scholarly journals Ring-like oligomers of Synaptotagmins and related C2 domain proteins

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Maria N Zanetti ◽  
Oscar D Bello ◽  
Jing Wang ◽  
Jeff Coleman ◽  
Yiying Cai ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Synaptic Transmission ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Structural Aspect ◽  
C2 Domain ◽  
Early Step ◽  
Vesicle Docking ◽  
Ring Assembly ◽  
Synaptotagmin 1

We recently reported that the C2AB portion of Synaptotagmin 1 (Syt1) could self-assemble into Ca2+-sensitive ring-like oligomers on membranes, which could potentially regulate neurotransmitter release. Here we report that analogous ring-like oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and extended Synaptotagmins (E-Syts). Evidently, circular oligomerization is a general and conserved structural aspect of many C2 domain proteins, including Synaptotagmins. Further, using electron microscopy combined with targeted mutations, we show that under physiologically relevant conditions, both the Syt1 ring assembly and its rapid disruption by Ca2+ involve the well-established functional surfaces on the C2B domain that are important for synaptic transmission. Our data suggests that ring formation may be triggered at an early step in synaptic vesicle docking and positions Syt1 to synchronize neurotransmitter release to Ca2+ influx.

scholarly journals Reconstituted synaptotagmin I mediates vesicle docking, priming, and fusion

2011 ◽  
Vol 195 (7) ◽  
pp. 1159-1170 ◽  
Author(s):  
Zhao Wang ◽  
Huisheng Liu ◽  
Yiwen Gu ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Transmission ◽  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Cytoplasmic Domain ◽  
Snare Proteins ◽  
Antagonist Activity ◽  
Vesicle Docking ◽  
Synaptotagmin I ◽  
Target Membrane

The synaptic vesicle protein synaptotagmin I (syt) promotes exocytosis via its ability to penetrate membranes in response to binding Ca2+ and through direct interactions with SNARE proteins. However, studies using full-length (FL) membrane-embedded syt in reconstituted fusion assays have yielded conflicting results, including a lack of effect, or even inhibition of fusion, by Ca2+. In this paper, we show that reconstituted FL syt promoted rapid docking of vesicles (<1 min) followed by a priming step (3–9 min) that was required for subsequent Ca2+-triggered fusion between v- and t-SNARE liposomes. Moreover, fusion occurred only when phosphatidylinositol 4,5-bisphosphate was included in the target membrane. This system also recapitulates some of the effects of syt mutations that alter synaptic transmission in neurons. Finally, we demonstrate that the cytoplasmic domain of syt exhibited mixed agonist/antagonist activity during regulated membrane fusion in vitro and in cells. Together, these findings reveal further convergence of reconstituted and cell-based systems.

scholarly journals Structural elements that underlie Doc2β function during asynchronous synaptic transmission

2015 ◽  
Vol 112 (31) ◽  
pp. E4316-E4325 ◽  
Author(s):  
Renhao Xue ◽  
Jon D. Gaffaney ◽  
Edwin R. Chapman
Keyword(s):  
Plasma Membrane ◽  
Synaptic Transmission ◽  
Neurotransmitter Release ◽  
Lipid Binding ◽  
Slow Phase ◽  
Structural Elements ◽  
Excitatory Postsynaptic Current ◽  
Synaptotagmin 1 ◽  
Asynchronous Release

Double C2-like domain-containing proteins alpha and beta (Doc2α and Doc2β) are tandem C2-domain proteins proposed to function as Ca2+ sensors for asynchronous neurotransmitter release. Here, we systematically analyze each of the negatively charged residues that mediate binding of Ca2+ to the β isoform. The Ca2+ ligands in the C2A domain were dispensable for Ca2+-dependent translocation to the plasma membrane, with one exception: neutralization of D220 resulted in constitutive translocation. In contrast, three of the five Ca2+ ligands in the C2B domain are required for translocation. Importantly, translocation was correlated with the ability of the mutants to enhance asynchronous release when overexpressed in neurons. Finally, replacement of specific Ca2+/lipid-binding loops of synaptotagmin 1, a Ca2+ sensor for synchronous release, with corresponding loops from Doc2β, resulted in chimeras that yielded slower kinetics in vitro and slower excitatory postsynaptic current decays in neurons. Together, these data reveal the key determinants of Doc2β that underlie its function during the slow phase of synaptic transmission.

scholarly journals Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle fusion and replenishment in a dosage-dependent manner

2020 ◽  
Author(s):  
Zhuo Guan ◽  
Mónica C. Quiñones-Frías ◽  
Yulia Akbergenova ◽  
J. Troy Littleton
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Dependent Manner ◽  
Short Term ◽  
Sensor Model ◽  
Synaptotagmin 1 ◽  
Asynchronous Release ◽  
Synaptic Vesicle Fusion

