The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

Abstract

All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered release of neurotransmitters and hormones. Expansion of this small pore to release cargo is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins. Pore dilation relied on calcium-induced insertion of the tandem C2 domain hydrophobic loops of Syt1 into the membrane, previously shown to reorient the C2 domain. Mathematical modelling suggests that C2B reorientation rotates a bound SNARE complex so that it exerts force on the membranes in a mechanical lever action that increases the height of the fusion pore, provoking pore dilation to offset the bending energy penalty. We conclude that Syt1 exerts novel non-local calcium-dependent mechanical forces on fusion pores that dilate pores and assist neurotransmitter and hormone release.

Optimal Detection of Fusion Pore Dynamics Using Polarized Total Internal Reflection Fluorescence Microscopy

“…Because simultaneous monitoring of content release and lipid mixing shows that most fusion pores reseal after partial content release in a similar assay ( Stratton et al, 2016 ), the intensity changes observed by Kiessling et al (2010) more likely represent diffusion of the lipid labels from the SUV into the SBL with the SUV retaining an omega-shape until the lipids are dispersed into the SBL. This would be an interpretation consistent with observations of secretory granule exocytosis in neuroendocrine cells ( Taraska et al, 2003 ; Tran et al, 2007 ; Anantharam et al, 2010 ; Anantharam et al, 2012 ; Chiang et al, 2014 ; Karatekin, 2018 ; Shin et al, 2018 ), enveloped viral fusion events ( Melikyan et al, 1993a ; Melikyan et al, 1993b ; Melikyan et al, 1995 ; Cohen and Melikyan, 2004 ), and other reconstitutions with artificial membranes with sufficient resolution to monitor fusion pore dynamics ( Chanturiya et al, 1997 ; Lai et al, 2013 ; Wu et al, 2016 ; Wu et al, 2017 ; Wu et al, 2021 ), where pore flickering and slow changes in the -shape of the vesicle appear to be the norm.…”

Synaptotagmin-1 C2B domains cooperatively stabilize the fusion stalk via a master-servant mechanism

“…However, the molecular mechanism by which Syt1 drives membrane fusion is yet not completely understood. 13,20…”

“…However, the molecular mechanism by which Syt1 drives membrane fusion is yet not completely understood. 13,20 In the present work, we use enhanced molecular dynamics with an ad hoc collective variable to induce a fusion stalk between lipid bilayers. We demonstrate that Syt1-C2B domains cooperatively facilitate the formation of the fusion stalk, a Instituto de Histología y Embriología de Mendoza (IHEM) -Consejo Nacional de Investigaciones Cientícas y Técnicas (CONICET), Universidad Nacional de Cuyo (UNCuyo), 5500, Mendoza, Argentina.…”

DataSheet1.ZIP

“…Because simultaneous monitoring of content release and lipid mixing shows that most fusion pores reseal after partial content release in a similar assay (Stratton et al, 2016), the intensity changes observed by Kiessling et al (2010) more likely represent diffusion of the lipid labels from the SUV into the SBL with the SUV retaining an omega-shape until the lipids are dispersed into the SBL. This would be an interpretation consistent with observations of secretory granule exocytosis in neuroendocrine cells (Taraska et al, 2003;Tran et al, 2007;Anantharam et al, 2010;Anantharam et al, 2012;Chiang et al, 2014;Karatekin, 2018;Shin et al, 2018), enveloped viral fusion events (Melikyan et al, 1993a;Melikyan et al, 1993b;Melikyan et al, 1995;Cohen and Melikyan, 2004), and other reconstitutions with artificial membranes with sufficient resolution to monitor fusion pore dynamics (Chanturiya et al, 1997;Lai et al, 2013;Wu et al, 2016;Wu et al, 2017;Wu et al, 2021), where pore flickering and slow changes in the Ω-shape of the vesicle appear to be the norm.…”