Structure and Dynamics of the Fusion Pores in Live GH-Secreting Cells Revealed Using Atomic Force Microscopy

Abstract

Earlier studies in live pancreatic acinar cells identified new cellular structures at the cell plasma membrane called 'pits' and 'depressions', where membrane-bound secretory vesicles dock and fuse to release vesicular contents. In the current study, using atomic force microscopy we identify similar structures at the plasma membrane of GH-secreting cells of the pituitary and implicate their involvement in hormone release. Pits containing 100-200 nm in diameter depressions or fusion pores were identified in resting GH-secreting cells. Following stimulation of secretion the size of depression enlarged and gold-tagged GH antibody were found to bind to the pit structures in the stimulated GH cells. This study documents for the first time the presence of these structures and their involvement in secretions in a neuroendocrine cell.

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].…”

“…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. Following fusion of secretory vesicles at porosomes, only a 20-35% increase in porosome diameter is demonstrated [1][2][3]. It is therefore reasonable to conclude that secretory vesicles "transiently" dock and fuse at the base of porosomes, instead of complete merger of the secretory vesicle membrane at the site.…”

“…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.…”