Abstract
ABSTRACTCa2+-dependent regulation of fusion pore dilation and closure is a key mechanism determining the output of cellular secretion. We have recently described ‘fusion-activated’ Ca2+ entry (FACE) following exocytosis of lamellar bodies in alveolar type II cells. FACE regulates fusion pore expansion and facilitates secretion. However, the mechanisms linking this locally restricted Ca2+ signal and fusion pore expansion were still elusive. Here, we demonstrate that synaptotagmin-7 (Syt7) is expressed on lamellar bodies and links FACE and fusion pore dilation. We directly assessed dynamic changes in fusion pore diameters by analysing diffusion of fluorophores across fusion pores. Expressing wild-type Syt7 or a mutant Syt7 with impaired Ca2+-binding to the C2 domains revealed that binding of Ca2+ to the C2A domain facilitates FACE-induced pore dilation, probably by inhibiting translocation of complexin-2 to fused vesicles. However, the C2A domain hampered Ca2+-dependent exocytosis of lamellar bodies. These findings support the hypothesis that Syt7 modulates fusion pore expansion in large secretory organelles and extend our picture that lamellar bodies contain the necessary molecular inventory to facilitate secretion during the exocytic post-fusion phase. Moreover, regulating Syt7 levels on lamellar bodies appears to be essential in order that exocytosis is not impeded during the pre-fusion phase.
Highlights
Ca2+ is the key element in regulated exocytosis controlling multiple steps during the exocytic pre- and post-fusion stages
The ubiquitously expressed synaptotagmin-7 (Syt7) acts as a high-affinity Ca2+ sensor (Bhalla et al, 2005) and has been implicated in regulation of fusion pore dynamics in exocytosis of large vesicles in non-neuronal cells including insulin-secreting granules of b-pancreatic cells (Gao et al, 2000), large dense-core vesicles in PC12 cells (Zhang et al, 2010) and lysosomes (Jaiswal et al, 2004)
Synaptotagmin-7 is expressed in isolated alveolar type II (ATII) cells and localised on the lamellar body membrane Several molecules have been identified as regulating fusion pore transitions in a Ca2+-dependent manner, with synaptotagmins being among the best studied (Jackson and Chapman, 2008)
Summary
Ca2+ is the key element in regulated exocytosis controlling multiple steps during the exocytic pre- and post-fusion stages. We have recently described a ‘fusion-activated’ Ca2+-entry (FACE) during the post-fusion stage of lamellar body exocytosis in primary alveolar type II (ATII) cells. FACE occurs through P2X4 receptors on the membrane of lamellar bodies upon fusion of lamellar bodies with the plasma membrane and opening of the fusion pore. The resulting rise in Ca2+ is restricted in time and space to the onset and site of vesicle fusion. FACE drives expansion of the initial fusion pore and subsequent surfactant release (Miklavc et al, 2011). The molecular links between FACE, the localised increase in Ca2+ around the fused
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