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.

Secretion machinery at the cell plasma membrane

“…Hence for clarity, the term "porosome" was assigned to this newly discovered structure at the cell plasma membrane, where secretory vesicles transiently dock and fuse to release their contents. In the past decade, studies demonstrate that porosomes are present in all secretory cells examined, such as acinar cells of the exocrine pancreas, neurons, chromaffin cells, mast cells, growth hormone cells of the pituitary gland, β-cells of the endocrine pancreas [1][2][3][4][5][6]33], and recently in astrocytes (personal observation). Furthermore, the porosome has been isolated, its composition determined [4,6], and it has been both structurally and functionally reconstituted in artificial lipid membrane [5,6].…”

“…1) [1][2][3][4][5][6] as permanent (not induced) and dynamic structures at the cell plasma membrane, brought to light a molecular understanding of the secretory process in cells [33]. Old questions such as the appearance of partially empty vesicles following cell secretion were answered [33].…”

“…Porosomes in pancreatic acinar (Fig. 1A-1D) [4][5][6] or growth hormone-secreting cells are permanent structures at the cell plasma membrane, with a 100 to 180 nm-diameter opening. Membrane-bound secretory vesicles ranging in size from 0.2 to 1.2 μm in diameter dock and fuse at porosomes to release vesicular contents.…”

“…Secretion is a precisely regulated and universal cellular process, involving delivery of intracellular products to the extra-cellular space. Cellular cargo destined for secretion are packaged and stored in membranous sacs or vesicles, which on demand dock and fuse at specialized plasma membrane structures called porosomes [1][2][3][4][5][6]. The establishment of continuity between secretory vesicle membrane and the porosome membrane requires calcium and the assembly of a molecular complex [7] formed when specific proteins [8][9][10][11] in opposing bilayers interact.…”

“…The extent of vesicle swelling dictates the amount of vesicular contents expelled [13]. In the past two decades, besides discovery of the porosome [1][2][3][4][5][6], the discovery of SNAREs [14][15][16], the molecular assembly [7,17 •• ] and disassembly [18,19 • ] of the membrane-associated t-/v-SNARE complex [7,17 •• ,18], in addition to the molecular mechanism of secretory vesicle swelling [20][21][22] required for the regulated release of intravesicular contents, has been determined using, among other approaches, the AFM.…”