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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) Time lapse series of fluorescence micrographs showing a single fusion event of a large unilamellar vesicle containing full length synaptobrevin 2 with a pore-spanning membrane with reconstituted ΔN-acceptor complex. (top) Oregon Green DHPE fluorescence of the pore-spanning membranes; (bottom) Texas Red DHPE fluorescence of the vesicle. The yellow circles in (a) shows the region of interest (ROI) that was used for reading out the time-resolved changes in the relative fluorescence intensities depicted in (C); the white circles in the images highlight the region, where the vesicle docks and fuses and only serves as a guide to the eye. Scale bar: 5 μm. (B) Schematic drawing of the postulated fusion states at the time of image recording. Membranes are colored according to their incorporated fluorescent dye. (C) Time resolved changes in the relative fluorescence intensity of Oregon Green DHPE (green) and Texas Red DHPE (red) during the shown fusion event. Dashed blue lines serve as a guide to the eye highlighting the distinct levels of intensity. Letters (a–f) correspond to the fluorescence images in (A). For further details see text and Supplementary Fig. 7.
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f3: (A) Time lapse series of fluorescence micrographs showing a single fusion event of a large unilamellar vesicle containing full length synaptobrevin 2 with a pore-spanning membrane with reconstituted ΔN-acceptor complex. (top) Oregon Green DHPE fluorescence of the pore-spanning membranes; (bottom) Texas Red DHPE fluorescence of the vesicle. The yellow circles in (a) shows the region of interest (ROI) that was used for reading out the time-resolved changes in the relative fluorescence intensities depicted in (C); the white circles in the images highlight the region, where the vesicle docks and fuses and only serves as a guide to the eye. Scale bar: 5 μm. (B) Schematic drawing of the postulated fusion states at the time of image recording. Membranes are colored according to their incorporated fluorescent dye. (C) Time resolved changes in the relative fluorescence intensity of Oregon Green DHPE (green) and Texas Red DHPE (red) during the shown fusion event. Dashed blue lines serve as a guide to the eye highlighting the distinct levels of intensity. Letters (a–f) correspond to the fluorescence images in (A). For further details see text and Supplementary Fig. 7.

Mentions: Pore-spanning membrane patches of various sizes ranging from 10–150 μm in diameter were obtained by GUV spreading as visualized by the Oregon Green DHPE fluorescence (Fig. 3A,a). For the detection of single vesicle fusion events, membrane patches with a size larger than 40 × 40 μm2 were selected on the porous surface. After addition of full length synaptobrevin 2 containing LUVs doped with Texas Red DHPE, time series for both lipid dyes were recorded simultaneously (Supplementary Movie 1). A characteristic time series of movie frames of a single vesicle fusion event that basically captures all observables in these experiments is depicted in Fig. 3. The time dependent fluorescence intensity changes of Oregon Green DHPE and Texas Red DHPE upon docking and fusion of a single vesicle to the planar membrane was monitored by defining a region of interest (ROI, yellow colored circle in Fig. 3A,a) around the center of mass of the docked vesicle and integrating the fluorescence intensity as a function of time. The obtained time traces of the two fluorophores including all characteristic features of a single vesicle fusion event are depicted in Fig. 3C. Prior to vesicle docking a low Texas Red DHPE (TR) and a high Oregon Green DHPE (OG) fluorescence is observed (Fig. 3C,a) When a single vesicle approaches the surface and docks, a sudden increase in TR fluorescence occurs, while the OG fluorescence remains unaffected (Fig. 3C,b). Upon onset of fusion (Fig. 3C,c) the TR intensity shows a transient increase, while concomitantly a small transient decrease in the OG fluorescence intensity is observed, which is a result of the Förster resonance energy transfer (FRET) between the TR and OG dye as the lipids of the outer leaflet of the vesicle (Texas Red DHPE) start mixing with the lipids of the outer leaflet of the pore-spanning membrane (Oregon Green DHPE) (Fig. 3C,c). After this first transient changes of TR and OG fluorescence, the TR fluorescence intensity drops to and then remains at an intensity that is well above baseline level but lower than that of the originally docked vesicle. Simultaneously, an increase in OG fluorescence intensity is observed. These