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Visualization of SNARE-Mediated Hemifusion between Giant Unilamellar Vesicles Arrested by Myricetin
Heo, Paul,Park, Joon-Bum,Shin, Yeon-Kyun,Kweon, Dae-Hyuk Frontiers Media S.A. 2017 Frontiers in molecular neuroscience Vol.10 No.-
<P>Neurotransmitters are released within a millisecond after Ca<SUP>2+</SUP> arrives at an active zone. However, the vesicle fusion pathway underlying this synchronous release is yet to be understood. At the center of controversy is whether hemifusion, in which outer leaflets are merged while inner leaflets are still separated, is an on-pathway or off-pathway product of Ca<SUP>2+</SUP>-triggered exocytosis. Using the single vesicle fusion assay, we recently demonstrated that hemifusion is an on-pathway intermediate that immediately proceeds to full fusion upon Ca<SUP>2+</SUP> triggering. It has been shown that the flavonoid myricetin arrests soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-mediated vesicle fusion at hemifusion, but that the hemifused vesicles spontaneously convert to full fusion when the myricetin clamp is removed by the enzyme laccase. In the present study, we visualized SNARE-mediated hemifusion between two SNARE-reconstituted giant unilamellar vesicles (GUVs) arrested by myricetin. The large size of the GUVs enabled us to directly image the hemifusion between them. When two merging GUVs were labeled with different fluorescent dyes, GUV pairs showed asymmetric fluorescence intensities depending on the position on the GUV pair consistent with what is expected for hemifusion. The flow of lipids from one vesicle to the other was revealed with fluorescence recovery after photobleaching (FRAP), indicating that the two membranes had hemifused. These results support the hypothesis that hemifusion may be the molecular status that primes Ca<SUP>2+</SUP>-triggered millisecond exocytosis. This study represents the first imaging of SNARE-driven hemifusion between GUVs.</P>
Green fluorescence protein-based content-mixing assay of SNARE-driven membrane fusion
Heo, Paul,Kong, Byoungjae,Jung, Young-Hun,Park, Joon-Bum,Shin, Jonghyeok,Park, Myungseo,Kweon, Dae-Hyuk Academic Press 2017 Biochemical and biophysical research communication Vol. No.
<P><B>Abstract</B></P> <P>Soluble <I>N</I>-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available <I>in vitro</I> assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regarding the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening.</P> <P><B>Highlights</B></P> <P> <UL> <LI> SNARE proteins drive membrane fusion and open a pore for cargo release. </LI> <LI> Biotinylated GFP and DyStrp was used as the reporter pair of fusion pore opening. </LI> <LI> Procedure for efficient SNARE reconstitution and reporter encapsulation was established. </LI> <LI> The FRET pair reported opening of a large fusion pore bigger than 5 nm. </LI> <LI> The assay was robust and provided information of stoichiometry of vesicle fusion. </LI> </UL> </P>