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Observing isolated synaptic vesicle association and fusion ex vivo

Nature Protocols (2024)Cite this article

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Abstract

Here, we present a protocol for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and using them in a single-vesicle assay to examine their association and fusion with plasma membrane mimic vesicles. This is a Protocol Extension, building on our previous protocol, which used a purely synthetic system comprised of reconstituted proteins in liposomes. We also describe the generation of a peptide based on the vesicular glutamate transporter, which is essential in the isolation process of glutamatergic synaptic vesicles. This method uses easily accessible reagents to generate fusion-competent glutamatergic synaptic vesicles through immunoisolation. The generation of the vGlut peptide can be accomplished in 6 d, while the isolation of the synaptic vesicles by using the peptide can be accomplished in 2 d, with an additional day to fluorescently label the synaptic vesicles for use in a single-vesicle hybrid fusion assay. The single-vesicle fusion assay can be accomplished in 1 d and can unambiguously delineate synaptic vesicle association, dissociation, Ca2+-independent and Ca2+-dependent fusion modalities. This assay grants control of the synaptic vesicle environment while retaining the complexity of the synaptic vesicles themselves. This protocol can be adapted to studies of other types of synaptic vesicles or, more generally, different secretory or transport vesicles. The workflow described here requires expertise in biochemistry techniques, in particular, protein purification and fluorescence imaging. We assume that the laboratory has protein-purification equipment, including chromatography systems.

Key points

  • This protocol describes an immunoisolation-based approach for isolating functionally intact glutamatergic synaptic vesicles from whole-mouse brain tissue and their use in a single-vesicle assay to examine their association and fusion with plasma membrane mimic vesicles.

  • Isolated vesicles are amenable to a range of functional studies. In addition to the fusion assay described in this protocol, the vesicles can be used for cryo-electron microscopy and cryo-electron tomography.

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Fig. 1: Slide preparation.
Fig. 2: Example SEC chromatogram of a vGlut peptide preparation.
Fig. 3: Isolation scheme of ISVs and example verification by electron microscopy.
Fig. 4: Example western blot analysis of the isolation probed with antibodies against vGlut, vGAT and synaptophysin.
Fig. 5: Example of ISV association with PM vesicles that mimic the plasma membrane.

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Data availability

All imaging data and post-processed files are available in the Stanford Digital Repository at https://doi.org/10.25740/wc823yz5804.

Code availability

MATLAB analysis scripts for the single-vesicle fusion experiments are available on GitHub (https://github.com/brungerlab/single_molecule_matlab_scripts)31. The smCamera software (Taekjip Ha’s laboratory, Johns Hopkins University, Baltimore, MD) was used for acquisition and is available at https://github.com/Ha-SingleMoleculeLab (ref. 32).

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Acknowledgements

We thank R. Jahn and Z. Farsi for stimulating discussions and introducing us to synaptic vesicle isolation techniques. This research was supported by the National Institutes of Health (RO1MH63105 to A.T.B.) and by a grant from the Stanford ADRC Zaffaroni Alzheimer’s Disease Translational Research Program to A.T.B. (ADRC grant no. 5P50AG047366-02).

Author information

Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA

    Jeremy Leitz, Chuchu Wang, Luis Esquivies, John J. Peters, Nisha Gopal, Richard A. Pfuetzner, Austin L. Wang & Axel T. Brunger

  2. Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA

    Jeremy Leitz, Chuchu Wang, Luis Esquivies, John J. Peters, Nisha Gopal, Richard A. Pfuetzner, Austin L. Wang & Axel T. Brunger

  3. Department of Structural Biology, Stanford University, Stanford, CA, USA

    Jeremy Leitz, Chuchu Wang, Luis Esquivies, John J. Peters, Nisha Gopal, Richard A. Pfuetzner, Austin L. Wang & Axel T. Brunger

  4. Department of Photon Science, Stanford University, Stanford, CA, USA

    Jeremy Leitz, Chuchu Wang, Luis Esquivies, John J. Peters, Nisha Gopal, Richard A. Pfuetzner, Austin L. Wang & Axel T. Brunger

  5. Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA

    Jeremy Leitz, Chuchu Wang, Luis Esquivies, John J. Peters, Nisha Gopal, Richard A. Pfuetzner, Austin L. Wang & Axel T. Brunger

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Contributions

J.L. performed the experiments and designed the hybrid fusion assay with input from A.T.B. J.L. and R.A.P. designed the vGlut peptide. J.L. and J.J.P. developed the custom Matlab software to analyze the fusion assay data. J.L., J.J.P. and C.W. generated LP2. A.L.W., L.E. and C.W. optimized the purification protocol for the vGlut peptide. L.E., A.L.W. and R.A.P. produced the proteins used in the hybrid fusion assay. N.G., C.W. and J.L. performed western blots. J.L., L.E. and A.T.B. wrote the paper.

Corresponding author

Correspondence to Axel T. Brunger.

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Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Pietro De Camilli, Shyam Krishnakumar, Tae-Young Yoon and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

Related links

Key references using this protocol

Wang, C. et al. Nature (2024): https://doi.org/10.1038/s41586-024-07610-x

Leitz, J. et al. Cell Rep. 43, 114026 (2024): https://doi.org/10.1016/j.celrep.2024.114026

Brunger, A. T. & Leitz, J. J. Mol. Biol. 435, 167853 (2023): https://doi.org/10.1016/j.jmb.2022.167853

Peters, J. J. et al. J. Struct. Biol. 214, 107851 (2022): https://doi.org/10.1016/j.jsb.2022.107851

This protocol is an extension to: Nat. Protoc. 8, 1–16 (2013): https://doi.org/10.1038/nprot.2012.134

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Leitz, J., Wang, C., Esquivies, L. et al. Observing isolated synaptic vesicle association and fusion ex vivo. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01014-x

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