fluorescence time traces can be explained with the formation of an intermediate hemifused state (Fig. 3C,d). In the hemifused state, the outer leaflets merge, while the inner leaflet of the TR-doped vesicle remains unaffected. Thus, after a first merger of the outer leaflets, the TR fluorescence intensity drops to an intermediate intensity level, as Texas Red DHPE lipids located in the outer leaflet of the vesicle diffuse into the pore-spanning membrane. Simultaneously, Oregon Green DHPE lipids diffuse into the outer leaflet of the vesicle and move away from the porous surface into the third dimension resulting in an increase in OG fluorescence intensity. The magnitude of the observed OG fluorescence increase depends on the position of the fusing vesicle. Fig. 4 shows two single fusion events at two different positions of the pore-spanning membrane. If the vesicle fuses in the center of the pore-spanning membrane (Fig. 4A) only a small increase in fluorescence intensity is observed as a result of the three dimensional vesicle structure atop the pore-spanning membrane at the hemifusion state. From geometric considerations taking the average membrane area of the outer membrane leaflet of the vesicle (~0.3 μm2) and the area of both leaflets of the pore-spanning membrane in the ROI (~1.5 μm2) into account, an OG intensity increase of about 20% is expected, which agrees well with the observed increase in OG fluorescence (Fig. 4A). However, if the vesicle fuses at the pore rim, where the OG fluorescence of the planar membrane is quenched owing to the close contact with the underlying gold surface31, the OG fluorescence gets significantly increased. This is a result of the Oregon Green DHPE lipids diffusing into the outer leaflet of the vesicle with a dimension of several hundred nanometers. As the OG lipids move significantly away from the gold covered surface, fluorescence quenching, which occurs at distances within about 15 nm from the gold surface, becomes almost zero31. Instead de-quenching and surface-enhanced fluorescence is observed, which is found at tens of nanometers beyond the range of quenching41 resulting in an increase in OG fluorescence significantly larger than the calculated 20% (Fig. 4B).


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) Time lapse series of fluorescence micrographs showing a single fusion event of a large unilamellar vesicle containing full length synaptobrevin 2 with a pore-spanning membrane with reconstituted ΔN-acceptor complex. (top) Oregon Green DHPE fluorescence of the pore-spanning membranes; (bottom) Texas Red DHPE fluorescence of the vesicle. The yellow circles in (a) shows the region of interest (ROI) that was used for reading out the time-resolved changes in the relative fluorescence intensities depicted in (C); the white circles in the images highlight the region, where the vesicle docks and fuses and only serves as a guide to the eye. Scale bar: 5 μm. (B) Schematic drawing of the postulated fusion states at the time of image recording. Membranes are colored according to their incorporated fluorescent dye. (C) Time resolved changes in the relative fluorescence intensity of Oregon Green DHPE (green) and Texas Red DHPE (red) during the shown fusion event. Dashed blue lines serve as a guide to the eye highlighting the distinct levels of intensity. Letters (a–f) correspond to the fluorescence images in (A). For further details see text and Supplementary Fig. 7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: (A) Time lapse series of fluorescence micrographs showing a single fusion event of a large unilamellar vesicle containing full length synaptobrevin 2 with a pore-spanning membrane with reconstituted ΔN-acceptor complex. (top) Oregon Green DHPE fluorescence of the pore-spanning membranes; (bottom) Texas Red DHPE fluorescence of the vesicle. The yellow circles in (a) shows the region of interest (ROI) that was used for reading out the time-resolved changes in the relative fluorescence intensities depicted in (C); the white circles in the images highlight the region, where the vesicle docks and fuses and only serves as a guide to the eye. Scale bar: 5 μm. (B) Schematic drawing of the postulated fusion states at the time of image recording. Membranes are colored according to their incorporated fluorescent dye. (C) Time resolved changes in the relative fluorescence intensity of Oregon Green DHPE (green) and Texas Red DHPE (red) during the shown fusion event. Dashed blue lines serve as a guide to the eye highlighting the distinct levels of intensity. Letters (a–f) correspond to the fluorescence images in (A). For further details see text and Supplementary Fig. 7.
