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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

left: Time resolved intensity courses of Oregon Green DHPE (green) and Texas Red DHPE (red).(A) Docking of a vesicle, (B) hemifusion event, (C,D) full fusion events (C) with and (D) without resolved hemifusion state. The dotted vertical lines illustrate the docking time (tdock) and hemifusion time (themi). The horizontal dashed blue lines serve as guide to the eye highlighting the distinct levels of intensity. right: Schematics of the scenarios envisioned at the end of the time series. For further details see also Supplementary Fig. 7.
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f5: left: Time resolved intensity courses of Oregon Green DHPE (green) and Texas Red DHPE (red).(A) Docking of a vesicle, (B) hemifusion event, (C,D) full fusion events (C) with and (D) without resolved hemifusion state. The dotted vertical lines illustrate the docking time (tdock) and hemifusion time (themi). The horizontal dashed blue lines serve as guide to the eye highlighting the distinct levels of intensity. right: Schematics of the scenarios envisioned at the end of the time series. For further details see also Supplementary Fig. 7.

Mentions: To obtain statistically meaningful data, all vesicles docked onto the pore-spanning membranes were automatically detected by a tracking algorithm, counted and the time traces of the fluorescence intensities were read out from the ROI with 3 pixels around the center of mass of each docked vesicle. As demonstrated in Fig. 3, three different states of the vesicle can be distinguished (Fig. 5A–D, Supplementary Fig. 7). If the TR fluorescence intensity increases in one step to a constant higher value while there is no change in OG fluorescence, the vesicle has only docked (Fig. 5A, docking). The vesicle then either remains docked onto the membrane until the end of the time series or detaches within the observation period indicated by a drop in TR fluorescence intensity in one step to baseline level. Characteristic for the onset of a fusion event following docking of the vesicle is in all cases a simultaneous decrease in TR and an increase in OG fluorescence intensity even if the FRET peak of the OG fluorescence is not observable (Supplementary Fig. 7).


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)

left: Time resolved intensity courses of Oregon Green DHPE (green) and Texas Red DHPE (red).(A) Docking of a vesicle, (B) hemifusion event, (C,D) full fusion events (C) with and (D) without resolved hemifusion state. The dotted vertical lines illustrate the docking time (tdock) and hemifusion time (themi). The horizontal dashed blue lines serve as guide to the eye highlighting the distinct levels of intensity. right: Schematics of the scenarios envisioned at the end of the time series. For further details see also Supplementary Fig. 7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: left: Time resolved intensity courses of Oregon Green DHPE (green) and Texas Red DHPE (red).(A) Docking of a vesicle, (B) hemifusion event, (C,D) full fusion events (C) with and (D) without resolved hemifusion state. The dotted vertical lines illustrate the docking time (tdock) and hemifusion time (themi). The horizontal dashed blue lines serve as guide to the eye highlighting the distinct levels of intensity. right: Schematics of the scenarios envisioned at the end of the time series. For further details see also Supplementary Fig. 7.
Mentions: To obtain statistically meaningful data, all vesicles docked onto the pore-spanning membranes were automatically detected by a tracking algorithm, counted and the time traces of the fluorescence intensities were read out from the ROI with 3 pixels around the center of mass of each docked vesicle. As demonstrated in Fig. 3, three different states of the vesicle can be distinguished (Fig. 5A–D, Supplementary Fig. 7). If the TR fluorescence intensity increases in one step to a constant higher value while there is no change in OG fluorescence, the vesicle has only docked (Fig. 5A, docking). The vesicle then either remains docked onto the membrane until the end of the time series or detaches within the observation period indicated by a drop in TR fluorescence intensity in one step to baseline level. Characteristic for the onset of a fusion event following docking of the vesicle is in all cases a simultaneous decrease in TR and an increase in OG fluorescence intensity even if the FRET peak of the OG fluorescence is not observable (Supplementary Fig. 7).

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