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Resolving single membrane fusion events on planar pore-spanning membranes.

Schwenen LL, Hubrich R, Milovanovic D, Geil B, Yang J, Kros A, Jahn R, Steinem C - Sci Rep (2015)

Bottom Line: As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4.Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy.Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion.

View Article: PubMed Central - PubMed

Affiliation: Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstr. 2, 37077 Göttingen, Germany.

ABSTRACT
Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic.

No MeSH data available.


Related in: MedlinePlus

(A) Schematic drawing of the model system for SNARE mediated membrane fusion based on pore-spanning membranes. SNARE proteins are shown in red (syntaxin 1A), green (SNAP25) and blue (synaptobrevin 2). Membranes are depicted as thick orange lines. (B) Scanning electron micrograph of a porous silicon nitride substrate. Scale bar: 3 μm. (C) Pore-spanning membrane patch obtained from spreading a giant unilamellar vesicle composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) and doped with 1 mol % Oregon Green DHPE on a gold/6-mercapto-1-hexanol-functionalized substrate. Scale bar: 20 μm.
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f1: (A) Schematic drawing of the model system for SNARE mediated membrane fusion based on pore-spanning membranes. SNARE proteins are shown in red (syntaxin 1A), green (SNAP25) and blue (synaptobrevin 2). Membranes are depicted as thick orange lines. (B) Scanning electron micrograph of a porous silicon nitride substrate. Scale bar: 3 μm. (C) Pore-spanning membrane patch obtained from spreading a giant unilamellar vesicle composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) and doped with 1 mol % Oregon Green DHPE on a gold/6-mercapto-1-hexanol-functionalized substrate. Scale bar: 20 μm.

Mentions: SNARE proteins are the well-known key players in neuronal membrane fusion and an excellent test case for the development and evaluation of the envisioned membrane fusion system. In synaptic transmission three SNARE proteins, namely syntaxin 1, SNAP25 and synaptobrevin 2 mediate the fundamental steps of membrane fusion30. Syntaxin 1 and SNAP25 are localized in the mainly planar presynaptic membrane, with which synaptobrevin 2 containing synaptic vesicles can fuse. To mimic this in vivo situation, we established a reconstituted membrane system as depicted in Fig. 1A. SNARE complexes composed of syntaxin 1A, SNAP25a and a soluble fragment of synaptobrevin 2 are embedded in a planar pore-spanning membrane by spreading SNARE-containing giant unilamellar vesicles (GUVs) on a gold-coated functionalized regular pore array in silicon nitride with pore diameters of 1.2 μm (Fig. 1B), while the fusing vesicles contain synaptobrevin 2. The success of pore-spanning membrane formation can be readily observed by fluorescence microscopy with a characteristic fluorescence pattern (Fig. 1C). The fluorescence pattern arises from the fact that the fluorescence of the membrane is quenched on the gold covered pore rims owing to the close contact of the membrane to the support. No quenching occurs in the freestanding parts of the membrane resulting in the pore pattern in the fluorescence images, which allows to observe each individual pore-spanning membrane31.


Resolving single membrane fusion events on planar pore-spanning membranes.

Schwenen LL, Hubrich R, Milovanovic D, Geil B, Yang J, Kros A, Jahn R, Steinem C - Sci Rep (2015)

(A) Schematic drawing of the model system for SNARE mediated membrane fusion based on pore-spanning membranes. SNARE proteins are shown in red (syntaxin 1A), green (SNAP25) and blue (synaptobrevin 2). Membranes are depicted as thick orange lines. (B) Scanning electron micrograph of a porous silicon nitride substrate. Scale bar: 3 μm. (C) Pore-spanning membrane patch obtained from spreading a giant unilamellar vesicle composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) and doped with 1 mol % Oregon Green DHPE on a gold/6-mercapto-1-hexanol-functionalized substrate. Scale bar: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4499801&req=5

f1: (A) Schematic drawing of the model system for SNARE mediated membrane fusion based on pore-spanning membranes. SNARE proteins are shown in red (syntaxin 1A), green (SNAP25) and blue (synaptobrevin 2). Membranes are depicted as thick orange lines. (B) Scanning electron micrograph of a porous silicon nitride substrate. Scale bar: 3 μm. (C) Pore-spanning membrane patch obtained from spreading a giant unilamellar vesicle composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) and doped with 1 mol % Oregon Green DHPE on a gold/6-mercapto-1-hexanol-functionalized substrate. Scale bar: 20 μm.
Mentions: SNARE proteins are the well-known key players in neuronal membrane fusion and an excellent test case for the development and evaluation of the envisioned membrane fusion system. In synaptic transmission three SNARE proteins, namely syntaxin 1, SNAP25 and synaptobrevin 2 mediate the fundamental steps of membrane fusion30. Syntaxin 1 and SNAP25 are localized in the mainly planar presynaptic membrane, with which synaptobrevin 2 containing synaptic vesicles can fuse. To mimic this in vivo situation, we established a reconstituted membrane system as depicted in Fig. 1A. SNARE complexes composed of syntaxin 1A, SNAP25a and a soluble fragment of synaptobrevin 2 are embedded in a planar pore-spanning membrane by spreading SNARE-containing giant unilamellar vesicles (GUVs) on a gold-coated functionalized regular pore array in silicon nitride with pore diameters of 1.2 μm (Fig. 1B), while the fusing vesicles contain synaptobrevin 2. The success of pore-spanning membrane formation can be readily observed by fluorescence microscopy with a characteristic fluorescence pattern (Fig. 1C). The fluorescence pattern arises from the fact that the fluorescence of the membrane is quenched on the gold covered pore rims owing to the close contact of the membrane to the support. No quenching occurs in the freestanding parts of the membrane resulting in the pore pattern in the fluorescence images, which allows to observe each individual pore-spanning membrane31.

Bottom Line: As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4.Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy.Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion.

View Article: PubMed Central - PubMed

Affiliation: Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstr. 2, 37077 Göttingen, Germany.

ABSTRACT
Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic.

No MeSH data available.


Related in: MedlinePlus