Synaptotagmin oligomerization is essential for calcium control of regulated exocytosis

Abstract

Regulated exocytosis, which underlies many intercellular signaling events, is a tightly controlled process often triggered by calcium ion(s) (Ca). Despite considerable insight into the central components involved, namely, the core fusion machinery [soluble -ethylmaleimide-sensitive factor attachment protein receptor (SNARE)] and the principal Ca sensor [C2-domain proteins like synaptotagmin (Syt)], the molecular mechanism of Ca-dependent release has been unclear. Here, we report that the Ca-sensitive oligomers of Syt1, a conserved structural feature among several C2-domain proteins, play a critical role in orchestrating Ca-coupled vesicular release. This follows from pHluorin-based imaging of single-vesicle exocytosis in pheochromocytoma (PC12) cells showing that selective disruption of Syt1 oligomerization using a structure-directed mutation (F349A) dramatically increases the normally low levels of constitutive exocytosis to effectively occlude Ca-stimulated release. We propose a parsimonious model whereby Ca-sensitive oligomers of Syt (or a similar C2-domain protein) assembled at the site of docking physically block spontaneous fusion until disrupted by Ca Our data further suggest Ca-coupled vesicular release is triggered by removal of the inhibition, rather than by direct activation of the fusion machinery.

Synaptotagmin oligomers are necessary and can be sufficient to form a Ca2+‐sensitive fusion clamp

“…The ability of Syt1 to bind PIP 2 is unaffected by the F349A mutation and correspondingly, we observed robust docking of the Syt1 349 ‐vSUVs (Table ). Interestingly, these vesicles diffused freely until they fused (Fig.…”

“…We further find that the precise positioning of these halfzippered SNARE complexes is determined by an underlying ring of the calcium-sensor protein Synapto-tagmin1 (Syt1), analogous to the in vitro ring-like oligomers observed with purified Syt1 protein in the presence of PIP 2 or analogous compounds [2][3][4]. This follows from the finding that the symmetrical organization of the fusion machinery under synaptic vesicles is lost or prevented by an engineered point mutation (F349A) in the polymerizing Syt1 C2B domain that destabilizes the oligomers without affecting any other known molecular properties [5].…”

“…As the Syt1 rings assembled on phospholipid surfaces are disassembled by Ca 2+ , we hypothesized that the assembly of a Syt ring provides the core mechanism of this fusion clamp, and its disassembly by Ca 2+ enables synchronous vesicle release . Indeed, expressing the ring‐destabilizing F349A mutant in neuroendocrine (PC12) cells dominantly and dramatically increases spontaneous release , suggesting that Syt1 oligomers play a necessary role in providing a reversible fusion clamp.…”

Symmetrical organization of proteins under docked synaptic vesicles

“…To test this hypothesis, we tested the effect of a Syt1 mutant (F349A), which is designed to specifically destabilize the Syt1 oligomers . As reported earlier, this F349A mutation supports vesicle docking and fusion in PC12 cells, but failed to clamp the vesicles . We over‐expressed the Syt1 F349A mutant, along with VAMP2‐4X‐pHluorin and generated 3105 reconstructions of docked SVs from 141 tomograms.…”

Complexin suppresses spontaneous exocytosis by capturing the membrane-proximal regions of VAMP2 and SNAP25

“…In the absence of Syt1, spontaneous release events are increased and evoked exocytosis is impaired (DiAntonio and Schwarz, 1994;Geppert et al, 1994;Littleton et al, 1994;Pang et al, 2006). Syt1 contains two cytosolic C2 domains (C2A, C2B), which bind Ca 2+ , anionic phospholipids, and SNAREs, likely forming oligomeric assemblies and restraining SNAREpins to arrest the prefusion stage in the absence of Ca 2+ (Bello et al, 2018;Brose et al, 1992;Davletov and Sudhof, 1993;de Wit et al, 2009;Li et al, 2019;Perin et al, 1990;Zhou et al, 2017). In the presence of Ca 2+ , the C2 domains deform the membrane, exerting force on the SNAREpins and membrane fusion is triggered (Chapman and Davis, 1998;Fernandez-Chacon et al, 2001;Hui et al, 2009;Martens et al, 2007;Wang et al, 2016).…”