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

Content Provider	Europe PMC
Author	Mühlenbrock, Peter Sari, Merve Steinem, Claudia
Abstract	Neuronal 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.
Related Links	https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC8071798&blobtype=pdf
ISSN	01757571
Journal	European Biophysics Journal [Eur Biophys J]
Volume Number	50
DOI	10.1007/s00249-020-01479-0
PubMed Central reference number	PMC8071798
Issue Number	2
PubMed reference number	33320298
e-ISSN	14321017
Language	English
Publisher	Springer International Publishing
Publisher Date	2020-12-15
Publisher Place	Gewerbestrasse 11, Cham, Ch 6330, Switzerland
Access Restriction	Open
Rights License	Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020
Subject Keyword	Content mixing Fluorescence microscopy Lipid mixing Model membranes SNAREs
Content Type	Text
Resource Type	Article
Subject	Medicine Biophysics