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

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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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.

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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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Conformation and Membrane Position of the Region Linking the Two C2 Domains in Synaptotagmin 1 by Site-Directed Spin Labeling†

Biochemistry ◽

10.1021/bi801470m ◽

2008 ◽

Vol 47 (47) ◽

pp. 12380-12388 ◽

Cited By ~ 19

Author(s):

Hao Huang ◽

David S. Cafiso

Keyword(s):

Spin Labeling ◽

C2 Domains ◽

Synaptotagmin 1 ◽

Site Directed Spin Labeling

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Differential regulation of synchronous versus asynchronous neurotransmitter release by the C2 domains of synaptotagmin 1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1000606107 ◽

2010 ◽

Vol 107 (33) ◽

pp. 14869-14874 ◽

Cited By ~ 34

Author(s):

M. Yoshihara ◽

Z. Guan ◽

J. T. Littleton

Keyword(s):

Neurotransmitter Release ◽

Differential Regulation ◽

C2 Domains ◽

Synaptotagmin 1

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

eLife ◽

10.7554/elife.56469 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

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.

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A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

The Journal of Cell Biology ◽

10.1083/jcb.202008121 ◽

2021 ◽

Vol 220 (4) ◽

Cited By ~ 1

Author(s):

Lei Li ◽

Haowen Liu ◽

Mia Krout ◽

Janet E. Richmond ◽

Yu Wang ◽

...

Keyword(s):

Neurotransmitter Release ◽

Release Kinetics ◽

Sensor System ◽

C2 Domains ◽

Loosely Coupled ◽

C Elegans ◽

Membrane Tethering ◽

Synaptotagmin 1 ◽

Tm Domain ◽

Fast Release

Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.

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Transmembrane tethering of synaptotagmin to synaptic vesicles controls multiple modes of neurotransmitter release

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1420312112 ◽

2015 ◽

Vol 112 (12) ◽

pp. 3793-3798 ◽

Cited By ~ 19

Author(s):

Jihye Lee ◽

J. Troy Littleton

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Neurotransmitter Release ◽

Temporal Dynamics ◽

Transgenic Animals ◽

Vesicle Fusion ◽

Full Length ◽

Dependent Manner ◽

Membrane Phospholipids ◽

C2 Domains

Synaptotagmin 1 (Syt1) is a synaptic vesicle integral membrane protein that regulates neurotransmitter release by activating fast synchronous fusion and suppressing slower asynchronous release. The cytoplasmic C2 domains of Syt1 interact with SNAREs and plasma membrane phospholipids in a Ca2+-dependent manner and can substitute for full-length Syt1 in in vitro membrane fusion assays. To determine whether synaptic vesicle tethering of Syt1 is required for normal fusion in vivo, we performed a structure-function study with tethering mutants at the Drosophila larval neuromuscular junction. Transgenic animals expressing only the cytoplasmic C2 domains or full-length Syt1 tethered to the plasma membrane failed to restore synchronous synaptic vesicle fusion, and also failed to clamp spontaneous vesicle release. In addition, transgenic animals with shorter, but not those with longer, linker regions separating the C2 domains from the transmembrane segment abolished Syt1’s ability to activate synchronous vesicle fusion. Similar defects were observed when C2 domain alignment was altered to C2B-C2A from the normal C2A-C2B orientation, leaving the tether itself intact. Although cytoplasmic and plasma membrane-tethered Syt1 variants could not restore synchronous release in syt1 null mutants, they were very effective in promoting fusion through the slower asynchronous pathway. As such, the subcellular localization of Syt1 within synaptic terminals is important for the temporal dynamics that underlie synchronous and asynchronous neurotransmitter release.

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