scholarly journals Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Ying Lai ◽  
Jiajie Diao ◽  
Daniel J Cipriano ◽  
Yunxiang Zhang ◽  
Richard A Pfuetzner ◽  
...  
Keyword(s):  
Lipid Content ◽  
Point Contact ◽  
Vesicle Fusion ◽  
Neuronal Cultures ◽  
Spontaneous Release ◽  
Terminal Domain ◽  
Synaptotagmin 1 ◽  
Free Membrane ◽  
Synaptic Vesicle Fusion ◽  
Single Vesicle

Previously we showed that fast Ca2+-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle–vesicle lipid/content mixing assay (<xref ref-type="bibr" rid="bib5">Diao et al., 2012</xref>). When complexin-1 was included, a more pronounced Ca2+-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca2+-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca2+-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca2+-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca2+-triggered fusion and inhibition of spontaneous fusion.

scholarly journals Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion

eLife ◽  
2012 ◽  
Vol 1 ◽  
Author(s):  
Jiajie Diao ◽  
Patricia Grob ◽  
Daniel J Cipriano ◽  
Minjoung Kyoung ◽  
Yunxiang Zhang ◽  
...  
Keyword(s):  
Neurotransmitter Release ◽  
Heterogeneous Network ◽  
Point Contact ◽  
Synaptic Proteins ◽  
Complete Fusion ◽  
Cryo Electron Microscopy ◽  
Before And After ◽  
Synaptotagmin 1 ◽  
Synaptic Vesicle Fusion ◽  
Single Vesicle

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.

scholarly journals Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicholas A. Courtney ◽  
Huan Bao ◽  
Joseph S. Briguglio ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
Snare Proteins ◽  
Major Focus ◽  
C2 Domains ◽  
Releasable Pool ◽  
Readily Releasable Pool ◽  
Synaptotagmin 1 ◽  
Synaptic Vesicle Fusion ◽  
C2a Domain

Abstract Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

scholarly journals Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion

2021 ◽  
Author(s):  
Manindra Bera ◽  
Sathish Ramakrishnan ◽  
Jeff Coleman ◽  
Shyam S Krishnakumar ◽  
James E Rothman
Keyword(s):  
Specific Interaction ◽  
Vesicle Fusion ◽  
Assembly Process ◽  
Inhibitory Function ◽  
Physiological Relevance ◽  
Vesicular Release ◽  
Molecular Determinants ◽  
Synaptotagmin 1 ◽  
Single Vesicle

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al. 2020). Here using the same in vitro single-vesicle fusion assay, we establish the molecular details of the Complexin clamp and its physiological relevance. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins, analogous to the trans clamping model, enhances this functionality. The c-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Further, we find that Complexin likely contributes to rapid Ca2+-synchronized vesicular release by preventing un-initiated fusion rather than by directly facilitating vesicle fusion.

scholarly journals Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Vincent Huson ◽  
Marieke Meijer ◽  
Rien Dekker ◽  
Mirelle ter Veer ◽  
Marvin Ruiter ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Energy Barrier ◽  
Phorbol Esters ◽  
Release Kinetics ◽  
Vesicle Fusion ◽  
Tetanic Stimulation ◽  
Release Rates ◽  
Synaptotagmin 1 ◽  
Post Tetanic Potentiation ◽  
Synaptic Vesicle Fusion

Previously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca2+-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca2+-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1D232N) or increasing its hydrophobicity (Syt14W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt19Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1-independent mechanisms that lower the energy barrier and act additively with Syt1-dependent mechanisms to produce PTP by exerting multiplicative effects on release rates.

Synaptobrevin 1 mediates vesicle priming and evoked release in a subpopulation of hippocampal neurons

2014 ◽  
Vol 112 (6) ◽  
pp. 1559-1565 ◽  
Author(s):  
Johannes Zimmermann ◽  
Thorsten Trimbuch ◽  
Christian Rosenmund
Keyword(s):  
Plasma Membrane ◽  
Hippocampal Neurons ◽  
Vesicle Fusion ◽  
Snare Proteins ◽  
Spontaneous Release ◽  
Lower Efficiency ◽  
Readily Releasable Pool ◽  
Close Proximity ◽  
Α Helix ◽  
Synaptic Vesicle Fusion

The core machinery of synaptic vesicle fusion consists of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, the two t-SNAREs at the plasma membrane (SNAP-25, Syntaxin 1) and the vesicle-bound v-SNARE synaptobrevin 2 (VAMP2). Formation of the trans-oriented four-α-helix bundle between these SNAREs brings vesicle and plasma membrane in close proximity and prepares the vesicle for fusion. The t-SNAREs are thought to be necessary for vesicle fusion. Whether the v-SNAREs are required for fusion is still unclear, as substantial vesicle priming and spontaneous release activity remain in mammalian mass-cultured synaptobrevin/cellubrevin-deficient neurons. Using the autaptic culture system from synaptobrevin 2 knockout neurons of mouse hippocampus, we found that the majority of cells were devoid of any evoked or spontaneous release and had no measurable readily releasable pool. A small subpopulation of neurons, however, displayed release, and their release activity correlated with the presence and amount of v-SNARE synaptobrevin 1 expressed. Comparison of synaptobrevin 1 and 2 in rescue experiments demonstrates that synaptobrevin 1 can substitute for the other v-SNARE, but with a lower efficiency in neurotransmitter release probability. Release activity in synaptobrevin 2-deficient mass-cultured neurons was massively reduced by a knockdown of synaptobrevin 1, demonstrating that synaptobrevin 1 is responsible for the remaining release activity. These data support the hypothesis that both t- and v-SNAREs are absolutely required for vesicle priming and evoked release and that differential expression of SNARE paralogs can contribute to differential synaptic coding in the brain.

scholarly journals Post-tetanic potentiation lowers the energy barrier for synaptic vesicle fusion independently of Synaptotagmin-1

2020 ◽  
Author(s):  
Vincent Huson ◽  
Marieke Meijer ◽  
Rien Dekker ◽  
Mirelle ter Veer ◽  
Marvin Ruiter ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Energy Barrier ◽  
Phorbol Esters ◽  
Release Kinetics ◽  
Vesicle Fusion ◽  
Tetanic Stimulation ◽  
Release Rates ◽  
Synaptotagmin 1 ◽  
Post Tetanic Potentiation ◽  
Synaptic Vesicle Fusion

AbstractPreviously, we showed that modulation of the energy barrier for synaptic vesicle fusion boosts release rates supralinearly (Schotten, 2015). Here we show that mouse hippocampal synapses employ this principle to trigger Ca2+-dependent vesicle release and post-tetanic potentiation (PTP). We assess energy barrier changes by fitting release kinetics in response to hypertonic sucrose. Mimicking activation of the C2A domain of the Ca2+-sensor Synaptotagmin-1 (Syt1), by adding a positive charge (Syt1D232N) or increasing its hydrophobicity (Syt14W), lowers the energy barrier. Removing Syt1 or impairing its release inhibitory function (Syt19Pro) increases spontaneous release without affecting the fusion barrier. Both phorbol esters and tetanic stimulation potentiate synaptic strength, and lower the energy barrier equally well in the presence and absence of Syt1. We propose a model where tetanic stimulation activates Syt1 dependent and independent mechanisms that lower the energy barrier independently in an additive manner to produce PTP by multiplication of release rates.

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