Faculty Opinions recommendation of Microsecond dissection of neurotransmitter release: SNARE-complex assembly dictates speed and Ca²⁺ sensitivity.

Munc18-1 orchestrates SNARE complex assembly together with Munc13-1 to mediate neurotransmitter release. Munc18-1 binds to synaptobrevin, but the relevance of this interaction and its relation to Munc13 function are unclear. NMR experiments now show that Munc18-1 binds specifically and non-specifically to synaptobrevin. Specific binding is inhibited by a L348R mutation in Munc18-1 and enhanced by a D326K mutation designed to disrupt the ‘furled conformation’ of a Munc18-1 loop. Correspondingly, the activity of Munc18-1 in reconstitution assays that require Munc18-1 and Munc13-1 for membrane fusion is stimulated by the D326K mutation and inhibited by the L348R mutation. Moreover, the D326K mutation allows Munc13-1-independent fusion and leads to a gain-of-function in rescue experiments in Caenorhabditis elegans unc-18 nulls. Together with previous studies, our data support a model whereby Munc18-1 acts as a template for SNARE complex assembly, and autoinhibition of synaptobrevin binding contributes to enabling regulation of neurotransmitter release by Munc13-1.

Download Full-text

BETA- AND GAMMA-SYNUCLEINS MODULATE SYNAPTIC VESICLE-BINDING OF ALPHA-SYNUCLEIN

10.1101/2020.11.19.390419 ◽

2020 ◽

Author(s):

Kathryn E. Carnazza ◽

Lauren Komer ◽

André Pineda ◽

Yoonmi Na ◽

Trudy Ramlall ◽

...

Keyword(s):

Nervous System ◽

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Synaptic Vesicles ◽

Direct Interaction ◽

Alpha Synuclein ◽

Snare Complex ◽

Complex Assembly ◽

Mutual Compensation ◽

Conflicting Evidence

SUMMARYα-Synuclein (αSyn), β-synuclein (βSyn), and γ-synuclein (γSyn) are abundantly expressed in the vertebrate nervous system. αSyn functions in neurotransmitter release via binding to and clustering synaptic vesicles and chaperoning of SNARE-complex assembly. The functions of βSyn and γSyn are unknown. Functional redundancy of the three synucleins and mutual compensation when one synuclein is deleted have been proposed, but with conflicting evidence. Here, we demonstrate that βSyn and γSyn have a reduced affinity towards membranes compared to αSyn, and that direct interaction of βSyn or γSyn with αSyn results in reduced membrane binding of αSyn. Our data suggest that all three synucleins affect synapse function, but only αSyn mediates the downstream function of vesicle clustering and SNARE-complex assembly, while βSyn and γSyn modulate the activity of αSyn through regulating its binding to synaptic vesicles.

Download Full-text

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.

Download Full-text

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

eLife ◽

10.7554/elife.13696 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 45

Author(s):

Xiaoxia Liu ◽

Alpay Burak Seven ◽

Marcial Camacho ◽

Victoria Esser ◽

Junjie Xu ◽

...

Keyword(s):

Neurotransmitter Release ◽

Plasma Membranes ◽

Snare Complex ◽

Complex Assembly ◽

C2 Domains ◽

Vesicle Docking ◽

Synaptotagmin 1 ◽

Primed State ◽

Multiple Domains ◽

Stimulation Of

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Hemophagocytic lymphohistiocytosis caused by dominant-negative mutations in STXBP2 that inhibit SNARE-mediated membrane fusion

Blood ◽

10.1182/blood-2014-11-610816 ◽

2015 ◽

Vol 125 (10) ◽

pp. 1566-1577 ◽

Cited By ~ 64

Author(s):

Waldo A. Spessott ◽

Maria L. Sanmillan ◽

Margaret E. McCormick ◽

Nishant Patel ◽

Joyce Villanueva ◽

...

Keyword(s):

Membrane Fusion ◽

Hemophagocytic Lymphohistiocytosis ◽

Dominant Negative ◽

Snare Complex ◽

Complex Assembly ◽

Mutant Proteins ◽

Key Points ◽

Lymphocyte Cytotoxicity ◽

Dominant Negative Mutations

Key Points Monoallelic STXBP2 mutations affecting codon 65 impair lymphocyte cytotoxicity and contribute to hemophagocytic lymphohistiocytosis. Munc18-2R65Q/W mutant proteins function in a dominant-negative manner to impair membrane fusion and arrest SNARE-complex assembly.

Download Full-text

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.

Download Full-text

Evidence for a functional link between Rab3 and the SNARE complex

Journal of Cell Science ◽

10.1242/jcs.109.12.2875 ◽

1996 ◽

Vol 109 (12) ◽

pp. 2875-2884 ◽

Cited By ~ 2

Author(s):

L. Johannes ◽

F. Doussau ◽

A. Clabecq ◽

J.P. Henry ◽

F. Darchen ◽

...

Keyword(s):

Neurotransmitter Release ◽

Type A ◽

Snare Complex ◽

Mutant Protein ◽

Secretory Process ◽

Functional Link ◽

Mutant Proteins ◽

Aplysia Neurons ◽

Botulinum Type ◽

The Stability

Rab3 is a monomeric GTP-binding protein associated with secretory vesicles which has been implicated in the control of regulated exocytosis. We have exploited Rab3 mutant proteins to investigate the function of Rab3 in the process of neurotransmitter release from Aplysia neurons. A GTPase-deficient Rab3 mutant protein was found to inhibit acetylcholine release suggesting that GTP hydrolysis by Rab3 is rate-limiting in the exocytosis process. This effect was abolished by a mutation in the effector domain, and required the association of Rab3 with membranes. In order to determine the step at which Rab3 interferes with the secretory process, tetanus and botulinum type A neurotoxins were applied to Aplysia neurons pre-injected with the GTPase-deficient Rab3 mutant protein. These neurotoxins are Zn(2+)-dependent proteases that cleave VAMP/synaptobrevin and SNAP-25, two proteins which can form a ternary complex (termed the SNARE complex) with syntaxin and have been implicated in the docking of synaptic vesicles at the plasma membrane. The onset of toxin-induced inhibition of neurotransmitter release was strongly delayed in these cells, indicating that the mutant Rab3 protein led to the accumulation of a toxin-insensitive component of release. Since tetanus and botulinum type A neurotoxins cannot attack their targets, VAMP/synaptobrevin and SNAP-25, when the latter are engaged in the SNARE complex, we propose that Rab3 modulates the activity of the fusion machinery by controlling the formation or the stability of the SNARE complex.

Download Full-text