Studies in the early 90’s demonstrated for the first time the physical existence of fusion pores or porosomes as permanent structures at the plasma membrane in live secretory cells . Discovery of the porosome, and its function as the universal secretory machinery, is the major discovery in cell biology since discovery of the ribosome and determination of its involvement in protein synthesis. The atomic force microscope (AFM) allowed discovery of porosomes at the plasma membrane in live cells and their structure and dynamics uncovered for the first time at nm resolution and in real time in live pancreatic acinar cells undergoing secretion . The AFM images of porosomes in live cells strikingly resemble the proposed hypothetical model of the fusion pore published earlier , which was built on the concept of a lipid-enriched proteinaceous fusion pore having defined dimensions [26, 27]. Pancreatic acinar cells are polarized cells which secrete digestive enzymes following a meal. Unlike neurons or neuroendocrine cells, the pancreatic acinar cell is a slow secretory cell, secreting in minutes rather in seconds or milliseconds. The pancreatic acinar cell is a medium sized cell measuring 8–14 μm and is one of the well-characterized cells. Historically, the pancreatic acinar cell has been used in studies on cell secretion. These attributes may have been the primary reason for choosing the pancreatic acinar cell for cell secretion studies using the AFM leading to the discovery of the porosome [25, 28–31]. AFM studies carried out on isolated live pancreatic acinar cells in physiological buffer demonstrate at the apical plasma membranea group of circular ‘pits’ measuring 0.4–1.2 μm in diameter. Within each ‘pit’ typically 3–4 ‘depressions’, each measuring 100–150 nm in diameter, are present . The ‘depressions’ turned out to be the fusion pore or porosome, where secretory vesicles transiently dock and fuse to release intravesicular contents to outside of the cell [28–30]. Studies demonstrate that porosomes are present only at the apical plasma membrane in pancreatic acinar cells, where secretion is known to occur. The basolateral membranes of acinar cells are devoid of ‘pits’ or the porosome structures . When pancreatic acinar cells are exposed to secretagogues (secretory stimulants) such as carbamylcholine or mastoparan, there is a time-dependent increase (20–35%) in the diameter of porosomes followed by a return to their resting size following completion of secretion . The enlargement of porosome diameter and an increase in its relative depth is demonstrated and correlates with increased secretion . When cells are exposed to cytochalasin B, a fungal toxin that inhibits actin polymerization, it results in a 15–20% decrease in porosome size and a consequent 50–60% loss in secretion . These results suggested porosomes to be the long sought-after and elusive fusion pores. The involvement of actin in regulating both the structure and function of porosomes was further demonstrated from these studies. The membrane-bound secretory vesicles in exocrine pancreas, called zymogen granules (ZGs), contain the starch digesting enzyme amylase. Subsequent immuno-AFM studies determined the selective localization of gold-conjugated amylase antibody to porosomes, thereby conclusively demonstrating secretion to occur through these structures . These findings confirmed porosomes to be permanent fusion pore structures at the plasma membrane of live pancreatic acinar cells. Up to this point, only the topology of porosomes at the live cell surface was available. To determine the morphology of the porosome at the cytosolic compartment, pancreatic plasma membrane preparations suspended in physiological buffer solutions were used . When inside-out membrane preparations in buffer and adhering to a mica surface were imaged using the AFM, scattered circular disks measuring 0.5–1 μm in diameter (the pits) containing inverted cup-shaped structures -the porosomes, were demonstrated . Studies also demonstrated the association of secretory vesicles and the presence of t-SNAREs at the base of these cup-shaped structures, further confirming them to be porosomes, the cell’s secretory machinery . Subsequently, the structure of the porosome was further examined and confirmed using transmission electron microscopy (TEM) . TEM studies also demonstrated porosomes to possess a cup-shaped structure, with similar dimensions as determined by AFM. Using high-resolution TEM, it was demonstrated that porosomes possess a basket-like morphology, with three lateral rings and a number of vertically arranged ridges [29, 30]. Subsequently, the porosome was isolated and its composition determined. Studies demonstrated that SNAP-23, syntaxin 2, actin, α-fodrin, vimentin, the calcium channels β3 and α1c, together with the SNARE regulatory protein NSF, and the chloride ion channels ClC2 and ClC3, constitute the porosome complex . Using yeast 2-hybrid analysis, the presence and interaction of some of these proteins within the porosome complex has further been confirmed . In a recent study, the critical role of cholesterol on the structural integrity of the porosome complex has also been reported . The morphology and size of the immunoisolated porosome complex (protein backbone of the porosome) has also been determined, using both negative staining electron microscopy and AFM . The immunoisolated