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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

Fluorescence micrographs of pore-spanning membranes composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) on a 6-mercapto-1-hexanol functionalized gold covered porous silicon nitride surface.The images show fluorescence signals of (A) Atto488 DPPE, (0.01 mol%), (B) Atto647N-syntaxin 1 transmembrane domain (0.0055 mol%) and (C) an overlay of (A) and (B). Scale bars: 3 μm. Autocorrelation curves of the performed FCS measurements with excitation wavelengths of 488 nm (Atto488 DPPE, green, D) and 633 nm (Atto647N-syntaxin 1-TMD, red, E). The blue FCS curve in D was obtained on a gold covered pore rim showing that the fluorescence of Atto488 DPPE is significantly quenched and hence, the diffusion constant cannot be determined. Fitting eq. (4) to the autocorrelation curves provide diffusion coefficients of 7.7 ± 0.4 μm2/s (SD) for Atto488 DPPE and 3.4 ± 0.2 μm2/s (SD) for Atto647N-syntaxin 1-TMD. For Atto488-DPPE L was 200 nm, and for Atto647N-syntaxin 1A L was 250 nm. For each diffusion constant, at least 20 pore-spanning membranes from three independent reconstitutions were analyzed.
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f2: Fluorescence micrographs of pore-spanning membranes composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) on a 6-mercapto-1-hexanol functionalized gold covered porous silicon nitride surface.The images show fluorescence signals of (A) Atto488 DPPE, (0.01 mol%), (B) Atto647N-syntaxin 1 transmembrane domain (0.0055 mol%) and (C) an overlay of (A) and (B). Scale bars: 3 μm. Autocorrelation curves of the performed FCS measurements with excitation wavelengths of 488 nm (Atto488 DPPE, green, D) and 633 nm (Atto647N-syntaxin 1-TMD, red, E). The blue FCS curve in D was obtained on a gold covered pore rim showing that the fluorescence of Atto488 DPPE is significantly quenched and hence, the diffusion constant cannot be determined. Fitting eq. (4) to the autocorrelation curves provide diffusion coefficients of 7.7 ± 0.4 μm2/s (SD) for Atto488 DPPE and 3.4 ± 0.2 μm2/s (SD) for Atto647N-syntaxin 1-TMD. For Atto488-DPPE L was 200 nm, and for Atto647N-syntaxin 1A L was 250 nm. For each diffusion constant, at least 20 pore-spanning membranes from three independent reconstitutions were analyzed.

Mentions: Spreading of a GUV to form pore-spanning membranes is achieved by a proper functionalization of the porous silicon nitride substrate. The highly ordered pore array made of Si3N4 with pore diameters of 1.2 μm is gold coated at the top surface followed by functionalization with 6-mercapto-1-hexanol32. To examine whether the SNARE proteins reconstituted into GUVs according to the protocol described in the Methods Section, are successfully transferred into pore-spanning membranes, two different fluorescently labeled syntaxin derivatives were used. A synthetic transmembrane domain (TMD) labeled with Atto647N (Atto647N-syntaxin 1-TMD) as previously used for GUV experiments33, and a cysteine mutant of syntaxin 1A labeled with Alexa488 (Alexa488-syntaxin 1A). Both were reconstituted into GUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS)/cholesterol (5:2:1:2). After spreading of the GUVs, the fluorescence of the Atto647N-syntaxin 1-TMD (Fig. 2B) as well as that of Alexa488-syntaxin 1A (Supplementary Fig. 1B) was readily observed in the pore-spanning membranes indicating successful reconstitution of transmembrane peptides in pore-spanning bilayers. The fluorescence intensity of Atto647N-syntaxin 1-TMD was fully homogeneous throughout the free-standing parts of the pore-spanning membranes. However, the fluorescence of Alexa488-syntaxin 1A appeared to be slightly inhomogeneous, which might be explained by the tendency of syntaxin 1A to cluster, driven by homotypic protein-protein interactions3435.


