Exposure of pancreatic acinar cells to a secretagogue results in a time-dependent increase (20–45%) in both the diameter and relative depth of depressions (Fig. 3). Studies demonstrate that depressions return to resting size on completion of cell secretion (6, 7). No demonstrable change in pit size is detected following stimulation of secretion (6). Enlargement of the depression diameter and an increase in its relative depth following exposure to a secretagogue correlates with secretion. Additionally, exposure of pancreatic acinar cells to cytochalasin B, a fungal toxin that inhibits actin polymerization and secretion, results in a 15–20% decrease in depression size and a consequent 50–60% loss in secretion (6). Results from these experiments suggested depressions to be the secretory portals in pancreatic acinar cells. Furthermore, these studies demonstrated the involvement of actin in regulation of both the structure and function of depressions. Similarly, depression in resting GH cells measures 154  ±  4.5 nm (mean  ±  SE) in diameter, and following exposure to a secretagogue, results in a 40% increase in depression diameter (215  ±  4.6 nm; p  <  0.01), with no appreciable change in pit size (9). The enlargement of depression diameter during cell secretion and subsequent decrease accompanied by loss in secretion following exposure to actin depolymerizing agents (9) also suggested that they represent the secretory portal in GH cells. A direct determination that depressions are the secretory portals in cells, through which secretory products are expelled, was unequivocally demonstrated using immuno-AFM studies, initially in the exocrine pancreas (7) (Fig. 4), followed by studies in the GH cells of the pituitary (9). The localization at depressions of gold-conjugated antibody to secretory proteins finally provided the first direct evidence that secretion occurs through depressions. Moreover, AFM micrographs demonstrating the specific localization of gold-tagged amylase-specific antibodies (ZGs contain amylase) at depressions following stimulation of cell secretion (7, 13) conclusively demonstrated depressions to be the secretory portal in these cells. Similarly, in somatotrophs of the pituitary gland, gold-tagged growth hormone-specific antibody found to selectively localize at the depression openings following stimulation of secretion (9), established these sites too, to be the secretory portals in GH cells. Over the years, the term “fusion pore” has been loosely used to refer to plasma membrane dimples that originate following a secretory stimulus or to the continuity or channel established between opposing lipid membranes during membrane fusion. Therefore for clarity, the term “porosome” was assigned to the depression structures at the cell plasma membrane.

The porosome structure, at the cytosolic compartment of the plasma membrane in the exocrine pancreas ((14), Fig. 5) and in neurons ((10), Fig. 6), has also been determined at near nanometer resolution in live cells. To determine the morphology of porosomes at the cytosolic compartment of pancreatic acinar cells, isolated plasma membrane preparations in near physiological buffered solution have been imaged at ultrahigh resolution using the AFM. These studies reveal scattered circular disks measuring 0.5–1 μm in diameter, with inverted cup-shaped structures within (14). The inverted cups at the cytosolic compartment of isolated pancreatic plasma membrane preparations measure approximately 15 nm in height. On a number of occasions, ZGs ranging in size from 0.4 to 1 μm in diameter have been observed in association with one or more of the inverted cups, suggesting the circular disks to represent pits, and inverted cups porosomes, in inside-out pancreatic plasma membrane preparations. To further confirm that the cup-shaped structures are indeed porosomes, where secretory vesicles dock and fuse, immuno-AFM studies have been performed. As mentioned earlier, target membrane proteins SNAP-23 (61, 72) and syntaxin (62) (t-SNARE) and secretory VAMP v-SNARE or VAMP (60) are part of the conserved protein complex involved in fusion of opposing bilayers in the presence of calcium (58, 66, 67). Since ZGs dock and fuse at the plasma membrane to release vesicular contents, it was hypothesized that if the inverted cups or porosomes are the secretory sites, then plasma membrane-associated t-SNAREs should localize at the structure. The t-SNARE protein SNAP-23 had previously been reported in pancreatic acinar cells (72). A polyclonal monospecific SNAP-23 antibody recognizing a single 23 kDa protein in immunoblots of pancreatic plasma membrane fraction, when used in immuno-AFM studies, demonstrated selective localization to the base of the cup-shaped structures ((14); Fig. 7). These results confirm that the inverted cup-shaped structures in inside-out pancreatic plasma membrane preparations are indeed porosomes. The size and shape of the immunoisolated porosome complex has also been determined in exocrine pancreas ((14); Fig. 8), neurons ((10–12); Fig. 2), and astrocytes (73) using both negative staining EM and AFM. The immunoisolated porosome complex has further been structurally and functionally reconstituted into artificial liposomes and lipid bilayer membrane ((10–12, 14), Fig. 9). Transmission electron micrographs of pancreatic porosomes reconstituted into liposomes exhibit a 150–200 nm cup-shaped basket-like morphology, similar to their native