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High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis

Ramakrishnan, S; Gohlke, A; Li, F; Coleman, J; Xu, W; Rothman, JE; Pincet, F; (2018) High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis. Langmuir , 34 (20) pp. 5849-5859. 10.1021/acs.langmuir.8b00116. Green open access

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Abstract

In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO2 holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis.

Type: Article
Title: High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis
Open access status: An open access version is available from UCL Discovery
DOI: 10.1021/acs.langmuir.8b00116
Publisher version: https://doi.org/10.1021/acs.langmuir.8b00116
Language: English
Additional information: This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords: Content release, lipid mixing, holey silicon substrate, protein mobility, SNARE
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences > UCL Queen Square Institute of Neurology > Clinical and Experimental Epilepsy
URI: https://discovery.ucl.ac.uk/id/eprint/10121461
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