Fusion pore revealed from AFM studies
As reported earlier (Cho et al., 2002c
; Schneider et al., 1997), pancreatic acinar cells in physiological buffer reveal the presence of pits measuring 0.4–1 µm in diameter which contain "depressions" or fusion pores. Each fusion pore measured 
125–185 nm in diameter and 19–25 nm in relative depth (Fig. 1 a). Localization of gold-labeled anti-amylase with the AFM confirmed that amylase is located at the fusion pore after stimulation of secretion (Fig. 1, b–d and Cho et al., 2002c). To determine the morphology of the fusion pore at the cytosolic side, a pancreatic PM preparation was used. Isolated PM in buffer, when placed on freshly cleaved mica, tightly adheres to the mica surface, allowing imaging by the AFM. The PM preparations contain scattered, circular disks measuring 0.5–1 µm in diameter, with inverted cup-shaped structures within (Fig. 2). The inverted cups range in height from 10–15 nm. On several occasions, ZGs of 0.4–0.1 µm in diameter are found associated with one or more of the inverted cups (Fig. 2, a–c). Because ZGs are the next largest structures after the nucleus in pancreatic acinar cells, this suggests the circular disks are pits, and the inverted cup-like structures are the fusion pores. To determine if the cup-shaped structures in isolated PM preparations are the fusion pores, immunoAFM studies were performed. Because our studies (Fig. 1, b–d; and Cho et al., 2002c; Schneider et al., 1997) demonstrated that ZGs dock and fuse at the fusion pore to release vesicular contents, it is reasonable to suggest that PM-associated t-SNAREs may be located at the base of the fusion pore, i.e., the tip of the inverted cup-shaped structure. The t-SNARE protein, SNAP-23, has been identified and implicated in secretion from pancreatic acinar cells (Gaisano et. al., 1997). A polyclonal monospecific SNAP-23 antibody, recognizing a single 23 kDa band in the Western blot analysis of a pancreatic homogenate (Fig. 2 d), was used in all immunoAFM studies. When the SNAP-23 specific antibody was added to the PM preparation during imaging with the AFM, the antibody specifically localized to the base of the cup-shaped structure which is the tip of the inverted cup (Fig. 2, e and f). No antibody labeling of the structure was detected when preimmune serum was applied (data not shown). These results demonstrate that the inverted cup-shaped structures in isolated PM preparations are the fusion pores observed from its cytosolic side. To further confirm our AFM studies, and to have a better understanding of the fusion pore, TEM was done.
Transmission electron microscopy
Because fusion pores are relatively small structures (125–185 nm in diameter at its wide end and 10–40 nm at the base) and are present only at the apical PM of pancreatic acinar cells, it would be extremely rare to be able to take a cross section through the fusion pore along with any associated secretory vesicles. Even if rare, such structures were observed in electron micrographs of isolated cells or tissue preparations (Fig. 3). TEM studies confirm the fusion pore to have a cup-shaped structure, with similar dimensions as determined in all our AFM studies (Cho et al., 2002a,b,c,d; Schneider et al., 1997). Additionally, TEM results reveal that the fusion pore has a basket-like morphology, with three lateral and a number of vertically arranged ridges. A ring-like structure is also observed at the base of the fusion pore cup (Fig. 3).
Because these PM structures are stable, we hypothesized that, if membrane-bound secretory vesicles (ZGs) were to fuse at the base of the fusion pore, it should be possible to prepare isolated ZG-associated fusion pore complexes. To test this hypothesis, ZGs were isolated and the preparation was processed for TEM. Our TEM studies confirmed this hypothesis and reveal the isolation of the fusion pore associated with docked vesicles (Fig. 3). The PM and the ZG membrane are distinct and clearly visible in the isolated fusion pore-ZG complex. As observed in whole cells (Fig. 3, a and b), vertical structures originate from within the fusion pore complex (Fig. 3, c and d). These vertical structures appear attached to the fusion pore membrane (Figs. 3 and 4). The presence of such vertical ridges lining the fusion pore in NG108-15 nerve cells has been previously reported (Tojima, et al., 2000). In our EM micrograph the ridges or vertical rods appear supported by a central core rod. This arrangement may be similar to the nuclear pore having a plug at the center. In our AFM micrograph, however, the central rod is undetectable (Fig. 1 d), possibly due to its molecular size and softness. Thus, the combination of AFM and EM studies provides a better understanding of the fusion pore morphology. It needs to be mentioned that earlier studies report the association of t-SNAREs with secretory vesicles (Otto et al., 1997). This could be due to the co-isolation of the fusion pore (containing t-SNAREs) during secretory vesicle preparation.
Composition of the fusion pore complex
In a recent study, Cho et al. (2002e) used purified SNARE proteins and artificial lipid membranes to demonstrate that t- and v-SNAREs, located in opposing bilayers, interact in a circular array to form conducting pores (Fig. 4 e; Cho et al., 2002e). The 45–50 nm circular structure that is observed at the base of the fusion pore (Fig. 3 b) and the SNAP-23 immunoreactivity that is also localized at this site (Fig. 2 e) demonstrate the presence of t-SNAREs at the base of the fusion pore cup. To determine the association of other proteins with the fusion pore complex, immunoprecipitation studies on total pancreatic homogenates using SNAP-23 specific antibody were performed. The precipitated sample was resolved using 12.5% SDS-PAGE, transferred to a nitrocellulose membrane, and probed using various antibodies. In agreement with earlier findings in other tissues (Bennett, 1990; Cho et al., 2002c; Faigle et al., 2000; Goodson et al., 1997; Nakano et al., 2001; Ohyama et al., 2001; Rothman, 1994; Weber et al., 1998), our study demonstrates the association of SNAP-23 with syntaxin 2; with cytoskeletal proteins actin, 
-fodrin, and vimentin; and with calcium channels ß3 and 1c, together with the SNARE regulatory protein, NSF (Fig. 5). These studies demonstrate that the fusion pore is a cup-shaped lipoprotein basket at the cell PM where secretory vesicles dock and fuse to release vesicular contents. The base of the fusion pore complex is where t- and v-SNAREs interact in a circular array to form a pore, and hence we name the structure the "porosome". Purification and further characterization of the porosome is required to determine its complete biochemical composition. Furthermore, immunoTEM combined with immunoAFM will help determine the specific arrangement and localization of the various porosome-associated proteins.
ACKNOWLEDGEMENTS
This work was supported in part by research grants DK56212 and NS39918 to B.P.J. from the National Institutes of Health. S.-J.C. is a recipient of a National Institutes of Health postdoctoral fellowship award (DK60368).
Submitted on September 16, 2002; accepted for publication October 11, 2002.
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