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Biology Articles » Cell biology » Molecular Machinery and Mechanism of Cell Secretion » The Porosome

The Porosome
- Molecular Machinery and Mechanism of Cell Secretion

 

Earlier electrophysiological studies on mast cells suggested the existence of fusion pores at the cell plasma membrane (PM), which became continuous with the secretory vesicle membrane following stimulation of secretion (31). Studies on live secretory cells using the AFM revealed, for the first time, the physical existence of the fusion pore or porosome and determined its structure and dynamics in the exocrine pancreas (3, 4, 7, 8), in neuroendocrine cells (5, 6), and in neurons (15) at nanometer to subnanometer resolution and in real time.

Isolated live pancreatic acinar cells in physiological buffer, when imaged with the AFM (3, 4, 7, 8), reveal at the apical PM a group of circular pits measuring 0.4–1.2 µm in diameter, contain smaller depressions (Fig. 1Go). Each depression averages between 100 and 180 nm in diameter, and typically 3–4 depressions are located within a pit. The basolateral membrane of acinar cells is devoid of either pits or depressions. High-resolution AFM images of depressions in live cells further reveal a cone-shaped morphology. The depth of each depression cone measures 15–35 nm. Similarly, growth hormone (GH)-secreting cells of the pituitary gland, chromaffin cells, ß-cells of the endocrine pancreas, mast cells, and neurons, all possess depressions at their PM, 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 and relative depth, followed by a return to resting size on completion of secretion (3, 4, 7, 8). No demonstrable change in pit size is detected following stimulation of secretion (3). Enlargement of depression diameter and an increase in its relative depth after exposure to secretagogues correlate with increased secretion. Conversely, 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 (3). Results from these studies suggested depressions to be the fusion pores in pancreatic acinar cells. Furthermore, these studies demonstrate the involvement of actin in regulation of both the structure and function of depressions. Analogous to pancreatic acinar cells, examination of resting GH-secreting cells of the pituitary (5) and chromaffin cells of the adrenal medulla (6) also reveal the presence of pits and depressions at the cell PM (Fig. 2Go). The presence of porosomes in neurons, ß-cells of the endocrine pancreas, and in mast cells have also been demonstrated (Fig. 2Go; Refs. 14, 15). Depressions in resting GH cells measure 154 ± 4.5 nm (mean ± SE) in diameter. Exposure of GH cells to a secretagogue results in a 40% increase in depression diameter (215 ± 4.6 nm; P 5). The enlargement of depression diameter during secretion and its decrease accompanied by loss in secretion following exposure to actin depolymerizing agents (3) suggested depressions to be the fusion pores. However, a more direct determination that depressions are fusion pores was achieved using immuno-AFM studies (Fig. 3Go). AFM localization at depressions of gold-conjugated antibody to secretory proteins demonstrated secretion to occur through depressions (4, 5). The membrane-bound secretory vesicles in exocrine pancreas, called zymogen granules (ZGs), contain the starch-digesting enzyme amylase. AFM micrographs demonstrated localization of amylase-specific antibodies tagged with colloidal gold at depressions following stimulation of secretion (Fig. 3Go; Ref. 4). These studies confirm depressions to be the fusion pores, or porosomes, in pancreatic acinar cells, where membrane-bound secretory vesicles dock and fuse to release their contents. Similarly, in somatotrophs of the pituitary, gold-tagged growth hormone-specific antibody is found to selectively localize at depressions following stimulation of secretion (5), again identifying depressions in GH cells as fusion pores, or porosomes. The porosome at the cytosolic side of the plasma membrane in the exocrine pancreas (7) and in neurons (15) has also been imaged at near-nanometer resolution in live tissue in buffer.

To determine the morphology of the porosome at the cytosolic compartment of the cell, pancreatic PM preparations were used. Isolated PM in buffer, when placed on freshly cleaved mica, tightly adhere to the mica surface, enabling imaging by AFM. The PM preparations reveal scattered circular disks measuring 0.5–1 µm in diameter, with inverted cup-shaped structures within (7). The inverted cups range in height from 10 to 15 nm. In a number of studies, AFM micrographs reveal ZGs ranging in size from 0.4 to 1 µm in diameter, found associated with one or more of the inverted cups. This suggested the circular disks to be pits and the inverted cups to be fusion pores or porosomes. To further confirm the cup-shaped structures to be porosomes, immuno-AFM studies were performed on them. Because ZGs dock and fuse at the PM to release vesicular contents, it was hypothesized that, if porosomes are these sites, then PM-associated t-SNAREs should localize at the base of porosomes. The t-SNARE protein SNAP-23 has been identified and implicated in secretion from pancreatic acinar cells (32). A polyclonal monospecific SNAP-23 antibody recognizing a single 23-kDa band on Westerns of pancreatic PM fraction was used in such immuno-AFM studies. When the SNAP-23-specific antibody was added to the PM preparation during imaging with the AFM, the antibody selectively localized to the base of the cup-shaped structure, that is, the tip of the inverted cup. These results demonstrate that the inverted cup-shaped structures in the isolated PM preparations are the porosomes observed from the cytosolic compartment of the cell (7, 8). 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 (9, 17, 18, 29). Because membrane-bound secretory vesicles dock and fuse at porosomes to release vesicular contents, it suggests that t-SNAREs associate at the porosome complex. It was therefore no surprise that the t-SNARE protein SNAP-23, implicated in secretion from pancreatic acinar cells, was located at the tip of the inverted cup (i.e., the base of the porosome), where secretory vesicles transiently dock and fuse.

The structure of the porosome was further determined using transmission electron microscopy (TEM; Figs. 1Go and 2Go; Refs. 7, 8). TEM studies confirm the fusion pore to have a cup-shaped structure, with similar dimensions as determined from AFM measurement. Additionally, TEM micrographs reveal porosomes to possess a basket-like morphology, with three lateral and a number of vertically arranged ridges. A ring at the base of the complex is also identified (7). Because porosomes are found to be stable structures at the cell PM, it was hypothesized that, if ZGs were to fuse at the base of the structure, it would be possible to isolate ZG-associated porosomes. Indeed, TEM studies performed on isolated ZG preparations reveal porosomes associated with docked vesicles (7, 8). As observed in whole cells, vertical structures were found to originate from within the porosome complex and appear attached to its membrane. As discussed later in this review, studies using full-length recombinant SNARE proteins and artificial lipid membranes demonstrated that t- and v-SNAREs located in opposing bilayers interact in a circular array to form conducting pores (9). Similar circular structures observed at the base of the porosome and SNAP-23 immunoreactivity found to localize at this site, suggest that the t-SNAREs present at the base of porosomes are possibly arranged in a ring pattern.


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