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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 a pore-spanning membrane patch containing cholesterol-PEG12-(EIAALEK)4 (A) before and (B) after the addition of large unilamellar vesicles containing cholesterol-PEG12-(KIAALKE)4. All 26 Texas Red DHPE doped vesicles, marked with white circles, have docked onto the pore-spanning membranes at the end of the time series. Scale bars: 5 μm.
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f7: Fluorescence micrographs of a pore-spanning membrane patch containing cholesterol-PEG12-(EIAALEK)4 (A) before and (B) after the addition of large unilamellar vesicles containing cholesterol-PEG12-(KIAALKE)4. All 26 Texas Red DHPE doped vesicles, marked with white circles, have docked onto the pore-spanning membranes at the end of the time series. Scale bars: 5 μm.

Mentions: To compare the results of SNARE-mediated fusion with other SNARE mimetics and evaluate the influence of the planar membrane geometry on the overall fusion efficiency, we performed the same experiments with two well-established SNARE fusion mimetics44, i.e. the coiled coil forming lipidated peptides cholesterol-PEG12-(EIAALEK)4 and cholesterol-PEG12-(KIAALKE)4. Reconstituted into small unilamellar vesicles, the two vesicle populations containing either cholesterol-PEG12-(KIAALKE)4 or cholesterol-PEG12-(EIAALEK)4 fuse (Supplementary Fig. 5) as reported previously with very similar synthetic lipidated coiled coil forming peptides4546. Next, cholesterol-PEG12-(EIAALEK)4 was reconstituted into pore-spanning membranes, while cholesterol-PEG12-(KIAALKE)4 was reconstituted into LUVs using the exact same conditions as reported for the SNAREs. Upon addition of the LUVs to the pore-spanning membranes, vesicles dock onto the membrane (Fig. 7) and remain docked during the entire observation time. No fusion events were observed. Altogether, we analyzed 5 different pore-spanning membrane patches from two different membrane preparations and counted 159 docked vesicles. None of the docked vesicles fused during the observation time. Docking of the vesicles was, however highly specific and did not occur without the reconstitution of the lipopeptides demonstrating that the molecular recognition between the peptide sequences, i.e. the formation of a coiled coil structure takes place.


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 a pore-spanning membrane patch containing cholesterol-PEG12-(EIAALEK)4 (A) before and (B) after the addition of large unilamellar vesicles containing cholesterol-PEG12-(KIAALKE)4. All 26 Texas Red DHPE doped vesicles, marked with white circles, have docked onto the pore-spanning membranes at the end of the time series. Scale bars: 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Fluorescence micrographs of a pore-spanning membrane patch containing cholesterol-PEG12-(EIAALEK)4 (A) before and (B) after the addition of large unilamellar vesicles containing cholesterol-PEG12-(KIAALKE)4. All 26 Texas Red DHPE doped vesicles, marked with white circles, have docked onto the pore-spanning membranes at the end of the time series. Scale bars: 5 μm.
Mentions: To compare the results of SNARE-mediated fusion with other SNARE mimetics and evaluate the influence of the planar membrane geometry on the overall fusion efficiency, we performed the same experiments with two well-established SNARE fusion mimetics44, i.e. the coiled coil forming lipidated peptides cholesterol-PEG12-(EIAALEK)4 and cholesterol-PEG12-(KIAALKE)4. Reconstituted into small unilamellar vesicles, the two vesicle populations containing either cholesterol-PEG12-(KIAALKE)4 or cholesterol-PEG12-(EIAALEK)4 fuse (Supplementary Fig. 5) as reported previously with very similar synthetic lipidated coiled coil forming peptides4546. Next, cholesterol-PEG12-(EIAALEK)4 was reconstituted into pore-spanning membranes, while cholesterol-PEG12-(KIAALKE)4 was reconstituted into LUVs using the exact same conditions as reported for the SNAREs. Upon addition of the LUVs to the pore-spanning membranes, vesicles dock onto the membrane (Fig. 7) and remain docked during the entire observation time. No fusion events were observed. Altogether, we analyzed 5 different pore-spanning membrane patches from two different membrane preparations and counted 159 docked vesicles. None of the docked vesicles fused during the observation time. Docking of the vesicles was, however highly specific and did not occur without the reconstitution of the lipopeptides demonstrating that the molecular recognition between the peptide sequences, i.e. the formation of a coiled coil structure takes place.

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