In vitro single vesicle fusion study of Ca2+-triggered SNARE-mediated membrane fusion

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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Molecular mechanisms of atlastin-mediated ER membrane fusion revealed by a FRET-based single-vesicle fusion assay

Scientific Reports ◽

10.1038/s41598-017-09162-9 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 4

Author(s):

Kyung Tae Kim ◽

Yeojin Moon ◽

Yunsu Jang ◽

Kang Taek Lee ◽

Changwook Lee ◽

...

Keyword(s):

Membrane Fusion ◽

Molecular Mechanisms ◽

Vesicle Fusion ◽

Single Vesicle ◽

Er Membrane

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Single Vesicle Fusion Assay in Model Membranes to Study SNARE-medicated Membrane Fusion

10.18130/v39z5n ◽

2010 ◽

Author(s):

Marta Katarzyna Domanska

Keyword(s):

Membrane Fusion ◽

Vesicle Fusion ◽

Model Membranes ◽

Single Vesicle

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In vitro single vesicle fusion assays based on pore-spanning membranes: merits and drawbacks

European Biophysics Journal ◽

10.1007/s00249-020-01479-0 ◽

2020 ◽

Author(s):

Peter Mühlenbrock ◽

Merve Sari ◽

Claudia Steinem

Keyword(s):

Lipid Bilayers ◽

Vesicle Fusion ◽

In Vitro Assays ◽

Supported Lipid Bilayers ◽

Protein Receptors ◽

Membrane Components ◽

Interconnected Structure ◽

Single Vesicle ◽

Fusion Efficiency

AbstractNeuronal fusion mediated by soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) is a fundamental cellular process by which two initially distinct membranes merge resulting in one interconnected structure to release neurotransmitters into the presynaptic cleft. To get access to the different stages of the fusion process, several in vitro assays have been developed. In this review, we provide a short overview of the current in vitro single vesicle fusion assays. Among those assays, we developed a single vesicle assay based on pore-spanning membranes (PSMs) on micrometre-sized pores in silicon, which might overcome some of the drawbacks associated with the other membrane architectures used for investigating fusion processes. Prepared by spreading of giant unilamellar vesicles with reconstituted t-SNAREs, PSMs provide an alternative tool to supported lipid bilayers to measure single vesicle fusion events by means of fluorescence microscopy. Here, we discuss the diffusive behaviour of the reconstituted membrane components as well as that of the fusing synthetic vesicles with reconstituted synaptobrevin 2 (v-SNARE). We compare our results with those obtained if the synthetic vesicles are replaced by natural chromaffin granules under otherwise identical conditions. The fusion efficiency as well as the different fusion states observable in this assay by means of both lipid mixing and content release are illuminated.

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Plant Alkaloids Inhibit Membrane Fusion Mediated by Calcium and Fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 Fusion Peptides

Biomedicines ◽

10.3390/biomedicines9101434 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1434

Author(s):

Egor V. Shekunov ◽

Svetlana S. Efimova ◽

Natalia M. Yudintceva ◽

Anna A. Muryleva ◽

Vladimir V. Zarubaev ◽

...

Keyword(s):

Membrane Fusion ◽

Lipid Vesicles ◽

Vero Cells ◽

Vesicle Fusion ◽

Fusion Peptides ◽

Release Assay ◽

Plant Alkaloids ◽

Calcein Release ◽

Novel Coronavirus

To rationalize the antiviral actions of plant alkaloids, the ability of 20 compounds to inhibit calcium-mediated fusion of lipid vesicles composed of phosphatidylglycerol and cholesterol was investigated using the calcein release assay and dynamic light scattering. Piperine, tabersonine, hordenine, lupinine, quinine, and 3-isobutyl-1-methylxanthine demonstrated the most potent effects (inhibition index greater than 50%). The introduction of phosphatidylcholine into the phosphatidylglycerol/cholesterol mixture led to significant changes in quinine, hordenine, and 3-isobutyl-1-methylxanthine efficiency. Comparison of the fusion inhibitory ability of the tested alkaloids, and the results of the measurements of alkaloid-induced alterations in the physical properties of model membranes indicated a potent relationship between a decrease in the cooperativity of the phase transition of lipids and the ability of alkaloids to prevent calcium-mediated vesicle fusion. In order to use this knowledge to combat the novel coronavirus pandemic, the ability of the most effective compounds to suppress membrane fusion induced by fragments of MERS-CoV and SARS-CoV/SARS-CoV-2 fusion peptides was studied using the calcein release assay and confocal fluorescence microscopy. Piperine was shown to inhibit vesicle fusion mediated by both coronavirus peptides. Moreover, piperine was shown to significantly reduce the titer of SARS-CoV2 progeny in vitro in Vero cells when used in non-toxic concentrations.

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