the Parotid Glands of Two Species of Tamarins: Polarized Secretory Granules

“…In this process, the membranes surrounding the now empty granule remnants must take on the characteristics of the plasmalemma so that further granules recognize them as potential docking sites. In the pancreatic acinar cell, it has been shown by confocal immunofluorescence microscopy that protein attachment protein acceptors, such as syntaxin 2, extend from the plasmalemma into the membranes of fused granules (Pickett et al, 2005).…”