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
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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.
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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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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).
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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.
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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.
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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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DOI: https://doi.org/10.1038/s41596-024-01014-x
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