Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18

Lai, Ying; Choi, Ucheor B.; Leitz, Jeremy; Rhee, Hong Jun; Lee, Choong-Ku; Altas, Bekir; Zhao, Minglei; Pfuetzner, Richard A.; Wang, Austin L.; Brose, Nils; Rhee, Jeong−Seop; Brunger, Axel T.

doi:10.1016/j.neuron.2017.07.004

Cited by 208 publications

(305 citation statements)

References 68 publications

(92 reference statements)

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“…We incubated the SV vesicles for 30 min with 2 μM full-length Cpx (referred to as SV Cpx vesicles) before mixing with PM vesicles. As previously reported (12), inclusion of Cpx increased the amplitude of Ca 2+ -triggered fusion between SV Cpx and PM vesicles using the single vesicle fusion assay (Fig. S1).…”

Section: Effect Of Complexin-1 On Membrane Separation and Contact Sitesupporting

confidence: 86%

“…When mixed together, and Ca 2+ is added, SV and PM vesicles fuse as shown by a single vesicle fusion assay as previously reported (12,16) (Fig. S1): i.e., the SV and PM vesicles represent a minimal system for Ca 2+ -triggered fusion.…”

Section: Volta Phase Plate Electron Cryomicroscopy Of Functional Synasupporting

confidence: 77%

“…Imaging of Flexible Munc13 Bound to Membranes. Next, we preincubated PM vesicles with 0.5 μM C1C2BMUN fragment of Munc13 for 30 min (referred to as PM C1C2BMUN vesicles) and also included 0.5% diacylglycerol (DAG) in the preparation of the PM vesicles as previously described (12). The cryo-ET tomograms of the PM C1C2BMUN vesicles alone revealed several flexible filamentous densities protruding from them, roughly corresponding to the dimensions of the C1C2BMUN fragment based on its crystal structure (25) (Fig.…”

Section: Effect Of Complexin-1 On Membrane Separation and Contact Sitementioning

confidence: 99%

“…3A). The loosely bound C1C2BMUN fragments might involve binding to DAG in the membrane because the C1 domain of Munc13 binds to DAG (26,27), and both DAG and PIP2 were included in the preparation of the PM vesicles (12). The tighter bound C1C2BMUN fragments may additionally involve an interaction with the reconstituted complex of syntaxin-1A and SNAP-25A in the PM vesicles.…”

Section: Effect Of Complexin-1 On Membrane Separation and Contact Sitementioning

confidence: 99%

“…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).…”

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confidence: 99%

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Morphologies of synaptic protein membrane fusion interfaces

Gipson

Fukuda

Danev

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Effect Of Complexin-1 On Membrane Separation and Contact Sitesupporting

confidence: 86%

Section: Volta Phase Plate Electron Cryomicroscopy Of Functional Synasupporting

confidence: 77%

Section: Effect Of Complexin-1 On Membrane Separation and Contact Sitementioning

confidence: 99%

Section: Effect Of Complexin-1 On Membrane Separation and Contact Sitementioning

confidence: 99%

mentioning

confidence: 99%

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Morphologies of synaptic protein membrane fusion interfaces

Gipson

Fukuda

Danev

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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

et al. 2017

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Neural networks are optimized to detect temporal coincidence on the millisecond timescale. Here, we offer a synthetic hypothesis based on recent structural insights into SNAREs and the C2 domain proteins to explain how synaptic transmission can keep this pace. We suggest that an outer ring of up to six curved Munc13 ‘MUN’ domains transiently anchored to the plasma membrane via its flanking domains surrounds a stable inner ring comprised of synaptotagmin C2 domains to serve as a work‐bench on which SNAREpins are templated. This ‘buttressed‐ring hypothesis’ affords straightforward answers to many principal and long‐standing questions concerning how SNAREpins can be assembled, clamped, and then released synchronously with an action potential.

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Tomosyn guides SNARE complex formation in coordination with Munc18 and Munc13

Wang

et al. 2018

FEBS Letters

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As a SNARE binding protein, tomosyn has been reported to negatively regulate synaptic exocytosis via arresting syntaxin-1 and SNAP-25 into a nonfusogenic product that precludes synaptobrevin-2 entry, raising the question how the assembly of the SNARE complex is achieved. Here, we have investigated new functions of tomosyn in SNARE complex formation and SNARE-mediated vesicle fusion. Assisted by NSF/α-SNAP, syntaxin-1 escapes tomosyn arrest and assembles into the Munc18-1/syntaxin-1 complex. Munc13-1 then catalyzes the transit of syntaxin-1 from the Munc18-1/syntaxin-1 complex to the SNARE complex in a manner specific to synaptobrevin-2 but resistant to tomosyn. Our data suggest that tomosyn ensures SNARE assembly in a way amenable to tight regulation by Munc18-1 and Munc13-1.

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Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and Munc18

Cited by 208 publications

References 68 publications

Morphologies of synaptic protein membrane fusion interfaces

Morphologies of synaptic protein membrane fusion interfaces

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

Tomosyn guides SNARE complex formation in coordination with Munc18 and Munc13

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