Functional synergy between the Munc13 C-terminal C1 and C2 domains

Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca2+-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a ‘primed’ state that does not fuse but is ready for fast fusion upon Ca2+ influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion.

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Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP

eLife ◽

10.7554/elife.38880 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 21

Author(s):

Eric A Prinslow ◽

Karolina P Stepien ◽

Yun-Zu Pan ◽

Junjie Xu ◽

Josep Rizo

Keyword(s):

Complex Formation ◽

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Plasma Membranes ◽

Snare Complex ◽

Complex Assembly ◽

Multiple Factors ◽

Synaptotagmin 1 ◽

Snare Complex Formation

Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.

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Synaptotagmin-1–, Munc18-1–, and Munc13-1–dependent liposome fusion with a few neuronal SNAREs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019314118 ◽

2021 ◽

Vol 118 (4) ◽

pp. e2019314118

Author(s):

Karolina P. Stepien ◽

Josep Rizo

Keyword(s):

Neurotransmitter Release ◽

Plasma Membranes ◽

Snare Complex ◽

Complex Assembly ◽

Synaptotagmin 1 ◽

Low Levels ◽

Liposome Fusion ◽

Membrane Anchoring ◽

Fusion Efficiency

Neurotransmitter release is governed by eight central proteins among other factors: the neuronal SNAREs syntaxin-1, synaptobrevin, and SNAP-25, which form a tight SNARE complex that brings the synaptic vesicle and plasma membranes together; NSF and SNAPs, which disassemble SNARE complexes; Munc18-1 and Munc13-1, which organize SNARE complex assembly; and the Ca2+ sensor synaptotagmin-1. Reconstitution experiments revealed that Munc18-1, Munc13-1, NSF, and α-SNAP can mediate Ca2+-dependent liposome fusion between synaptobrevin liposomes and syntaxin-1–SNAP-25 liposomes, but high fusion efficiency due to uncontrolled SNARE complex assembly did not allow investigation of the role of synaptotagmin-1 on fusion. Here, we show that decreasing the synaptobrevin-to-lipid ratio in the corresponding liposomes to very low levels leads to inefficient fusion and that synaptotagmin-1 strongly stimulates fusion under these conditions. Such stimulation depends on Ca2+ binding to the two C2 domains of synaptotagmin-1. We also show that anchoring SNAP-25 on the syntaxin-1 liposomes dramatically enhances fusion. Moreover, we uncover a synergy between synaptotagmin-1 and membrane anchoring of SNAP-25, which allows efficient Ca2+-dependent fusion between liposomes bearing very low synaptobrevin densities and liposomes containing very low syntaxin-1 densities. Thus, liposome fusion in our assays is achieved with a few SNARE complexes in a manner that requires Munc18-1 and Munc13-1 and that depends on Ca2+ binding to synaptotagmin-1, all of which are fundamental features of neurotransmitter release in neurons.

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Polybasic patches in both C2 domains of Synaptotagmin-1 are required for evoked neurotransmitter release

10.1101/2021.07.05.451149 ◽

2021 ◽

Author(s):

Zhenyong Wu ◽

Lu Ma ◽

Nicholas A Courtney ◽

Jie Zhu ◽

Yongli Zhang ◽

...

Keyword(s):

Optical Tweezers ◽

Single Molecule ◽

Calcium Binding ◽

Neurotransmitter Release ◽

Transmembrane Domain ◽

Snare Complex ◽

Critical Feature ◽

C2 Domains ◽

Electrophysiological Recordings ◽

Synaptotagmin 1

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here we asked what critical feature of C2A may be responsible for its functional role, and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.

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Phosphatidylinositol 4,5 bisphosphate controls the cis and trans interactions of synaptotagmin 1

10.1101/582965 ◽

2019 ◽

Author(s):

S.B. Nyenhuis ◽

A. Thapa ◽

D. S. Cafiso

Keyword(s):

Neurotransmitter Release ◽

Plasma Membranes ◽

Full Length ◽

Membrane Insertion ◽

Membrane Interface ◽

Vesicle Membrane ◽

C2 Domains ◽

Synaptotagmin 1 ◽

Site Directed Spin Labeling ◽

Cis And Trans

AbstractSynaptotagmin 1 acts as the Ca2+-sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here we describe measurements on full-length membrane reconstituted synaptotagmin 1 using site-directed spin labeling where we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate (PIP2), even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+-binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS containing bilayers and will bind in a cis configuration to membranes containing PS even if a PIP2 membrane is presented in trans. The data are consistent with a bridging activity for Syt1 where the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

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All-atom molecular dynamics simulations of synaptic vesicle fusion I: a glimpse at the primed state

10.1101/2021.12.29.474428 ◽

2021 ◽

Author(s):

Josep Rizo ◽

Levent Sari ◽

Yife Qi ◽

Wonpil Im ◽

Milo M Lin

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Plasma Membranes ◽

Synaptotagmin 1 ◽

Primed State ◽

Dynamics Simulations ◽

Synaptic Vesicle Fusion ◽

Shed Light

Synaptic vesicles are primed into a state that is ready for fast neurotransmitter release upon Ca2+-binding to synaptotagmin-1. This state likely includes trans-SNARE complexes between the vesicle and plasma membranes that are bound to synaptotagmin-1 and complexins. However, the nature of this state and the steps leading to membrane fusion are unclear, in part because of the difficulty of studying this dynamic process experimentally. To shed light into these questions, we performed all-atom molecular dynamics simulations of systems containing trans-SNARE complexes between two flat bilayers or a vesicle and a flat bilayer with or without fragments of synaptotagmin-1 and/or complexin-1. Our results help visualize potential states of the release machinery en route to fusion, and suggest mechanistic features that may control the speed of release. In particular, the simulations suggest that the primed state contains almost fully assembled trans-SNARE complexes bound to the synaptotagmin-1 C2B domain and complexin-1 in a spring-loaded configuration where interactions of the C2B domain with the plasma membrane orient complexin-1 toward the vesicle, avoiding premature membrane merger but keeping the system ready for fast fusion upon Ca2+ influx.

