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Preparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores

Nature Protocols volume 8, pages 935–948 (2013)Cite this article

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Abstract

This protocol describes an assay that uses suspended nanomembranes called nanodiscs to analyze fusion events. A nanodisc is a lipid bilayer wrapped by membrane scaffold proteins. Fluorescent lipids and a protein that is part of a fusion machinery, VAMP2 in the example detailed herein, are included in the nanodiscs. Upon fusion of a nanodisc with a nonfluorescent liposome containing cognate proteins (for instance, the VAMP2 cognate syntaxin1/SNAP-25 complex), the fluorescent lipids are dispersed in the liposome and the increase in fluorescence, initially quenched in the nanodisc, is monitored on a plate reader. Because the scaffold proteins restrain pore expansion, the fusion pore eventually reseals. A reducing agent, such as dithionite, which can quench the fluorescence of accessible lipids, can then be used to determine the number of fusion events. A fluorescence-based approach can also be used to monitor the release of encapsulated cargo. From data on the total cargo release and the number of the much faster lipid-mixing events, the researcher may determine the amount of cargo released per fusion event. This assay requires 3 d for preparation and 4 h for data acquisition and analysis.

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Figure 1: A nanodisc and a liposome-nanodisc fusion assay.
Figure 2: Nanodisc preparation and characterization.
Figure 3: Lipid-mixing and dithionite assays.
Figure 4: Content-release assays.
Figure 5: Analysis of content release.

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Acknowledgements

This work was supported by a ANR-09-Blanc-0129 grant to F.P., US National Institutes of Health (NIH) grant DK027044 to J.E.R. and a Partner University Funds exchange grant between the Yale University and Ecole Normale Supérieure laboratories. We thank T. Melia for many helpful discussions.

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Author notes

  1. Lei Shi and Kevin Howan: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, School of Medicine, Yale University, New Haven, Connecticut, USA

    Lei Shi, James E Rothman & Frédéric Pincet

  2. Département de Physique, Laboratoire de Physique Statistique, Ecole Normale Supérieure (ENS), Unité Mixte de Recherche (UMR) 8550, Centre National de la Recherche Scientifique (CNRS), Universités Pierre et Marie Curie and Paris Diderot, Paris, France

    Kevin Howan, Yong Jian Wang & Frédéric Pincet

  3. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Qing-Tao Shen

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  1. Lei Shi
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Contributions

L.S., J.E.R. and F.P. designed the research. L.S., Y.J.W. and K.H. optimized the detailed protocol. Q.-T. Shen performed the electron microscopy. L.S., K.H. and F.P. analyzed the experiments. L.S., K.H., Y.J.W. and F.P. wrote the paper.

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Correspondence to Frédéric Pincet.

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The authors declare no competing financial interests.

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Shi, L., Howan, K., Shen, QT. et al. Preparation and characterization of SNARE-containing nanodiscs and direct study of cargo release through fusion pores. Nat Protoc 8, 935–948 (2013). https://doi.org/10.1038/nprot.2013.048

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