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Simultaneous lipid and content mixing assays for in vitro reconstitution studies of synaptic vesicle fusion

Nature Protocols volume 12, pages 2014–2028 (2017)Cite this article

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Abstract

This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca2+-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNARE synaptobrevin (with or without the Ca2+ sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE–Biotin) and Cy5–streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored. This protocol is based on a method developed to study yeast vacuolar fusion. In contrast to other protocols used to study the release machinery, this assay incorporates N-ethylmaleimide sensitive factor (NSF) and α-SNAP, which disassemble syntaxin-1 and SNAP-25 heterodimers. As a result, fusion requires Munc18-1, which binds to the released syntaxin-1, and Munc13-1, which, together with Munc18-1, orchestrates SNARE complex assembly. The protocol can be readily adapted to investigation of other types of intracellular membrane fusion by using appropriate alternative proteins. Total time required for one round of the assay is 4 d.

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Figure 1: Working model of neurotransmitter release.
Figure 2: Diagram summarizing the overall approach followed in the lipid and content mixing assays described here.
Figure 3: Typical results obtained in our reconstitution experiments.
Figure 4: Analysis of liposome size distribution by DLS.

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Acknowledgements

We thank M. Zick and W. Wickner for discussions on how to set up the simultaneous lipid and content mixing assays. This work was supported by grant I-1304 from the Welch Foundation (to J.R.) and by NIH Research Project Award R35 NS097333 (to J.R.), and continues work supported previously by NIH grants NS037200 and NS049044 (to J.R.).

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Authors and Affiliations

  1. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Xiaoxia Liu, Alpay Burak Seven, Junjie Xu, Victoria Esser, Lijing Su & Josep Rizo

  2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Xiaoxia Liu, Alpay Burak Seven, Junjie Xu, Victoria Esser, Lijing Su & Josep Rizo

  3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Xiaoxia Liu, Alpay Burak Seven, Junjie Xu, Victoria Esser, Lijing Su & Josep Rizo

  4. Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

    Cong Ma

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Contributions

X.L. and A.B.S. developed the protocol described here. C.M. and L.S. developed the earlier protocol that used different lipid and content mixing assays. J.X. and V.E. provided purified Munc13-1 fragments. J.R. helped to develop the protocol and coordinated the project. X.L. and J.R. wrote the manuscript.

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Correspondence to Xiaoxia Liu or Josep Rizo.

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The authors declare no competing financial interests.

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Supplementary Methods

Expression and purification of recombinant proteins. (PDF 156 kb)

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Liu, X., Seven, A., Xu, J. et al. Simultaneous lipid and content mixing assays for in vitro reconstitution studies of synaptic vesicle fusion. Nat Protoc 12, 2014–2028 (2017). https://doi.org/10.1038/nprot.2017.068

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