Our current understanding of secretory vesicle fusion with the plasma membrane is derived from morphological (1-4), electrophysiological (5-10), and biochemical studies (1, 11-14). Morphological studies have been performed at both light and electron microscopic levels, primarily on chemically fixed or quick-frozen tissues. Ultrastructural studies on quick-freeze and freeze-fractures of stimulated mast cells demonstrate the presence of depressions, approximately 100 nm in diameter, which invaginate, subsequently making contact and fusing with the secretory vesicle membrane. The result is the formation of a continuous channel connecting the granule interior with the extracellular space (2). Electron microscopy of quick-freeze and fracture of the neuromuscular junctions following stimulation of the nerves demonstrates the presence of 30- to >150-nm diameter openings at the presynaptic plasma membrane (15). Similarly, patch-clamp experiments have shown that secretory granules can transiently fuse with the plasma membrane and release their secretory products (3, 4, 7, 16, 17). Capacitance measurements of mast cells following stimulation of secretion demonstrate the release of secretory products from a secretory vesicle that do not undergo complete fusion (7). Patch-clamp measurements of the activity of individual fusion pores in mast cells further suggest the presence of a pore through the bilayered plasma membrane that becomes continuous with the secretory vesicle membrane following stimulation of secretion (9).
Although these studies have revealed a wealth of information on exocytosis, little is known about plasma membrane components and the dynamics of their involvement in the secretory process of a living cell. Using the BioScope atomic force microscope (BAFM) (18) and a newly designed perfusion chamber mounted on an inverted microscope, we have been able to observe and study the involvement of a new group of plasma membrane structures in living cells. These structures were unidentifiable earlier in fixed or frozen tissue preparations, probably due to membrane perturbations during processing. The crucial advantage of using the atomic force microscope in our study was that live cells could be imaged at nanometer resolution (19-23) as they underwent exocytosis. Pancreatic acinar cells were chosen for the present study due to the presence of a slow secretory process (minutes) (1, 24, 25), compared with neuroendocrine cells (seconds to milliseconds) (26) or neurons (microseconds) (12, 27). In such a slow secretory cell, one is more likely to identify the sequence of events leading to the fusion of secretory vesicles with the plasma membrane, which are unidentifiable in a fast secretor. When isolated acinar cells (Fig. 1) are exposed to a secretagogue, they release their secretory contents to the extracellular space in membrane-bound vesicles called zymogen granules (ZGs). One of the major components of the secreted material is amylase, which has been measured in our study to estimate the percent of total secretion from these cells (24). Mastoparan, an amphiphilic tetradecapeptide from wasp venom, is known to stimulate heterotrimeric Gi and Go proteins (28, 29). Recently, we have demonstrated the involvement of Gi and Go proteins in secretion from the rat exocrine pancreas (unpublished observation). Exposure of pancreatic acinar cells to Mas7, an active mastoparan analog, results in stimulation of secretion. The inactive mastoparan analog Mas17 has no effect on secretion from isolated pancreatic acinar cells. Time-course studies of amylase secretion and BAFM imaging of the plasma membrane were carried out on stimulated and unstimulated cells.
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