Mentions: Pore-spanning membrane patches of various sizes ranging from 10–150 μm in diameter were obtained by GUV spreading as visualized by the Oregon Green DHPE fluorescence (Fig. 3A,a). For the detection of single vesicle fusion events, membrane patches with a size larger than 40 × 40 μm2 were selected on the porous surface. After addition of full length synaptobrevin 2 containing LUVs doped with Texas Red DHPE, time series for both lipid dyes were recorded simultaneously (Supplementary Movie 1). A characteristic time series of movie frames of a single vesicle fusion event that basically captures all observables in these experiments is depicted in Fig. 3. The time dependent fluorescence intensity changes of Oregon Green DHPE and Texas Red DHPE upon docking and fusion of a single vesicle to the planar membrane was monitored by defining a region of interest (ROI, yellow colored circle in Fig. 3A,a) around the center of mass of the docked vesicle and integrating the fluorescence intensity as a function of time. The obtained time traces of the two fluorophores including all characteristic features of a single vesicle fusion event are depicted in Fig. 3C. Prior to vesicle docking a low Texas Red DHPE (TR) and a high Oregon Green DHPE (OG) fluorescence is observed (Fig. 3C,a) When a single vesicle approaches the surface and docks, a sudden increase in TR fluorescence occurs, while the OG fluorescence remains unaffected (Fig. 3C,b). Upon onset of fusion (Fig. 3C,c) the TR intensity shows a transient increase, while concomitantly a small transient decrease in the OG fluorescence intensity is observed, which is a result of the Förster resonance energy transfer (FRET) between the TR and OG dye as the lipids of the outer leaflet of the vesicle (Texas Red DHPE) start mixing with the lipids of the outer leaflet of the pore-spanning membrane (Oregon Green DHPE) (Fig. 3C,c). After this first transient changes of TR and OG fluorescence, the TR fluorescence intensity drops to and then remains at an intensity that is well above baseline level but lower than that of the originally docked vesicle. Simultaneously, an increase in OG fluorescence intensity is observed. These fluorescence time traces can be explained with the formation of an intermediate hemifused state (Fig. 3C,d). In the hemifused state, the outer leaflets merge, while the inner leaflet of the TR-doped vesicle remains unaffected. Thus, after a first merger of the outer leaflets, the TR fluorescence intensity drops to an intermediate intensity level, as Texas Red DHPE lipids located in the outer leaflet of the vesicle diffuse into the pore-spanning membrane. Simultaneously, Oregon Green DHPE lipids diffuse into the outer leaflet of the vesicle and move away from the porous surface into the third dimension resulting in an increase in OG fluorescence intensity. The magnitude of the observed OG fluorescence increase depends on the position of the fusing vesicle. Fig. 4 shows two single fusion events at two different positions of the pore-spanning membrane. If the vesicle fuses in the center of the pore-spanning membrane (Fig. 4A) only a small increase in fluorescence intensity is observed as a result of the three dimensional vesicle structure atop the pore-spanning membrane at the hemifusion state. From geometric considerations taking the average membrane area of the outer membrane leaflet of the vesicle (~0.3 μm2) and the area of both leaflets of the pore-spanning membrane in the ROI (~1.5 μm2) into account, an OG intensity increase of about 20% is expected, which agrees well with the observed increase in OG fluorescence (Fig. 4A). However, if the vesicle fuses at the pore rim, where the OG fluorescence of the planar membrane is quenched owing to the close contact with the underlying gold surface31, the OG fluorescence gets significantly increased. This is a result of the Oregon Green DHPE lipids diffusing into the outer leaflet of the vesicle with a dimension of several hundred nanometers. As the OG lipids move significantly away from the gold covered surface, fluorescence quenching, which occurs at distances within about 15 nm from the gold surface, becomes almost zero31. Instead de-quenching and surface-enhanced fluorescence is observed, which is found at tens of nanometers beyond the range of quenching41 resulting in an increase in OG fluorescence significantly larger than the calculated 20% (Fig. 4B).

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