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Inhibition of Vesicular Secretion in Both Neuronal and Nonneuronal Cells by a Retargeted Endopeptidase Derivative ofClostridium botulinum Neurotoxin Type A

Infection and Immunity ◽

10.1128/iai.68.5.2587-2593.2000 ◽

2000 ◽

Vol 68 (5) ◽

pp. 2587-2593 ◽

Cited By ~ 53

Author(s):

John A. Chaddock ◽

John R. Purkiss ◽

Lorna M. Friis ◽

Janice D. Broadbridge ◽

Michael J. Duggan ◽

...

Keyword(s):

Neurotransmitter Release ◽

Botulinum Neurotoxin ◽

Type A ◽

Cell Types ◽

Snare Complex ◽

Cell Binding ◽

Vesicle Docking ◽

Botulinum Neurotoxin Type A ◽

Clostridial Neurotoxins ◽

Dose Dependent

ABSTRACT Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of specific components of the vesicle docking/fusion complex, the SNARE complex. A derivative of the type A neurotoxin fromClostridium botulinum (termed LHN/A) that retains catalytic activity can be prepared by proteolysis. The LHN/A, however, lacks the putative native binding domain (HC) of the neurotoxin and is thus unable to bind to neurons and effect inhibition of neurotransmitter release. Here we report the chemical conjugation of LHN/A to an alternative cell-binding ligand, wheat germ agglutinin (WGA). When applied to a variety of cell lines, including those that are ordinarily resistant to the effects of neurotoxin, WGA-LHN/A conjugate potently inhibits secretory responses in those cells. Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopeptidase-dependent cleavage of the natural botulinum neurotoxin type A substrate. These data confirm that the function of the HC domain of C. botulinumneurotoxin type A is limited to binding to cell surface moieties. The data also demonstrate that the endopeptidase and translocation functions of the neurotoxin are effective in a range of cell types, including those of nonneuronal origin. These observations lead to the conclusion that a clostridial endopeptidase conjugate that can be used to investigate SNARE-mediated processes in a variety of cells has been successfully generated.

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Synaptotagmin-1: A Multi-Functional Protein that Mediates Vesicle Docking, Priming, and Fusion

Current Protein and Peptide Science ◽

10.2174/1389203722666210325110231 ◽

2021 ◽

Vol 22 ◽

Author(s):

Najeeb Ullah ◽

Ezzouhra El Maaiden ◽

Md. Sahab Uddin ◽

Ghulam Md Ashraf

Keyword(s):

Plasma Membrane ◽

Secretory Granules ◽

Secretory Vesicle ◽

Neuroendocrine Cells ◽

Snare Complex ◽

Secretory Vesicles ◽

Functional Protein ◽

Vesicle Docking ◽

Synaptotagmin 1

: The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca2+ sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca2+-influx promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

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Sec1p Binds to SNARE Complexes and Concentrates at Sites of Secretion

The Journal of Cell Biology ◽

10.1083/jcb.146.2.333 ◽

1999 ◽

Vol 146 (2) ◽

pp. 333-344 ◽

Cited By ~ 220

Author(s):

Chavela M. Carr ◽

Eric Grote ◽

Mary Munson ◽

Frederick M. Hughson ◽

Peter J. Novick

Keyword(s):

Fluorescent Protein ◽

Snare Complex ◽

The Other ◽

Complex Assembly ◽

Vesicle Docking ◽

Neuronal Protein ◽

The Core ◽

Target Membrane ◽

Green Fluorescent ◽

And Function

Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.

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Microsecond Dissection of Neurotransmitter Release: SNARE-Complex Assembly Dictates Speed and Ca2+ Sensitivity

Neuron ◽

10.1016/j.neuron.2014.04.020 ◽

2014 ◽

Vol 82 (5) ◽

pp. 1088-1100 ◽

Cited By ~ 36

Author(s):

Claudio Acuna ◽

Qingchen Guo ◽

Jacqueline Burré ◽

Manu Sharma ◽

Jianyuan Sun ◽

...

Keyword(s):

Neurotransmitter Release ◽

Snare Complex ◽

Complex Assembly

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Synaptotagmin-1 C2B domain interacts simultaneously with SNAREs and membranes to promote membrane fusion

eLife ◽

10.7554/elife.14211 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 40

Author(s):

Shen Wang ◽

Yun Li ◽

Cong Ma

Keyword(s):

Membrane Fusion ◽

Neurotransmitter Release ◽

Functional Significance ◽

Underlying Mechanism ◽

C2 Domains ◽

Membrane Deformation ◽

Synaptotagmin 1 ◽

Bottom Face ◽

Liposome Fusion

Synaptotagmin-1 (Syt1) acts as a Ca2+ sensor for neurotransmitter release through its C2 domains. It has been proposed that Syt1 promotes SNARE-dependent fusion mainly through its C2B domain, but the underlying mechanism is poorly understood. In this study, we show that the C2B domain interacts simultaneously with acidic membranes and SNARE complexes via the top Ca2+-binding loops, the side polybasic patch, and the bottom face in response to Ca2+. Disruption of the simultaneous interactions completely abrogates the triggering activity of the C2B domain in liposome fusion. We hypothesize that the simultaneous interactions endow the C2B domain with an ability to deform local membranes, and this membrane-deformation activity might underlie the functional significance of the Syt1 C2B domain in vivo.

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