Solution Single Vesicle Fusion Assay Reveals PIP2 Mediated Sequential Actions of Synaptotagmin-1 onto SNAREs

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.

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Molecular Determinants of Complexin Clamping in Reconstituted Single-Vesicle Fusion

10.1101/2021.07.05.451112 ◽

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.

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Synergistic roles of Synaptotagmin-1 and complexin in calcium-regulated neuronal exocytosis

eLife ◽

10.7554/elife.54506 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 2

Author(s):

Sathish Ramakrishnan ◽

Manindra Bera ◽

Jeff Coleman ◽

James E Rothman ◽

Shyam S Krishnakumar

Keyword(s):

Principal Components ◽

Synaptic Vesicles ◽

Vesicle Fusion ◽

Regulatory Proteins ◽

Snare Proteins ◽

Synaptotagmin 1 ◽

Single Vesicle

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca2+-regulated fusion. We find that under each vesicle, Synaptotagmin-1 oligomers bind and clamp a limited number of ‘central’ SNARE complexes via the primary interface and introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to arrest the remaining free ‘peripheral’ SNAREpins to produce a stably clamped vesicle. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles.

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Independent Yet Synergistic Roles of Synaptotagmin-1 and Complexin in Calcium Regulated Neuronal Exocytosis

10.1101/2019.12.16.878686 ◽

2019 ◽

Author(s):

Sathish Ramakrishnan ◽

Manindra Bera ◽

Jeff Coleman ◽

James E. Rothman ◽

Shyam S. Krishnakumar

Keyword(s):

Principal Components ◽

Synaptic Vesicles ◽

Vesicle Fusion ◽

Regulatory Proteins ◽

Snare Proteins ◽

Binding Interface ◽

Synaptotagmin 1 ◽

Single Vesicle

ABSTRACTCalcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved, namely the vesicular fusion machinery (SNARE proteins) and the regulatory proteins (Synaptotagmin-1 and Complexin) are well-known. Here, we use a reconstituted single-vesicle fusion assay to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act independently but synergistically to establish Ca2+-regulated fusion. Under physiologically-relevant conditions, we find that Synaptotagmin-1 oligomers bind and clamp a limited number of ‘central’ SNARE complexes via the primary binding interface, to introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to independently arrest the remaining free ‘peripheral’ SNAREpins to produce stably clamped vesicles. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles.

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The synaptotagmin 1 linker may function as an electrostatic zipper that opens for docking but closes for fusion pore opening

Biochemical Journal ◽

10.1042/bj20130949 ◽

2013 ◽

Vol 456 (1) ◽

pp. 25-33 ◽

Cited By ~ 21

Author(s):

Ying Lai ◽

Xiaochu Lou ◽

Yongseok Jho ◽

Tae-Young Yoon ◽

Yeon-Kyun Shin

Keyword(s):

Vesicle Fusion ◽

Fusion Pore ◽

Linker Region ◽

Synaptotagmin 1 ◽

Pore Opening ◽

Single Vesicle

We used the single-vesicle-fusion assay to dissect the function of the Syt1 linker region, and our results suggest that the Syt1 linker region might have some capacity to extend for docking and fold to facilitate pore opening.

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Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system

Nature Protocols ◽

10.1038/nprot.2012.134 ◽

2012 ◽

Vol 8 (1) ◽

pp. 1-16 ◽

Cited By ~ 59

Author(s):

Minjoung Kyoung ◽

Yunxiang Zhang ◽

Jiajie Diao ◽

Steven Chu ◽

Axel T Brunger

Keyword(s):

Vesicle Fusion ◽

Vesicle Content ◽

Single Vesicle

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

Nature Communications ◽

10.1038/s41467-019-12015-w ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 13

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.

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Doc2b promotes GLUT4 exocytosis by activating the SNARE-mediated fusion reaction in a calcium- and membrane bending–dependent manner

Molecular Biology of the Cell ◽

10.1091/mbc.e12-11-0810 ◽

2013 ◽

Vol 24 (8) ◽

pp. 1176-1184 ◽

Cited By ~ 35

Author(s):

Haijia Yu ◽

Shailendra S. Rathore ◽

Eric M. Davis ◽

Yan Ouyang ◽

Jingshi Shen

Keyword(s):

Glucose Transporter ◽

Fusion Reaction ◽

Vesicle Fusion ◽

Dependent Manner ◽

Membrane Bilayer ◽

Protein Receptors ◽

Synaptotagmin 1 ◽

Membrane Bending ◽

Glucose Transporter Glut4 ◽

Dependent Mechanism

The glucose transporter GLUT4 plays a central role in maintaining body glucose homeostasis. On insulin stimulation, GLUT4-containing vesicles fuse with the plasma membrane, relocating GLUT4 from intracellular reservoirs to the cell surface to uptake excess blood glucose. The GLUT4 vesicle fusion reaction requires soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) as the core fusion engine and a group of regulatory proteins. In particular, the soluble C2-domain factor Doc2b plays a key role in GLUT4 vesicle fusion, but its molecular mechanism has been unclear. Here we reconstituted the SNARE-dependent GLUT4 vesicle fusion in a defined proteoliposome fusion system. We observed that Doc2b binds to GLUT4 exocytic SNAREs and potently accelerates the fusion kinetics in the presence of Ca2+. The stimulatory activity of Doc2b requires intact Ca2+-binding sites on both the C2A and C2B domains. Using electron microscopy, we observed that Doc2b strongly bends the membrane bilayer, and this membrane-bending activity is essential to the stimulatory function of Doc2b in fusion. These results demonstrate that Doc2b promotes GLUT4 exocytosis by accelerating the SNARE-dependent fusion reaction by a Ca2+- and membrane bending–dependent mechanism. Of importance, certain features of Doc2b function appear to be distinct from how synaptotagmin-1 promotes synaptic neurotransmitter release, suggesting that exocytic Ca2+ sensors may possess divergent mechanisms in regulating vesicle fusion.

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