Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system

Kyoung, Minjoung; Zhang, Yunxiang; Diao, Jiajie; Chu, Steven; Brunger, Axel T.

doi:10.1038/nprot.2012.134

Cited by 100 publications

(155 citation statements)

References 42 publications

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“…To study the molecular mechanisms of Cpx, several reconstituted systems have been developed (19,(39)(40)(41)(42). As we reported previously, the C-terminal domain is important for suppression of Ca 2+ -independent fusion in a single-vesicle content mixing assay with reconstituted full-length neuronal SNAREs, and synaptotagmin-1 (19,43,44). Our fusion assay (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1 and 5 and Fig. S1) we used the same membrane compositions and protein densities as previously described (19,44). SV vesicles were reconstituted with both synaptotagmin-1 and synaptobrevin-2, whereas PM vesicles were reconstituted with syntaxin-1A and SNAP-25A.…”

Section: Methodsmentioning

confidence: 99%

“…The surface of the quartz slides was passivated by coating the surface with PEG molecules, which alleviated nonspecific binding of vesicles. The same protocol and quality controls (surface coverage and nonspecific binding) were used as described previously (39,44), except that PEG-SVA (Laysan Bio) instead of mPEG-SCM (Laysan Bio) was used because it has a longer half-life. The surface was functionalized by inclusion of biotin-PEG (Laysan Bio) during pegylation.…”

Section: Methodsmentioning

confidence: 99%

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C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Gong

Lai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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In presynaptic nerve terminals, complexin regulates spontaneous "mini" neurotransmitter release and activates Ca 2+ -triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using singleparticle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca 2+ -independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Gong

Lai

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Furthermore, a bulk lipid-mixing assay cannot distinguish between effects caused by differences in docking, hemifusion, or complete fusion. Our single-vesicle alternating-laser excitation (ALEX) lipid-mixing method (discussed below) can distinguish between docking and hemifusion/fusion (38), although it still cannot distinguish between hemifusion and complete fusion (35)(36)(37).…”

Section: Resultsmentioning

confidence: 99%

“…Using the purified large α-Syn oligomers, we first tested whether large α-Syn oligomers inhibited neuronal SNARE-mediated lipid mixing (32,33) with an in vitro lipid-mixing assay using proteoliposomes (34). Lipid mixing in such a bulk assay may result from hemifusion as well as fusion (35)(36)(37), but in any case requires trans-SNARE complex formation. Furthermore, a bulk lipid-mixing assay cannot distinguish between effects caused by differences in docking, hemifusion, or complete fusion.…”

Section: Resultsmentioning

confidence: 99%

Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking

Choi

Kim

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

238

237

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Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.

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SNARE‐Reconstituted Liposomes as Controllable Zeptoliter Nanoreactors for Macromolecules

Lai

Wang

et al. 2017

Adv. Biosys.

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store enzymes, proteins, DNA, and various drug molecules. [2,3] These vesicles contain an aqueous lumen confined by a lipid bilayer, which acts as a permeable barrier to generate confined volumes ranging from zeptoliters (10 −21 L) to attoliters (10 −18 L). [4] Liposomes provide feasible fluidic solutions especially in areas where biocompatibility and miniaturization are critical. The zeptoliter-scale cavity creates a high effective concentration for encapsulated molecules. As a biomedical tool, liposomes exhibit little to no toxicity to biological organization and can preserve drug molecules in a stable environment. For these reasons, catalyst-loaded liposomes for intracellular enzymatic therapy are expected to be the next generation drug delivery systems. [5] Here, we show that liposomes can provide a suitable environment for reactions involving molecules smaller than the membrane's threshold pore size. Moreover, we demonstrate that protein-reconstituted liposomes, called proteoliposomes, can function as two-compartment nanoreactors for large macromolecules through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-mediated membrane fusion in a fast and regulated manner.While liposomes are seen as ideal nanocontainers, [3,6] their lipid membranes are impermeable to macromolecules, ions, and small molecules, essentially making the inner lumen a closed space. To overcome this limitation, scientists have tested the use of porous liposomes generated by membrane phase changes at the lipid phase transition temperature [7] or by pore formation proteins. [8] Porous liposomes are promising technology for nanoreactors since molecules smaller than the threshold pore size can enter the lumen to trigger reactions on demand.We demonstrate the size-selective permeability of porous liposomes through the protection of encapsulated DNAzyme, a catalytic DNA system isolated by in vitro selection methods, from degradation. [9] The experimental scheme is shown in Figure 2a. Cy3/Cy5 dual-labeled DNAzyme complex ( Figure S2, Supporting Information), whose self-cleavage activity depends on the presence of UO 2 2+ , [10] is encapsulated in liposomes composed of dimyristoyl phospatidylcholine (DMPC) lipid molecules. Due to the close proximity of donor (Cy3) and acceptor (Cy5) molecules, the initial Förster (fluorescence) resonance energy transfer (FRET) efficiency, E, is high Liposomes are synthetic phospholipid vesicles containing an aqueous lumen confined by a lipid bilayer. These vesicles have been used as nanoreactors because they offer confined environments that can result in significant changes to chemistry. However, a major limitation of using liposomes as nanocontainers is the impermeability of their lipid membranes. To overcome this, scientists have tested the use of porous liposomes generated by membrane phase changes or by pore formation proteins. In this study, the selective permeability of porous liposomes to molecules smaller than the threshold pore size is demonstrated for triggering int...

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Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system

Cited by 100 publications

References 42 publications

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking

SNARE‐Reconstituted Liposomes as Controllable Zeptoliter Nanoreactors for Macromolecules

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