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Biology Articles » Cell biology » Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cells » Porosome: Structure and function

Porosome: Structure and function
- Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cells

To determine the morphology of the porosome at the cytosolic side 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 m in diameter, with inverted cup-shaped structures within (Fig. 7). The inverted cups range in height from 10-15 nm. On a number of occasions, zymogen granules (ZGs) ranging in size from 0.4-1 m in diameter were found associated with one or more of the inverted cups (Fig. 7A-C). This suggested the circular disks to be pits, and the inverted cups to be fusion pores or porosomes. To determine if the cup-shaped structures in isolated PM preparations are indeed porosomes, immuno- AFM studies were carried out. Since ZGs, the membrane-bound secretory vesicles in exocrine pancreas are known to 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 (tip of the inverted cups). The t-SNARE protein SNAP-23, has been identified and implicated in secretion from pancreatic acinar cells [7]. A polyclonal monospecific SNAP-23 antibody recognizing a single 23kDa band in Western blot analysis of pancreatic PM fraction (Fig. 7D) was used in 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, which is the tip of the inverted cup (Fig. 7E and F). No antibody labeling of the structure was detected when preimmune serum was applied. These results demonstrate that the inverted cup-shaped structures in isolated PM preparations are the porosomes observed from its cytosolic side [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, 10]. Since membrane-bounded secretory vesicles dock and fuse at porosomes to release vesicular contents, suggested t-SNAREs to associate at the porosome complex. It was therefore no surprise, that the t-SNARE protein SNAP-23, implicated in secretion from pancreatic acinar cells [8], was located at the tip of the inverted cup (i.e., the base of the porosome) where secretory vesicles dock and fuse. The structure of the porosome was further demonstrated using transmission electron microscopy (TEM) [8, 11] (Fig. 8). TEM studies confirm the fusion pore to have a cup-shaped structure, with similar dimensions as determined from AFM measurement [2-5]. Additionally, TEM micrographs reveal porosomes to possess a basket-like morphology, with three lateral and a number of vertically arranged ridges [8, 11]. A ring at the base of the complex is also identified. Since porosomes are found to be stable structures at the cell PM, it was hypothesized that if ZGs, the membrane-bounded secretory vesicles in exocrine pancreas were to fuse at the base of the structure, it would be possible to isolate ZG-associated porosomes. Indeed, TEM of isolated ZG preparations reveal the presence of porosomes associated with docked vesicles [8] (Fig. 9). As observed in whole cells, vertical structures were found to originate from within the porosome complex and appear attached to its membrane. The presence of vertical ridges lining the porosome, have also been reported in NG108-15 nerve cells [6]. As discussed in further detail later in this review, studies using 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 [12]. Since similar 45-50 nm circular structures are observed at the base of the porosome, and SNAP-23 immunoreactivity is found to localize at this site, suggests that the t-SNAREs present at the base of porosomes are possibly arranged in a ring pattern.


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