Enlargement of depression diameter following exposure of acinar cells to a secretagogue correlated with increased secretion. Additionally, actin-depolymerizing agents known to inhibit secretion (19) resulted in decreased depression size and accompanied loss in secretion. These studies suggested depressions to be the fusion pores. However, a more direct determination of the function of depressions was required. Combining the use of a gold-conjugated antibody to a specific vesicular secretory protein with AFM provides the means to determine if secretion occurs at depressions (8, 7). The membrane-bound secretory vesicles in exocrine pancreas contain the starch-digesting enzyme amylase. By using amylase-specific immunogold AFM studies, localization of amylase at depressions following stimulation of secretion was demonstrated (Fig. 4) (8). These studies confirm depressions to be the fusion pores in pancreatic acinar cells, where membrane-bound secretory vesicles dock and fuse to release vesicular contents (Fig. 5). Similarly in somatotrophs of the pituitary, gold-tagged GH-specific antibodies were found to be selectively localized at depressions following stimulation of secretion (7), again confirming depressions to be fusion pores.
Composition of the fusion pore
Although the molecular composition of the fusion pore (depression) remains to be established, our studies on the role of actin in the regulation of depression structure and dynamics clearly suggests actin to be a major component of the fusion pore complex. Target membrane proteins SNAP25 and syntaxin (t-SNARE) and secretory vesicle-associated membrane protein (v-SNARE) are part of a conserved protein complex involved in fusion of opposing bilayers (17, 20). Since membrane-bound secretory vesicles dock and fuse at depressions to release vesicular contents, it is reasonable to suggest that plasma membrane-associated t-SNAREs are part of the fusion pore complex. In the past decade, a number of studies demonstrated the involvement of cytoskeletal proteins in exocytosis, some directly interacting with SNAREs (3, 8, 10, 11, 14, 15). Actin and microtubule-based cytoskeleton have been implicated in intracellular vesicle traffic (11). Fodrin, which was previously implicated in exocytosis (3), has recently been shown to directly interact with SNAREs (14). Recent studies demonstrate that -fodrin regulates exocytosis through its interaction with the syntaxin family of proteins (14). The COOH-terminal coiled coil region of syntaxin interacts with -fodrin, a major component of the submembranous cytoskeleton. Similarly, vimentin filaments interact with SNAP23/25 and control the availability of free SNAP23/25 for assembly of the SNARE complex (10). Additionally, our recent studies (unpublished observations) demonstrate a direct interaction between actin and SNAREs. Results from these studies suggest that vimentin, -fodrin, actin, and SNAREs may all be part of the fusion pore complex. However, purification and further biochemical characterization of the fusion pore are required to determine its composition. 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 is its binding site, and VAMP is bound to myosin V in a calcium-independent manner (15). Further interaction of myosin V with syntaxin requires calcium and calmodulin. Studies suggest that VAMP acts as a myosin V receptor on secretory vesicles and regulates formation of the SNARE complex (15). Furthermore, interaction of VAMP with synaptophysin and myosin V has been demonstrated (8).