Morphologies of synaptic protein membrane fusion interfaces

Abstract

Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca 2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca 2+ -triggered release. T he process of vesicle trafficking is central for transporting materials both inside and outside of cells and is essential for maintaining cellular homeostasis (1, 2). Synaptic vesicle fusion releases neurotransmitter molecules into the synaptic cleft upon an action potential-a fast (<5 ms) and highly regulated process. The neuronal SNARE proteins synaptobrevin-2/VAMP2 and syntaxin-1A are anchored in the synaptic vesicle and plasma membranes, respectively, whereas SNAP-25 is peripherally associated with the plasma membrane. The N-to C-terminal zippering of the trans SNARE complex between synaptic and plasma membranes provides the force for membrane juxtaposition and fusion (3), but SNAREs alone are not Ca 2+ -sensitive. Synaptotagmins are membrane-tethered proteins containing two Ca 2+ -binding C2 domains, and a subset of the 16 isoforms of mammalian synaptotagmins act as Ca 2+ sensors for neurotransmitter release: e.g., synaptotagmin-1 (Syt1) is the Ca 2+ sensor for evoked synchronous neurotransmitter release (4). The system is exquisitely fine-tuned to increase the probability of fusion between synaptic vesicles and the plasma membrane by orders of magnitude upon Ca 2+ binding to Syt1. Synaptotagmins simultaneously interact with anionic phospholipid membranes (5) and the neuronal SNARE complex (6, 7). The SNARE complex also interacts with complexin (Cpx), a small soluble protein that both activates evoked release and regulates spontaneous release (8, 9). Moreover, Cpx forms a tripartite complex with the SNARE complex and Syt1 (10). This tripartite complex is part of the prefusion, "primed" state of the synaptic vesicle fusion machinery. Both Munc13 and Munc18 are important for priming, and, at the molecular level, they facilitate proper SNARE complex assembly (11, 12).Although high-resol...

The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis

“…5d). Consistent with this model of the primed state, constitutive insertion of Cpx into the SNARE complex locks release 42 , and inclusion of Cpx increases the separation between membranes in a reconstituted system as observed by electron cryo-tomography 43 .…”

Hypothesis – buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission

“…In such cases, there will be no inner Syt ring or Syt buttress to the SV to clamp the SNAREpins, so fusion will follow constitutively. This extension of our hypothesis suggests that each constitutive fusion event will involve only a handful of SNARE complexes and associated stoichiometric MUN‐containing chaperones as recently found by super‐resolution imaging of constitutive fusion at the PM 70.…”

Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18

“…Electron cryo-tomography studies revealed that nearly all associated SV-PM vesicle pairs fused upon Ca 2+ -addition within 1 min (Gipson et al, 2017). Moreover, the lack of C1C2BMUN fragments at a considerable number of contact sites before Ca 2+ addition is consistent with the notion that Munc13 catalyzed proper SNARE complex assembly and then dissociated from some of the primed pre-fusion complexes, prior to Ca 2+ triggering.…”