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

Pika Miklavc , Susanne Albrecht , Oliver H. Wittekindt , Peter Schullian , Thomas Haller , Paul Dietl (1)

1 Institute of Physiology

Exocytosis proceeds through prefusion stages such as hemifusion, but hemifusion is still an elusive intermediate of unknown duration. Using darkfield and fluorescence microscopy in alveolar type II (ATII) cells containing large secretory vesicles ("lamellar bodies", LBs), we show that exocytotic fusion events were accompanied by a mostly bi-phasic scattered (darkfield) light intensity decrease (SLID) originating from the vesicle border. Correlation with the diffusional behavior 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, an 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.

Mots clés: Life Sciences

Pika Miklavc, Susanne Albrecht, Oliver H. Wittekindt, Peter Schullian, Thomas Haller, et al.. Existence of exocytotic hemifusion intermediates with a life time up to seconds in type II pneumocytes. Biochemical Journal, 2009, 424 (1), pp.7-14. ⟨10.1042/BJ20091094⟩. ⟨hal-00479230⟩