structure observed in cells and when co-isolated with a ZG preparation (14). To test the functionality of the isolated porosome complex, purified porosomes obtained from exocrine pancreas or neurons have been reconstituted in lipid membrane of the electrophysiological bilayer setup (EPC9) and exposed to isolated ZGs (Fig. 9) or synaptic vesicle preparations. Electrical activity of the porosome-reconstituted membrane as well as the transport of vesicular contents from the cis to the trans compartments of the bilayer chambers when monitored demonstrated that the lipid membrane-reconstituted porosomes are indeed functional (10, 14), since in the presence of calcium, isolated secretory vesicles dock and fuse to transfer intravesicular contents from the cis to the trans compartment of the bilayer chamber. ZGs fused with the porosome-reconstituted bilayer as demonstrated by an increase in capacitance and conductance and a time-dependent transport of the ZG enzyme amylase from cis to the trans compartment of the bilayer chamber. Amylase was detected using immunoblot analysis of the buffer in the cis and trans compartments of the bilayer chambers, using immunoblot analysis. In the pancreas, chloride channel activity present in the reconstituted porosome complex is critical to porosome function, since the chloride channel blocker DIDS inhibits porosome function (Fig. 9). Similarly, the structure and biochemical composition of the neuronal porosome and the docking and fusion of synaptic vesicles at the neuronal porosome complex have also been demonstrated. AFM, EM, and electron density measurements followed by contour mapping, and 3D topography of the neuronal porosome, have further provided an understanding of the arrangement of proteins at nanometer resolution within the complex ((12), Fig. 9). Results from these studies demonstrate that proteins at the central plug of the porosome interact with proteins at the periphery of the complex, conforming to its eightfold symmetry (Fig. 2d, e). Furthermore, at the center of the porosome complex representing the porosome base, where synaptic vesicles dock and transiently fuse, SNARE proteins are assembled in a ring conformation. In neurons, the SNARE ring is composed of merely three SNARE pairs (74, 75) having a 1–1.5 nm in diameter channel, for the express release of neurotransmitters from synaptic vesicles via the porosome to the synaptic cleft. These studies demonstrate that porosomes are permanent structures at the presynaptic membrane of nerve terminals, where synaptic vesicles transiently dock and fuse to release neurotransmitters. Photon correlation spectroscopy (PCS) of isolated porosome complexes further confirms that neuronal porosomes measure on average 14–15 nm (Fig. 2i). In PCS measurements, the size distribution of isolated porosome complexes is obtained from plots of the relative intensity of light scattered by particles of known sizes and a calculation of their correlation function. Negative staining EM performed using low electron dose in a Tecnai 20 electron microscope operating at 200 kV demonstrated that proteins at the central plug of the porosome complex interact with proteins at the periphery of the structure (12). Similar to AFM micrographs, approximately eight interconnected protein densities are observed at the lip of the porosome complex in electron micrographs (Fig. 2). The eight interconnected protein densities are also connected to the central plug, via spoke-like structures. Electron density and contour maps, together with resultant 3D topology profiles of the porosome complex, provide further details of the circular arrangement of proteins and their connection to the central plug via distinct spokes (Fig. 2e). The contour map of proteins within the neuronal porosome complex has been determined using published procedures (76–79). These results have demonstrated the arrangement of proteins at the nanometer scale within the neuronal porosome complex. The next level of understanding of this supramolecular structure requires electron crystallography of isolation complexes, which are currently in progress. Studies demonstrate that the porosome complex constitutes: SNAP, syntaxin, the cytoskeletal proteins actin, α-fodrin, and vimentin, calcium channels β3 and α1c, together with the SNARE regulatory protein NSF (13, 14). Chloride ion channels ClC2 and ClC3 have also been identified as part of the porosome complex, and as previously stated, are critical for porosome function. Isoforms of the various other proteins identified within the porosome complex have been demonstrated using 2D-BAC gels electrophoresis. For example, three isoforms each of the calcium ion channel and vimentin are found in porosomes. Using yeast two-hybrid analysis and immunoisolation, studies confirm the presence and direct interaction between some of these proteins with t-SNAREs within the porosome complex (80). In addition to these proteins, studies report that the neuronal porosome assembly requires membrane cholesterol (11). Results from recent studies (11) demonstrate a significant inhibition in interactions between porosome-associated t-SNAREs and calcium channels following depletion of membrane cholesterol. Since calcium is critical to SNARE-induced membrane fusion, the loss of interaction between SNAP-25, Syntaxin-1, and calcium channels at the neuronal porosome complex would seriously compromise or even abrogate neurotransmission at the nerve terminal.