Enhanced Expansion and Reduced Kiss-and-Run Events in Fusion Pores Steered by Synaptotagmin-1 C2B Domains

Abstract

The fusion pore controls the release of exocytotic vesicle contents through a precise orchestration of lipids from the fusing membranes and proteins. There is a major lipid reorganization during the different stages in life of the fusion pore (membrane fusion, nucleation, and expansion) that can be scrutinized thermodynamically. In this work, using umbrella sampling simulations we describe the expansion of the fusion pore. We have calculated free energy profiles to drive a nascent, just nucleated, fusion pore to its expanded configuration. We have quantified the effects on the free energy of one and two Synaptotagmin-1 C2B domains in the cytosolic space. We show that C2B domains cumulatively reduce the cost for expansion, favoring the system to evolve toward full fusion. Finally, by conducting thousands of unbiased molecular dynamics simulations, we show that C2B domains significantly decrease the probability of kiss-and-run events.

Enhanced sampling for lipid-protein interactions during membrane dynamics

“…Starting from membrane bending and ending with the expansion of the fusion pore, this schematic curve follows the path of pore nucleation through the fusion stalk. The shape of the estimated free energy profile is based on results by Caparotta et al (2020b), di Bartolo and Masone (2022), di Bartolo et al (2022) and is in good agreement with previous studies on the energetics of the fusion pore (Lipowsky, 2022;Kawamoto et al, 2015;Smirnova et al, 2015;Gorai et al, 2021;Dhara et al, 2020;François-Martin et al, 2017;Markvoort and Marrink, 2011).…”

“…As widely accepted, the energetics along the membrane fusion paths towards the fusion pore (Fig. 2) highly depends on the lipid composition of the interacting bilayers (di Bartolo et al, 2022;Kawamoto and Shinoda, 2014;Fuertes et al, 2011;Aeffner et al, 2012;Cunill-Semanat and Salgado, 2019) as well as their hydration level for different inter-membrane distances (di Bartolo and Masone, 2022;Caparotta et al, 2020b;Poojari et al, 2021;Smirnova et al, 2015;Smirnova et al, 2019;Wu et al, 2021).…”

“…Also, di Bartolo and Masone (2022) revealed a cooperative mechanism between a pair of C2B domains that significantly decrease the energy costs for the formation of the fusion stalk and the first traversing water channel. In another study, di Bartolo et al (2022) demonstrated that C2B domains reduce the energy cost for fusion pore expansion and the probability of kiss-and-run events.…”

“…3a shows a 3D time-averaged density of a fusion pore from an enhanced molecular dynamics trajectory. The fusion pore for this figure was induced with the collective variable ξe (di Bartolo et al, 2022;Hub, 2021) (freely available on GitHub: https://github.com/lautarodibartolo/ FusionPore), and the trajectory density was generated using GROmaps (Briones et al, 2019). Fig.…”

“…Hence, membrane fusion is thought to be mediated by protein fusogens that perturb their organization and dynamics (Pattnaik, Meher and Chakraborty, 2018;Joardar, Pattnaik and Chakraborty, 2022). Key proteins for evoked neurotransmitter release such as the synaptotagmin-1 C2B domains (Gruget et al, 2020) collaborate to overcome the energetic barriers for the fusion stalk (Di Bartolo and Masone, 2022) and fusion pore expansion (Di Bartolo et al, 2022;Nyenhuis et al, 2021;Das et al, 2020), possibly inducing PIP2 clusters (Caparotta et al, 2020b). Another significant example is myoblast fusion, where Myomaker and Myomerger promote the hemifusion diaphragm (Golani et al, 2021;Whitlock and Chernomordik, 2021).…”