Existence of exocytotic hemifusion intermediates with a lifetime of up to seconds in type II pneumocytes

Exocytosis proceeds through prefusion stages such as hemifusion, but hemifusion is still an elusive intermediate of unknown duration. Using darkfield and fluorescence microscopy in ATII (alveolar type II) cells containing large secretory vesicles (LBs; lamellar bodies), we show that exocytotic fusion events were accompanied by a mostly biphasic SLID (scattered light intensity decrease) originating from the vesicle border. Correlation with the diffusional behaviour of fluorescence markers for either content or membrane mixing revealed that the onset of the fast second phase of SLID corresponded to fusion pore formation, which was followed by vesicle swelling. In contrast, a slow first phase of SLID preceded pore formation considerably but could still be accompanied by diffusion of farnesylated DsRed, all inner plasma membrane leaflet marker, or Nile Red. We conclude that hemifusion is an exocytotic intermediate that may last for several seconds. SLID is a new, non-invasive approach by which a prefusion phase, including hemifusion, can be continuously recorded and distinguished from fusion pore formation and postfusion vesicle swelling.