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

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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The neuronal calcium sensor Synaptotagmin-1 and SNARE proteins cooperate to dilate fusion pores

10.1101/623827 ◽

2019 ◽

Author(s):

Zhenyong Wu ◽

Nadiv Dharan ◽

Sathish Thiyagarajan ◽

Ben O’Shaughnessy ◽

Erdem Karatekin

Keyword(s):

Membrane Fusion ◽

Snare Complex ◽

Snare Proteins ◽

Fusion Pore ◽

Calcium Sensor ◽

Fusion Reactions ◽

Synaptotagmin 1 ◽

Pore Opening ◽

Pore Dilation ◽

Fusion Pores

ABSTRACTAll membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered vesicular release of neurotransmitters and hormones. Expansion of this small pore to release cargo molecules is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins, beyond its established role in coupling calcium influx to fusion pore opening. Our results suggest that fusion pore dilation by Syt1 requires interactions with SNAREs, PI(4,5)P2, and calcium. Pore opening was abolished by a mutation of the tandem C2 domain (C2AB) hydrophobic loops of Syt1, suggesting that their calcium-induced insertion into the membrane is required for pore opening. We propose that loop insertion is also required for pore expansion, but through a distinct mechanism. Mathematical modelling suggests that membrane insertion re-orients the C2 domains bound to the SNARE complex, rotating the SNARE complex so as to exert force on the membranes in a mechanical lever action that increases the intermembrane distance. The increased membrane separation provokes pore dilation to offset a bending energy penalty. We conclude that Syt1 assumes a critical role in calcium-dependent fusion pore dilation during neurotransmitter and hormone release.SIGNIFICANCE STATEMENTMembrane fusion is a fundamental biological process, required for development, infection by enveloped viruses, fertilization, intracellular trafficking, and calcium-triggered release of neurotransmitters and hormones when cargo-laden vesicles fuse with the plasma membrane. All membrane fusion reactions proceed through an initial, nanometer-sized fusion pore which can flicker open-closed multiple times before expanding or resealing. Pore expansion is required for efficient cargo release, but underlying mechanisms are poorly understood. Using a combination of single-pore measurements and quantitative modeling, we suggest that a complex between the neuronal calcium sensor Synaptotagmin-1 and the SNARE proteins together act as a calcium-sensitive mechanical lever to force the membranes apart and enlarge the pore.

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

eLife ◽

10.7554/elife.03756 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 56

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.

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Molecular origins of synaptotagmin 1 activities on vesicle docking and fusion pore opening

Scientific Reports ◽

10.1038/srep09267 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 14

Author(s):

Ying Lai ◽

Xiaochu Lou ◽

Jiajie Diao ◽

Yeon-Kyun Shin

Keyword(s):

Fusion Pore ◽

Vesicle Docking ◽

Synaptotagmin 1 ◽

Pore Opening

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

Biophysical Journal ◽

10.1016/j.bpj.2011.11.2731 ◽

2012 ◽

Vol 102 (3) ◽

pp. 499a

Author(s):

Jea-Yeol Kim ◽

Bong-Kyu Choi ◽

Mal-Gi Choi ◽

Sun-Ae Kim ◽

Yeon-Kyun Shin ◽

...

Keyword(s):

Vesicle Fusion ◽

Synaptotagmin 1 ◽

Single Vesicle

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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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Hypotonicity and peptide discharge from a single vesicle

AJP Cell Physiology ◽

10.1152/ajpcell.00303.2008 ◽

2008 ◽

Vol 295 (3) ◽

pp. C624-C631 ◽

Cited By ~ 19

Author(s):

Jernej Jorgačevski ◽

Matjaž Stenovec ◽

Marko Kreft ◽

Aleksandar Bajić ◽

Boštjan Rituper ◽

...

Keyword(s):

Time Course ◽

Single Cells ◽

Vesicle Fusion ◽

Fusion Pore ◽

Pituitary Hormone ◽

Secretory Vesicles ◽

Hormone Release ◽

Prolactin Release ◽

Average Pore ◽

Single Vesicle

Neuroendocrine secretory vesicles discharge their cargo in response to a stimulus, but the nature of this event is poorly understood. We studied the release of the pituitary hormone prolactin by hypotonicity, because this hormone also contributes to osmoregulation. In perfused rat lactotrophs, hypotonicity resulted in a transient increase followed by a sustained depression of prolactin release, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently labeled atrial natriuretic peptide cargo from ∼2% of vesicles/cell. In contrast, KCl-induced depolarization resulted in a response of ∼10% of vesicles/cell, with different unloading/loading time course of the two fluorescent probes. In cell-attached studies, discrete changes in membrane capacitance were recorded in both unstimulated and stimulated conditions, reflecting single vesicle fusion/fissions with the plasma membrane. In stimulated cells, the probability of occurrence of full fusion events was low and unchanged, whereas over 95% of fusion events were transient, with the open fusion pore probability, the average pore dwell-time, the frequency of occurrence, and the fusion pore conductance increased. Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that, in hypotonicity-stimulated lactotrophs, transient vesicle fusion mediates hormone release.

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