Depressions Are Probably the Exocytic Fusion Pores.
It is clearly suggested from these studies that the plasma membrane-associated depressions within pits may be involved in the exo/endocytic process. To determine whether the changes in depressions observed following stimulation of secretion are due to secretory vesicle fusion or to compensatory endocytosis, two hypothetical scenarios can be envisioned. One is that secretory vesicles dock and fuse with the plasma membrane away from the depressions, while the depressions facilitate compensatory endocytosis. The other is that secretory vesicles dock and fuse at the depressions, resulting in distention of the depression depth and diameter as demonstrated in our study. Since we do not observe any change in distance between depressions and pits following stimulation of secretion, this further argues against the first possibility, where incorporation of secretory vesicle with the plasma membrane would result in a transient increase in distance. It is likely that depressions may be the "fusion pores" in acinar cells. Since ZGs range in size from 0.1 to 1 µm in diameter (unpublished observation), the total fusion of a vesicle at a depression would extend the diameter of the depression severalfold larger than what is observed following stimulation of secretion. We therefore postulate that secretory vesicles transiently fuse at these plasma membrane depressions and release their contents at the apical membrane face. Furthermore, it could be argued that since no decrease from control levels in depression diameter or depth was observed throughout all times examined following stimulation of secretion, the depressions should be discounted as possible endocytic components. A hypothetical model involving depressions acting as transient docking and fusion stations of secretory vesicles is depicted schematically in Fig. 5. These plasma membrane-associated depressions are probably the so-called "exocytic fusion pores" observed in mast cells and at the neuromuscular junction when freeze-fracture electron microscopy (2-4) is used and as suggested by electrophysiological measurements (5-9). The BAFM has allowed us for the first time to observe, in real time, the dynamics of the plasma membrane-associated depressions involved in exocytosis in living cells.
Although a relative increase in depth of depressions is identifiable following stimulation of secretion (Fig. 3b, middle panel), absolute depth measurements are not possible due to limitation in access of the BAFM cantilever into the depressions' interior. Future development of finer cantilevers may make absolute depth measurements of the depressions possible. The measured size of the imaged depressions and pits may represent different elasticity at the plasma membrane due to differences in lipid content. Following stimulation of secretion, the detected dilation at depressions may be due to an increase in lipid content at the site. No irreversible fusion events were detected. The reason may be that the study was performed at room temperature or due to the nature of the secretagogue or both. At room temperature only very slow and transient fusion may occur, in contrast to exocytosis at 37°C.
A group of large pore-like structures ranging from 0.25 to 1 µm in diameter appear intermittently at the apical cell surface, and they do not change significantly in shape or size following stimulated secretion. The presence and function of these large-diameter pores is unknown at this time.
Actin Involvement in Depression Dynamics.
Earlier studies by Bauduin et al. (37) demonstrated that high concentrations of cytochalasin B inhibit secretion from the exocrine pancreas. Previous studies in pancreatic acinar cells and other cell types also demonstrated a requirement for actin filaments in exocytosis (37-41). A recent study by Muallem et al. (42) further demonstrates this role of actin and implicates its participation at a late step of exocytosis in the exocrine pancreas. The Muallem et al. (42) study demonstrates that high concentrations of actin-depolymerizing proteins inhibit exocytosis by agonists of several downstream effectors, suggesting that a minimal cytoskeletal structure is required for this process. From results of this study, the authors suggest actin participation in the secretory process to be a step close to or downstream of ZG docking at the plasma membrane. These studies further support our findings that actin regulates depressions, which may be the exocytic fusion pores, regulating amylase release. Our study's demonstration of an increase in amylase release during dilation of the depressions (Mas7 exposure) and a decrease in amylase release following constriction of the depressions (cytochalasin B exposure) further argues in favor of depressions being the exocytic fusion pores in live acinar cells.
FOOTNOTES
§ To whom reprint requests should be addressed at: Section of Gastroenterology, Department of Surgery, BML 365, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510-8062. e-mail:
Bhanu.Jena@Yale.edu
.
Abbreviations: BAFM, BioScope atomic force microscope; ZG, zymogen granule.
ACKNOWLEDGEMENTS
We thank Joseph F. Hoffman, James D. Jamieson, Gerhard H. Giebisch, and Fred J. Sigworth for valuable advice and discussion. We thank Digital Instruments for their continued technical support and Birgit Heil for help in preparing the hypothetical model. This work was supported by grants from the National Institutes of Health and the Ohse Award (to B.P.J.), the National Institutes of Health and the Whitaker Foundation (to J.P.G.), and a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to S.W.S.).
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