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)

Fluorescence micrographs of pore-spanning membranes composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) on a 6-mercapto-1-hexanol functionalized gold covered porous silicon nitride surface.The images show fluorescence signals of (A) Atto488 DPPE, (0.01 mol%), (B) Atto647N-syntaxin 1 transmembrane domain (0.0055 mol%) and (C) an overlay of (A) and (B). Scale bars: 3 μm. Autocorrelation curves of the performed FCS measurements with excitation wavelengths of 488 nm (Atto488 DPPE, green, D) and 633 nm (Atto647N-syntaxin 1-TMD, red, E). The blue FCS curve in D was obtained on a gold covered pore rim showing that the fluorescence of Atto488 DPPE is significantly quenched and hence, the diffusion constant cannot be determined. Fitting eq. (4) to the autocorrelation curves provide diffusion coefficients of 7.7 ± 0.4 μm2/s (SD) for Atto488 DPPE and 3.4 ± 0.2 μm2/s (SD) for Atto647N-syntaxin 1-TMD. For Atto488-DPPE L was 200 nm, and for Atto647N-syntaxin 1A L was 250 nm. For each diffusion constant, at least 20 pore-spanning membranes from three independent reconstitutions were analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Fluorescence micrographs of pore-spanning membranes composed of DOPC/POPE/POPS/cholesterol (5:2:1:2) on a 6-mercapto-1-hexanol functionalized gold covered porous silicon nitride surface.The images show fluorescence signals of (A) Atto488 DPPE, (0.01 mol%), (B) Atto647N-syntaxin 1 transmembrane domain (0.0055 mol%) and (C) an overlay of (A) and (B). Scale bars: 3 μm. Autocorrelation curves of the performed FCS measurements with excitation wavelengths of 488 nm (Atto488 DPPE, green, D) and 633 nm (Atto647N-syntaxin 1-TMD, red, E). The blue FCS curve in D was obtained on a gold covered pore rim showing that the fluorescence of Atto488 DPPE is significantly quenched and hence, the diffusion constant cannot be determined. Fitting eq. (4) to the autocorrelation curves provide diffusion coefficients of 7.7 ± 0.4 μm2/s (SD) for Atto488 DPPE and 3.4 ± 0.2 μm2/s (SD) for Atto647N-syntaxin 1-TMD. For Atto488-DPPE L was 200 nm, and for Atto647N-syntaxin 1A L was 250 nm. For each diffusion constant, at least 20 pore-spanning membranes from three independent reconstitutions were analyzed.
Mentions: Spreading of a GUV to form pore-spanning membranes is achieved by a proper functionalization of the porous silicon nitride substrate. The highly ordered pore array made of Si3N4 with pore diameters of 1.2 μm is gold coated at the top surface followed by functionalization with 6-mercapto-1-hexanol32. To examine whether the SNARE proteins reconstituted into GUVs according to the protocol described in the Methods Section, are successfully transferred into pore-spanning membranes, two different fluorescently labeled syntaxin derivatives were used. A synthetic transmembrane domain (TMD) labeled with Atto647N (Atto647N-syntaxin 1-TMD) as previously used for GUV experiments33, and a cysteine mutant of syntaxin 1A labeled with Alexa488 (Alexa488-syntaxin 1A). Both were reconstituted into GUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS)/cholesterol (5:2:1:2). After spreading of the GUVs, the fluorescence of the Atto647N-syntaxin 1-TMD (Fig. 2B) as well as that of Alexa488-syntaxin 1A (Supplementary Fig. 1B) was readily observed in the pore-spanning membranes indicating successful reconstitution of transmembrane peptides in pore-spanning bilayers. The fluorescence intensity of Atto647N-syntaxin 1-TMD was fully homogeneous throughout the free-standing parts of the pore-spanning membranes. However, the fluorescence of Alexa488-syntaxin 1A appeared to be slightly inhomogeneous, which might be explained by the tendency of syntaxin 1A to cluster, driven by homotypic protein-protein interactions3435.

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