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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

Statistical analysis of the fusion events on the pore rim attached membranes (A–C) and on the freestanding parts (D–F). (A,D) Number of vesicles that only dock, dock and fuse but stay in the hemifused state and vesicles that fully fuse with the membrane. (B,E) Histogram of the docking time tdock. The red lines are results of fitting eq. (1) to the data. (C,F) Histogram of the hemifusion time themi. The red line is the result of fitting eq. (2) to the data.
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f6: Statistical analysis of the fusion events on the pore rim attached membranes (A–C) and on the freestanding parts (D–F). (A,D) Number of vesicles that only dock, dock and fuse but stay in the hemifused state and vesicles that fully fuse with the membrane. (B,E) Histogram of the docking time tdock. The red lines are results of fitting eq. (1) to the data. (C,F) Histogram of the hemifusion time themi. The red line is the result of fitting eq. (2) to the data.

Mentions: For the statistical analysis, 2030 single vesicle events from 48 time series on individual membrane patches and 10 different reconstitutions were analyzed. We distinguished those events that were observed on the pore rims and those that were found on the freestanding parts of the pore-spanning membranes. 1492 docked vesicles (100%) were found on the pore rims and analyzed, from which 51% progressed to fusion. 62% of the fusing vesicles proceeded to full fusion, while the remaining 38% stayed in an intermediate hemifused state (Fig. 6A). Almost the same results were obtained for those events observed on the freestanding membrane parts. From 538 docked vesicles (100%) on the freestanding parts of the pore-spanning membranes, 47% progressed to fusion, while 53% remained in the docked state. 56% of the fusing vesicles proceeded to full fusion, while 44% remained in the hemifused state (Fig. 6D). From the vesicles that only docked, ⅓ detached during the observation window of 300 s, while ⅔ remained docked during the entire time period. These results indicate that even though there might be a difference in the diffusion behavior of the membrane components within the freestanding pore-spanning membranes and those on the pore rims, this does not considerably influence the ratios of the different observed fusion states.


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)

Statistical analysis of the fusion events on the pore rim attached membranes (A–C) and on the freestanding parts (D–F). (A,D) Number of vesicles that only dock, dock and fuse but stay in the hemifused state and vesicles that fully fuse with the membrane. (B,E) Histogram of the docking time tdock. The red lines are results of fitting eq. (1) to the data. (C,F) Histogram of the hemifusion time themi. The red line is the result of fitting eq. (2) to the data.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Statistical analysis of the fusion events on the pore rim attached membranes (A–C) and on the freestanding parts (D–F). (A,D) Number of vesicles that only dock, dock and fuse but stay in the hemifused state and vesicles that fully fuse with the membrane. (B,E) Histogram of the docking time tdock. The red lines are results of fitting eq. (1) to the data. (C,F) Histogram of the hemifusion time themi. The red line is the result of fitting eq. (2) to the data.
Mentions: For the statistical analysis, 2030 single vesicle events from 48 time series on individual membrane patches and 10 different reconstitutions were analyzed. We distinguished those events that were observed on the pore rims and those that were found on the freestanding parts of the pore-spanning membranes. 1492 docked vesicles (100%) were found on the pore rims and analyzed, from which 51% progressed to fusion. 62% of the fusing vesicles proceeded to full fusion, while the remaining 38% stayed in an intermediate hemifused state (Fig. 6A). Almost the same results were obtained for those events observed on the freestanding membrane parts. From 538 docked vesicles (100%) on the freestanding parts of the pore-spanning membranes, 47% progressed to fusion, while 53% remained in the docked state. 56% of the fusing vesicles proceeded to full fusion, while 44% remained in the hemifused state (Fig. 6D). From the vesicles that only docked, ⅓ detached during the observation window of 300 s, while ⅔ remained docked during the entire time period. These results indicate that even though there might be a difference in the diffusion behavior of the membrane components within the freestanding pore-spanning membranes and those on the pore rims, this does not considerably influence the ratios of the different observed fusion states.

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