In vitro system capable of differentiating fast Ca 2+ -triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release
Open accessAbstract
Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca(2+) triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca(2+) injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca(2+) injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca(2+) sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca(2+) triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce…
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Neuronal SNAREs and Syt1 represent a minimal system for Ca2+-triggered membrane fusion.
“Neuronal SNAREs and Syt1 represent a minimal system for Ca2+-triggered membrane fusion.”
From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [81]
The presence and purity of reconstituted proteins was confirmed by SDS-PAGE, and the directionality of the membrane proteins facing outward was assessed by chymotrypsin digestion followed by SDS-PAGE.
“As described previously81, the presence and purity of reconstituted proteins was confirmed by SDS-PAGE of the vesicle preparations, and the directionality of the membrane proteins (facing outward) was assessed by chymotrypsin digestion followed by SDS-PAGE.”
From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [81]
Recent evidence for the minimal system came from a reconstituted single vesicle-vesicle assay that discriminates among vesicle association, lipid mixing, and content mixing.
“Recent evidence for this notion came from a reconstituted single vesicle-vesicle assay that discriminates among vesicle association, lipid mixing, and content mixing37,38,81.”
From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [81]
The single vesicle-vesicle assay uses full-length Syt1 and monitors content mixing, a correlate for neurotransmitter release.
“Our single vesicle-vesicle assay thus uses full-length Syt1 and it monitors content mixing, a correlate for neurotransmitter release37,38,81.”
From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [81]
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Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis
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Self-citationMechanism of neurotransmitter release coming into focus
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Self-citationDifferent regions of synaptic vesicle membrane regulate VAMP2 conformation for the SNARE assembly
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- Authors
- Kyoung, M., Srivastava, A., Zhang, Y., Diao, J., Vrljic, M., Grob, P., Nogales, E., Chu, S., & Brunger, A. T.
- Affiliations
- Howard Hughes Medical Institute (US)
- QB3 (US)
- Lawrence Berkeley National Laboratory (US)
- Departments of Molecular and Cellular Physiology,
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2011-06-24
- Volume
- 108
- Issue
- 29
- Pages
- E304-13
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC3141984
- OpenAlex
- W2151858671
- Cited by
- 154
- References
- 6
- Citations by year
- 2026: 3 · 2025: 6 · 2024: 4 · 2023: 6 · 2022: 7 · 2021: 4 · 2020: 6 · 2019: 8 · 2018: 10 · 2017: 14 · 2016: 12 · 2015: 19 · 2014: 15 · 2013: 19 · 2012: 15
- FWCI
- 8.44
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute of Mental Health (R01-MH63105, R37MH063105, R01MH063105)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
- Keywords
- synaptic vesicle fusion, neurotransmitter release, calcium-triggered exocytosis, hemifusion, SNARE proteins, synaptotagmin-1, complexin, lipid exchange, readily releasable pool, fusion kinetics, cooperative calcium binding
- MeSH terms
- Animals, Calcium, Cell Line, Escherichia coli, Exocytosis, Fluorescence, Image Processing, Computer-Assisted, Lipids, Models, Biological, Nerve Tissue Proteins, Synaptic Vesicles, Neurotransmitter Agents, Spodoptera, SNARE Proteins, Synaptotagmin I, Rats, In Vitro Techniques
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