The Synaptotagmin-1 C2B Domain Is a Key Regulator in the Stabilization of the Fusion Pore

Abstract

Fusion pores serve as an effective mechanism to connect intracellular organelles and release vesicle contents during exocytosis. A complex lipid rearrangement takes place as membranes approximate, bend, fuse, and establish a traversing water channel to define the fusion pore, linking initially isolated chambers. Thermodynamically, the process is unfavorable and thought to be mediated by specialized proteins. In this work, we have developed a reaction coordinate to induce fusion pores from initially flat and parallel lipid bilayers and we have used it to describe the effects of the synaptotagmin-1 C2B domain during the process. We have obtained free-energy profiles of the whole lipid reorganization in biologically realistic membranes, going from planar and parallel bilayers through stalk hemifusion to water channel formation. Our results point to a lysine-rich polybasic region on synaptotagmin-1 C2B as the key to lipid reorganization control through the formation of phosphatidylinositol bisphosphate clusters that stabilize the fusion pore.

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

“…Here, using ξ f under the MARTINI 3 force-field, with two POPC:POPS:PIP 2 (87.5:10:2.5) lipid bilayers with ∼34 cytosolic water beads per nm 2 , we have shown that the necessary work to induce a fusion stalk between initially planar and parallel bilayers is ∼300 kJ/mol (see figure 2a), in good agreement with previous results of similar inter-membrane distances 5 . Smirnova et al demonstrated that increasing hydration levels, though larger inter-membrane distances, significantly increase the free energy cost for fusion stalk formation 26 .…”

“…PMF calculations with the new MARTINI 3 are in good agreement with previous results using the MARTINI 2.2 version 5 . A slight difference in the length of the pre-fusion region (ξ f <0.58) was although observed.…”

“…Specialized secretory cells experience regulated exocytosis as a response to physiological signals [1][2][3] . Mainly, sperm exocytosis (or acrosome reaction) is a regulated secretion needed to fertilize the egg that requires large membrane remodeling, membrane bending and fusion 2,[4][5][6][7] . While this collective process develops, multiple fusion pores spontaneously form between the acrosomal and plasma membranes, connecting the acrosome lumen to the extracellular milieu.…”

“…Synaptotagmin-1 (Syt1) is a vesicle-anchored protein known as a phospholipid binding machine 8 . Syt1 has been related to synaptic vesicle fusion 9 , fusion pore opening 10 , stabilization 5,11 and expansion 12,13 . Syt1 contains two C2 domains (C2A and C2B) with Ca 2+ binding loops, the latter (C2B) with a polybasic region KRLKKKKTTIKK (positions 321-332) that easily binds to negatively charged membrane patches, such as clusters of phosphatidylinositol-4,5-bisphosphate lipids (PI(4,5)P 2 , or simply PIP2), independently of Ca 2+ 14–18 .…”

Cholesterol plays a decisive role in tetraspanin assemblies during bilayer deformations

“…The MARTINI coarse-grained model 50 is widely used for proteinlipid molecular modelling 14,26,31,40,71 . In this particular reduced resolution space, beads are classified according to their mutual interactions as: polar, non-polar, apolar and charged.…”