porosome complex has also been reconstituted into artificial liposomes, and the liposome-reconstituted complex examined using TEM . Transmission electron micrographs of exocrine pancreatic tissue, and liposome-reconstituted porosome complex, demonstrate a 150–200 nm cupshaped basket-like structure as observed when the porosome is co-isolated with secretory vesicles. Reconstituted porosomes in lipid membranes of an electrophysiological bilayer apparatus demonstrated them to be functional . Addition of isolated ZGs to the cis compartment of the bilayer apparatus demonstrated a time-dependent increase in capacitance and conductance and the transfer of the ZG enzyme amylase from cis to the trans compartment of the bilayer chamber . Furthermore, the chloride channel inhibitor DIDS was found to inhibit current activity in the porosome-reconstituted bilayer. In summary, these studies demonstrated that the porosome in the exocrine pancreas is a 100–150 nm in diameter supramolecular cup-shaped lipoprotein structure at the cell plasma membrane, where secretory vesicles dock and fuse to release intravesicular contents to the cell’s exterior (30). Similar to the acinar cells of the exocrine pancreas, growth hormone (GH) secreting cells of the pituitary, chromaffin cells, β cells of the endocrine pancreas, mast cells, and neurons all possess porosomes at their plasma membrane, demonstrating porosomes to be the universal secretory machinery in cells. High resolution AFM examination of live GH cells , chromaffin cells , β-cells , mast cells , and neurons  demonstrated the presence of porosomes . Similar to pancreatic acinar cells, porosomes in GH cells, when stimulated by a secretagogue, results in a 40% increase in diameter, which is cytocholasin-sensitive, demonstrating the involvement of actin in its morphology and function . Analogous to the detection of amylase at the porosome opening in pancreatic acinar cells, gold-tagged growth hormone-specific antibody selectively localizes at porosomes following stimulation of GH secretion . Among the porosomes identified and studied in various secretory cells, the discovery of the neuronal porosome was truly outstanding and fascinating, due primarily to its small size. High resolution transmission electron microscopy and AFM examinations at 5–8 Å resolution revealed the morphology and distribution of neuronal porosomes at the presynaptic membrane . The dynamics of synaptic vesicles docked at the base of neuronal porosomes have also been successfully studied using AFM on isolated inside-out synaptosomal membrane preparations . Neuronal porosomes have further been isolated and their composition determined immunochemically . As in the acinar cells of the exocrine pancreas, isolated neuronal porosomes have been successfully reconstituted into lipid bilayers for both structural and functional analysis using AFM and electrophysiology. Increased capacitance of neuronal porosome-reconstituted membrane following exposure to synaptic vesicles demonstrated the functional reconstitution of isolated neuronal porosomes. Careful examination of the pre-synaptic membrane in boutons revealed the presence of 8–10 nm cup-shaped porosomes with 40–50 nm synaptic vesicles docked at its base . The porosomes appeared to have a plug-like structure at the center. Each synaptic vesicle was found docked to more than one porosome. Ultrahigh resolution AFM imaging revealed in 3D, the 8–10 nm in diameter porosomes having a distinct plug at the center, which was also confirmed by high resolution electron micrographs . Similar to the isolation of porosomes from the exocrine pancreas, neuronal porosomes were immunoisolated from detergent-solubilized synaptosome preparations. Electrophoretic resolution of the immunoisolates, and immunoblot analysis demonstrated the presence of SNAP-25, the P/Q-type calcium channel, actin, syntaxin-1, synaptotagmin-1, vimentin, the N-ethylmaleimide-sensitive factor (NSF), the chloride channel CLC-3 and the alpha subunit of the heterotrimeric GTP-binding Go . To determine if the complete porosome complex was immunoisolated, the immunoisolate has been reconstituted into lipid membrane prepared using brain dioleoylphosphatidylcholine (DOPC) and dioleylphosphatidylserine (DOPS) in a ratio of 7:3 . At low resolution, the AFM showed the immunoisolates to arrange in L- or V-shaped structures, which at higher resolution demonstrated the presence of porosomes in patches, similar to what was observed at the presynaptic membrane in intact synaptosomes . Similar to the observations made in electron and AFM micrographs of the presynaptic membrane in synaptosomes, the immunoisolated and lipid-reconstituted neuronal porosome demonstrated the presence of a 2 nm in diameter central plug [37[. These observations confirmed the isolated neuronal porosome to be complete.
New technological developments resulting in improved instrumentation and refined experimental procedures, have therefore resulted in the discovery of the 8–10 nm porosomes in neurons, at 5–8 Å resolution . The neuronal porosome although an order of magnitude smaller than those in the exocrine pancreas or in euroendocrine cells, possess many similarities both in structure and composition . Nature has designed the porosomes as the general secretory machinery, and has fine tuned it to suit various secretory processes in different cell types .