INTRODUCTION
The fusion of membrane-bound secretory vesicles at the cell plasma membrane (PM) and consequent expulsion of vesicular contents is a fundamental cellular process regulating basic physiological functions such as neurotransmission, enzyme secretion, or hormone release. Secretory vesicles dock and fuse at specific PM locations after secretory stimuli. Earlier electrophysiological studies on mast cells suggested the existence of fusion pores at the cell PM, which became continuous with the secretory vesicle membrane after stimulation of secretion (Monck et al., 1995
). Atomic force microscopy (AFM) has confirmed the existence of the fusion pore and its structure and dynamics in both exocrine (Schneider et al., 1997) and neuroendocrine cells (Cho et al., 2002a,b) at near nm resolution and in real time. Fusion pores in NG108-15 nerve cells have also been reported (Tojima et al., 2000).
Isolated live pancreatic acinar cells in physiological buffer, when imaged with the AFM (Cho et al., 2002c; Schneider et al., 1997), reveal at the apical PM a group of circular pits measuring 0.4–1.2 µm in diameter which contain smaller "depressions" (see Fig. 1 a). Each depression averages between 100 and 150 nm in diameter, and typically 3–4 depressions are located within a pit. The basolateral membrane of acinar cells is, however, devoid of either pits or depressions. High resolution AFM images of depressions in live cells further reveal a cone-shaped morphology (see Fig. 1 d). The depth of each depression cone measures 
15–30 nm. Similarly, both growth hormone (GH) secreting cells of the pituitary gland and chromaffin cells possess pits and depression structures in their PM (Cho et al., 2002a,b), suggesting their universal presence in secretory cells.
Exposure of pancreatic acinar cells to a secretagogue (mastoparan) results in a time-dependent increase (20–35%) in depression diameter, followed by a return to resting size after completion of secretion (Cho et al., 2002a,c; Schneider et al., 1997). No demonstrable change in pit size, however, is detected during this time (Schneider et al., 1997). Enlargement of depression diameter and an increase in its relative depth after exposure to secretagogues correlated with increased secretion. Conversely, exposure of pancreatic acinar cells to cytochalasin B, a fungal toxin that inhibits actin polymerization, resulted in a 15–20% decrease in depression size and a consequent 50–60% loss in secretagogue-induced secretion. Results from these studies suggested that depressions are the fusion pores in pancreatic acinar cells. Furthermore, these studies demonstrated the involvement of actin in regulation of the structure and function of depressions. Analogous to pancreatic acinar cells, examination of resting GH secreting cells of the pituitary (Cho et al., 2002a) and chromaffin cells of the adrenal medulla (Cho et al., 2002b) also revealed the presence of pits and depressions on the cell PM. Depressions in resting GH cells measured 154 ± 4.5 nm (mean ± SE) in diameter. Exposure of the GH cell to a secretagogue resulted in a 40% increase (215 ± 4.6 nm; p in pit size.
The enlargement of depression diameter during secretion and the known effect that actin depolymerizing agents decrease depression size and inhibit secretion (Schneider et al., 1997) suggested the depressions to be the fusion pores. However, a more direct determination of the function of depressions was required. Localization of a gold conjugated antibody to a specific vesicular secretory protein with AFM provided the ability to determine whether secretion occurred at depressions (Cho et al., 2002a,c). The membrane-bound secretory vesicles in exocrine pancreas contain the starch digesting enzyme amylase. The AFM was used to localize amylase-specific antibodies tagged with colloidal gold at "depressions" after stimulation of secretion (Cho et al., 2002c). These studies confirm that "depressions" are the fusion pores in pancreatic acinar cells where membrane-bound secretory vesicles dock and fuse to release vesicular contents. Similarly, in somatotrophs of the pituitary, gold-tagged GH-specific antibody was selectively localized at depressions after stimulation of secretion (Cho et al., 2002a), again identifying the depressions in GH cells as the fusion pores.
Although the molecular composition of the fusion pore is unknown, studies on the role of actin in the regulation of depression structure and dynamics (Schneider et al., 1997) clearly suggest that actin is a component of the fusion pore complex. Target membrane proteins SNAP-25 and syntaxin (t-SNARE) and secretory vesicle associated membrane protein (v-SNARE) are part of the conserved protein complex involved in fusion of opposing bilayers (Rothman, 1994; Weber et al., 1998). Because membrane-bound secretory vesicles dock and fuse at depressions to release vesicular contents, it is reasonable to suggest that PM-associated t-SNAREs are part of the fusion pore complex. In the last decade, a number of studies demonstrated the involvement of cytoskeletal proteins in exocytosis, some directly interacting with SNAREs (Bennett, 1990; Cho et al., 2002c; Faigle et al., 2000; Goodson et al., 1997; Nakano et al., 2001; Ohyama et al., 2001). The actin and microtubule-based cytoskeleton has been implicated in intracellular vesicle traffic (Goodson et al., 1997). Fodrin, which was previously implicated in exocytosis (Bennett, 1990), has recently been shown to interact directly with SNAREs (Nakano et al., 2001). Recent studies demonstrated that 
-fodrin regulates exocytosis through its interaction with the syntaxin family of proteins (Nakano et al., 2001). The C-terminal coiled-coil region of syntaxin interacts with -fodrin, a major component of the sub membranous cytoskeleton. Similarly, vimentin filaments interact with SNAP-23/25 and control the availability of free SNAP-23/25 for assembly of the SNARE complex (Faigle et al., 2000). Results from these studies suggest that vimentin, -fodrin, actin, and SNAREs may all be part of the fusion pore complex. Additional proteins such as v-SNARE (VAMP or synaptobrevin), synaptophysin, and myosin may associate when the fusion pore establishes continuity with the secretory vesicle membrane. The globular tail domain of myosin V contains a binding site for VAMP which is bound in a calcium independent manner (Ohyama et al., 2001). Further interaction of myosin V with syntaxin requires both calcium and calmodulin. It has been suggested that VAMP acts as a myosin V receptor on secretory vesicles and regulates formation of the SNARE complex (Ohyama et al., 2001). Interaction of VAMP with synaptophysin and myosin V was also observed by Prekeris and Terrian (1997). To understand the fusion pore in greater detail, its structure and biochemistry were further examined in the present study by using a combination of AFM, immunoAFM, electron microscopy (EM), and immunochemical analysis.
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