Observing isolated synaptic vesicle association and fusion ex vivo.

Autor: Leitz J; Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA.; Department of Structural Biology, Stanford University, Stanford, CA, USA.; Department of Photon Science, Stanford University, Stanford, CA, USA.; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA., Wang C; Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA.; Department of Structural Biology, Stanford University, Stanford, CA, USA.; Department of Photon Science, Stanford University, Stanford, CA, USA.; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA., Esquivies L; Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA.; Department of Structural Biology, Stanford University, Stanford, CA, USA.; Department of Photon Science, Stanford University, Stanford, CA, USA.; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA., Peters JJ; Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA.; Department of Structural Biology, Stanford University, Stanford, CA, USA.; Department of Photon Science, Stanford University, Stanford, CA, USA.; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA., Gopal N; Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA.; Department of Structural Biology, Stanford University, Stanford, CA, USA.; Department of Photon Science, Stanford University, Stanford, CA, USA.; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA., Pfuetzner RA; Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA.; Department of Structural Biology, Stanford University, Stanford, CA, USA.; Department of Photon Science, Stanford University, Stanford, CA, USA.; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA., Wang AL; Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA.; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA.; Department of Structural Biology, Stanford University, Stanford, CA, USA.; Department of Photon Science, Stanford University, Stanford, CA, USA.; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA., Brunger AT; Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA. brunger@stanford.edu.; Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA. brunger@stanford.edu.; Department of Structural Biology, Stanford University, Stanford, CA, USA. brunger@stanford.edu.; Department of Photon Science, Stanford University, Stanford, CA, USA. brunger@stanford.edu.; Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA. brunger@stanford.edu.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2024 Nov; Vol. 19 (11), pp. 3139-3161. Date of Electronic Publication: 2024 Jul 02.
DOI: 10.1038/s41596-024-01014-x
Abstrakt: 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, Ca 2+ -independent and Ca 2+ -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.
(© 2024. Springer Nature Limited.)
Databáze: MEDLINE
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