N-terminal domain of complexin independently activates calcium-triggered fusion

Lai, Ying; Choi, Ucheor B.; Zhang, Yunxiang; Zhao, Minglei; Pfuetzner, Richard A.; Wang, Austin L.; Diao, Jiajie; Brunger, Axel T.

doi:10.1073/pnas.1604348113

Cited by 54 publications

(56 citation statements)

References 78 publications

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“…Similarly, knockdown in cultured cortical neurons increases spontaneous release, although knockout of complexin in mice only affects spontaneous release depending on the particular neuronal cell type (12,16,27,28). A split pair of N-terminal and central domain fragments of Cpx is sufficient to activate Ca 2+ -triggered release using our reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery (30). Moreover, the Cpx accessory domain can be entirely eliminated, and the resulting split pair of Cpx constructs still activates Ca 2+ -triggered fusion (i.e., a clamping process is not required for activation) (30).…”

Section: Discussionmentioning

confidence: 74%

“…A split pair of N-terminal and central domain fragments of Cpx is sufficient to activate Ca 2+ -triggered release using our reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery (30). Moreover, the Cpx accessory domain can be entirely eliminated, and the resulting split pair of Cpx constructs still activates Ca 2+ -triggered fusion (i.e., a clamping process is not required for activation) (30). Cpx has two conformations when bound to the ternary SNARE complex (55,56), one of which induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of Cpx (56).…”

Section: Discussionmentioning

confidence: 81%

“…The accessory domain (amino acid residues 28-48) regulates spontaneous fusion in neurons and it suppresses Ca 2+ -independent fusion in reconstituted systems (12,14,19,(22)(23)(24)(25)(26)(27). Although the accessory domain is required for regulating spontaneous release, mutations of this domain do not affect the activating function of Cpx compared with wild-type neurons in rescue experiments with Cpx knockdown (28,29), and it can be entirely eliminated while still maintaining the activating function of Cpx in a reconstituted system (30). The α-helical central domain (amino acid residues 49-70) binds to the SNARE complex (31,32) and is essential for all functions of Cpx in all species studied to date, including priming (16,28,29,33), inhibiting spontaneous fusion (12,19,24,25,(34)(35)(36), and activation of Ca 2+ -triggered fusion (12-14, 19, 34, 36-38).…”

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confidence: 99%

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C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Gong

Lai

et al. 2016

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

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In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca 2+ -triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using singleparticle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca 2+ -independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

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Section: Discussionmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 81%

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confidence: 99%

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C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Gong

Lai

et al. 2016

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

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“…Moreover, the complex nature of cellular synapses consisting of dynamic and dense networks of protein-protein and protein-membrane interactions makes it difficult to study the synaptic fusion protein machinery in vivo. Therefore, we used a bottom-up approach where we reconstituted synaptic proteins into liposomes in a stepwise fashion: Synaptobrevin-2 and Syt1 were reconstituted into liposomes with a defined synthetic lipid composition that mimics synaptic vesicles (referred to as "SV vesicles"), and syntaxin-1A and SNAP-25A were reconstituted into liposomes with a lipid composition that mimics the plasma membrane obtained from lipid brain extracts (referred to as "PM vesicles") (19).…”

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confidence: 99%

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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“…Some characteristics of the in vivo function of complexin have successfully been reconstituted (Lai, et al, 2014;Giraudo, et al, 2009;Malsam, et al, 2012;Lai, et al, 2016); however, biochemical studies of complexin and SNARE proteins have not revealed a clear mechanism by which complexin inhibits spontaneous fusion. The EPR and NMR spectroscopy, fluorescence anisotropy, and TIRF microscopy to characterize the interaction between complexin, SNAREs, and the lipid bilayer.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of SNARE Mediated Membrane Fusion

Kreutzberger

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Regulated exocytosis is a process by which cells release neurotransmitters, peptides, proteins, and small molecules in response to a stimulus. This process is necessary for cell-cell communication in multi-cellular organisms. The final step of regulated exocytosis is membrane fusion, where the membrane of a secretory vesicle merges with the plasma membrane, opening a pore releasing soluble cargo. The molecular machinery that controls fusion is coupled to respond to intracellular calcium. The proteins involved include the SNARE proteins (the fusion machinery), the calcium sensor synaptotagmin, and the regulatory proteins complexin, Munc18, Munc13, and CAPS.I will show the use of planar supported bilayers as a model system to study membrane fusion of single vesicles (Chapter 3). This assay was used to investigate the activity of different plasma membrane target SNARE complexes (Chapter 4), the effect of membrane curvature (Chapter 5) and cholesterol (Chapter 6) on the fusion process, how complexin-1 interacts with the plasma membrane SNARE proteins (Chapter 7), and how the fusion process is coupled to calcium (Chapter 8). These results have led to a new model of membrane fusion. In the absence of calcium, secretory vesicle fusion is arrested in the presence of the SNARE proteins, Munc18, and complexin, but will fuse readily in the presence of calcium. Meanwhile, the protein CAPS or Munc13 act as a kinetic factors controlling the rate of fusion in response to a calcium stimulus.ii

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N-terminal domain of complexin independently activates calcium-triggered fusion

Cited by 54 publications

References 78 publications

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Morphologies of synaptic protein membrane fusion interfaces

Reconstitution of SNARE Mediated Membrane Fusion

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