Reconstituted fusion pore

Abstract

Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles.

FIGURE 1

AFM micrographs of fusion pores at the surface of apical plasma membrane in live pancreatic acinar cells and at the cytosolic side of isolated pancreatic plasma membrane preparations. (a) Several circular “pits” (yellow arrowheads) with fusion pores (red arrowheads) are seen in this AFM micrograph of the apical plasma membrane in live pancreatic acinar cell. (b) AFM micrograph of the cytosolic side of isolated pancreatic plasma membrane preparation depicting a “pit” (yellow arrowheads) containing several inverted cup-shaped fusion pores (red arrowhead) within, associated with a ZG (blue arrowhead). (c) The “pit” and inverted fusion pores in b is shown at higher magnification. (d) AFM micrograph of another “pit” with inverted fusion pores within, and associated with a ZG, is shown. Bar = 200 nm.

FIGURE 2

AFM micrographs of the fusion pore or porosome, as it appears at the cell surface and at the cytosolic side. (a) A pit with four fusion pores within, found at the apical surface in a live pancreatic acinar cell. (b) AFM micrograph of isolated plasma membrane preparation reveals the cytosolic end of a pit with inverted cup-shaped structures, the fusion pores. (c) A single fusion pore at the cell surface, and (d) as it appears at the cytosolic side of the cell membrane.

FIGURE 3

Transmission electron micrograph of a porosome associated with a docked secretory vesicle at the apical end of a pancreatic acinar cell. (a) Part of the apical end of a pancreatic acinar cell demonstrating within the green bordered square, the presence of a fusion pore or porosome and an associated zymogen granule (ZG), the electron dense secretory vesicle of the exocrine pancreas. (Bar = 400 nm; Fig 2 a only). The area within the green square in a has been enlarged to show the apical microvilli (MV) and a section through porosome and the ZG. Note the ZG membrane (ZGM) bilayer is attached directly to the base of the porosome cup. A higher magnification of the porosome in c depicts in further detail the porosome bilayer and cross section through the three protein rings, with the thicker ring (blue arrowhead) present close to the opening of the porosome to the outside. The third and the lowest ring away from the porosome opening is attached to the ZGM. (d) Yellow outline of the fusion pore (FP) or the porosome membrane shows the continuity with the apical plasma membrane (PM) at the apical end of the pancreatic acinar cell facing the lumen (L), and also defines the exact points of contact of the ZGM with the porosome membrane.

FIGURE 4

Transmission electron micrographs of porosomes (red arrowheads) at the apical end of pancreatic acinar cells. Note the thicker ring (blue arrowhead) present close to the opening of each porosome, shown in the enlarged images of the porosomes within insets having blue margins. Bar = 200 nm (not applicable to insets).

FIGURE 5

Transmission electron micrographs of zymogen granules (ZGs), co-isolated with fusion pores or porosomes. (a) Fusion pores associated at the surface of ZGs are shown. (Bar = 120 nm; top panel only). (b) At higher magnification details of the fusion pore, demonstrating the presence of separate plasma membrane (PM) and the ZG membrane (ZGM). Note the apically arranged ring complex of the fusion pore, similar to what is observed in electron micrographs of the fusion pore in intact cells (Fig. 2).

FIGURE 6

One (1D) and two dimensionally (2D) resolved proteins immunoisolated from solubilized pancreatic plasma membrane preparations, using a SNAP-23 specific antibody. The resolved proteins were silver stained and transferred to nitrocellulose membranes for immunoblot analysis. Note the identification in the immunoisolates of eight protein bands in a silver-stained SDS-PAGE 1D-resolved gel. (the far left panel in a). Immunoblot analysis of the 1D-resolved immunoisolated proteins demonstrate the presence of actin, vimentin, syntaxin-2, calcium channel, NSF, and chloride channels CLC-2 and CLC-3. (b) 2D resolution of the immunoisolated proteins, provide eleven spots in a silver stained gel. Immunoblot analysis of the 2D-resolved immunoisolated proteins further demonstrates the presence of isoforms of some of the proteins identified in a. (c) There appears to be at least three isoforms of the calcium channel: (d) one isoform of actin; (e) three isoforms of vimentin, (f) one NSF; (g) one each of the CLC-2; and (h) the CLC-3. Arrows pointing to lower molecular weights may represent proteolytic cleavage products.

FIGURE 7

Four separate sets of negatively stained electron micrographs and four sets of atomic force micrographs of the immunoisolated fusion pore or porosome complex. Note the similarity in size and morphology of the complex. Bar = 30 nm.

FIGURE 8

Negatively stained electron micrograph and atomic force micrograph of the immunoisolated fusion pore complex. (a) Negatively stained electron micrograph of an immunoisolated fusion pore complex from solubilized pancreatic plasma membrane preparations, using a SNAP-23 specific antibody. Note the three rings and the 10 spokes that originate from the inner smallest ring. This structure represents the protein backbone of the fusion pore complex, because the three rings and the vertical spikes are observed in electron micrographs of cells and fusion pores co-isolated with ZGs. Bar = 30 nm. (b) The electron micrograph of the fusion pore complex, cut out from a, and (c) an outline of the structure presented for clarity. (d–f) Atomic force micrograph of the isolated pore complex in near physiological buffer. Bar = 30 nm. Note the structural similarity of the complex, imaged both by TEM (g) and AFM (h). The TEM and AFM micrographs are superimposable (i).

FIGURE 9

Electron micrographs of reconstituted porosome or fusion pore complex in liposomes. showing a cup-shaped basket-like morphology. (a) A 500-nm vesicle with an incorporated porosome is shown. Note the spokes in the complex. The reconstituted complex at greater magnification is shown in b–d. Bar = 100 nm.

FIGURE 10

Lipid bilayer-reconstituted porosome complex is functional. (a) Schematic drawing of the bilayer setup for electrophysiological measurements. (b) Zymogen granules (ZGs) added to the cis side of the bilayer fuse with the reconstituted porosomes, as demonstrated by an increase in capacitance and current activities, and a concomitant time dependent release of amylase (a major ZG content) to the trans side of the membrane. The movement of amylase from the cis to the trans side of the chamber was determined by immunoblot analysis of the contents in the cis and the trans chamber over time. (c) As demonstrated by immunoblot analysis of the immunoisolated complex, electrical measurements in the presence and absence of chloride ion channel blocker DIDS, demonstrate the presence of chloride channels in association with the complex.