AbstractSynchronous neurotransmitter release is triggered by Ca2+ binding to the synaptic vesicle protein Synaptotagmin 1, while asynchronous fusion and short-term facilitation is hypothesized to be mediated by plasma membrane-localized Synaptotagmin 7 (SYT7). We generated mutations in Drosophila Syt7 to determine if it plays a conserved role as the Ca2+ sensor for these processes. Electrophysiology and quantal imaging revealed evoked release was elevated 2-fold. Syt7 mutants also had a larger pool of readily-releasable vesicles, faster recovery following stimulation, and robust facilitation. Syt1/Syt7 double mutants displayed more release than Syt1 mutants alone, indicating SYT7 does not mediate the residual asynchronous release remaining in the absence of SYT1. SYT7 localizes to an internal membrane tubular network within the peri-active zone, but does not enrich at release sites. These findings indicate the two Ca2+ sensor model of SYT1 and SYT7 mediating all phases of neurotransmitter release and facilitation is not applicable at Drosophila synapses.

scholarly journals A novel region in the CaV2.1 α1 subunit C-terminus regulates fast synaptic vesicle fusion and vesicle docking at the mammalian presynaptic active zone

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Matthias Lübbert ◽  
R Oliver Goral ◽  
Rachel Satterfield ◽  
Travis Putzke ◽  
Arn MJM van den Maagdenberg ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Physical Distance ◽  
Subunit C ◽  
Vesicle Docking ◽  
C Terminus ◽  
Synaptic Vesicle Release ◽  
Synaptic Vesicle Fusion ◽  
Α1 Subunit

In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by CaV2.1 calcium channels (CaV2.1) and is highly dependent on the physical distance between CaV2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of CaV2.1 through direct interactions with the CaV2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant CaV2.1 α1 subunits on a CaV2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of CaV2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for CaV2.1 abundance and coupling. Therefore, our work advances our molecular understanding of CaV2.1 regulation of neurotransmitter release in mammalian CNS synapses.

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

eLife ◽  
2019 ◽  
Vol 8 ◽  
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.

scholarly journals Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jason D Vevea ◽  
Edwin R Chapman
Keyword(s):  
Membrane Protein ◽  
Synaptic Vesicle ◽  
Protein Function ◽  
Neurotransmitter Release ◽  
Cell Biology ◽  
Hippocampal Neurons ◽  
Cell Types ◽  
Spontaneous Release ◽  
Vesicle Membrane ◽  
Synaptotagmin 1

The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.

scholarly journals Reconstructing essential active zone functions within a synapse

2021 ◽  
Author(s):  
Chao Tan ◽  
Shan Shan H Wang ◽  
Giovanni de Nola ◽  
Pascal S Kaeser
Keyword(s):  
Fusion Protein ◽  
Synaptic Vesicle ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Molecular Machines ◽  
Vesicle Docking ◽  
Active Zones ◽  
Ca2 Channels

Active zones are molecular machines that control neurotransmitter release through synaptic vesicle docking and priming, and through coupling of these vesicles to Ca2+ entry. The complexity of active zone machinery has made it challenging to determine which mechanisms drive these roles in release. Here, we induce RIM+ELKS knockout to eliminate active zone scaffolding networks, and then reconstruct each active zone function. Re-expression of RIM1-Zn fingers positioned Munc13 on undocked vesicles and rendered them release-competent. Reconstitution of release-triggering required docking of these vesicles to Ca2+ channels. Fusing RIM1-Zn to CaVbeta4-subunits sufficed to restore docking, priming and release-triggering without reinstating active zone scaffolds. Hence, exocytotic activities of the 80 kDa CaVbeta4-Zn fusion protein bypassed the need for megadalton-sized secretory machines. These data define key mechanisms of active zone function, establish that fusion competence and docking are mechanistically separable, and reveal that active zone scaffolding networks are not required for release.

scholarly journals Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release

2006 ◽  
Vol 176 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Anton Maximov ◽  
Ok-Ho Shin ◽  
Xinran Liu ◽  
Thomas C. Südhof
Keyword(s):  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Spontaneous Release ◽  
Dependent Protein Kinase ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis ◽  
Synaptotagmin 1 ◽  
Dependent Protein ◽  
Central Synapses

Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca2+. In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine97, and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine97 by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca2+-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.

scholarly journals A unique C2 domain at the C terminus of Munc13 promotes synaptic vesicle priming

2021 ◽  
Vol 118 (11) ◽  
pp. e2016276118
Author(s):  
Murugesh Padmanarayana ◽  
Haowen Liu ◽  
Francesco Michelassi ◽  
Lei Li ◽  
Daniel Betensky ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Neurotransmitter Release ◽  
Synaptic Vesicles ◽  
Genetic Screen ◽  
C2 Domain ◽  
Forward Genetic Screen ◽  
Molecular Events ◽  
C Terminus

Neurotransmitter release during synaptic transmission comprises a tightly orchestrated sequence of molecular events, and Munc13-1 is a cornerstone of the fusion machinery. A forward genetic screen for defects in neurotransmitter release in Caenorhabditis elegans identified a mutation in the Munc13-1 ortholog UNC-13 that eliminated its unique and deeply conserved C-terminal module (referred to as HC2M) containing a Ca2+-insensitive C2 domain flanked by membrane-binding helices. The HC2M module could be functionally replaced in vivo by protein domains that localize to synaptic vesicles but not to the plasma membrane. HC2M is broadly conserved in other Unc13 family members and is required for efficient synaptic vesicle priming. We propose that the HC2M domain evolved as a vesicle/endosome adaptor and acquired synaptic vesicle specificity in the Unc13ABC protein family.

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

eLife ◽  
2016 ◽  
Vol 5 ◽  
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.

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