A complete understanding of the molecular mechanisms governing vesicle formation requires quantitative assays and vesicle reconstitution using purified components. We describe a simple model membrane template for studying protein-mediated membrane remodeling and vesicle formation or fission that is amenable to both quantitative biochemical analysis and real-time imaging by epifluorescence microscopy. Supported bilayers with excess membrane reservoir (SUPER) templates are compositionally well-defined unilamellar membrane systems prepared on 2–5-μm silica beads under conditions that enable incorporation of excess membrane to form a loosely fitting bilayer that can be used to study membrane remodeling and fission. This protocol describes methods for SUPER template formation and characterization, as well as for the qualitative observation and quantitative measurement of vesicle formation and fission via microscopy and a simple sedimentation assay. SUPER templates can be prepared within 60 min. Results from either sedimentation-based or microscopy-based assays can be obtained within an additional 60 min.
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This work was funded by the US National Institutes of Health grants R01-GM42455 and R01-MH61345 to S.L.S. and fellowships from the Leukemia and Lymphoma Society to T.J.P. and from the German Research Foundation (DFG) and the American Heart Association to S.N. We thank Y.-W. Liu for her help in filming the procedures and M. Dadachov at Corpuscular for helpful discussions regarding silica bead preparation.
The authors declare no competing financial interests.
Membrane spill. For quality control of the SUPER templates, they can be transferred into a droplet of buffer on a clean, uncoated coverslip. As the templates come into contact with the glass surface, the excess membrane will expand away from the bead forming a supported bilayer that surrounds it. (MOV 462 kb)
Addition of SUPER templates to the reaction mixture for the sedimentation assay. Demonstration on how SUPER templates should be pipetted into the reaction mixture to avoid release of vesicles by shearing of the reservoir during the sedimentation assay. (MOV 1791 kb)
Real-time imaging of fission. Due to their large size SUPER templates are amenable to the real-time monitoring of protein-mediated membrane remodeling by fluorescence microscopy. SUPER templates were dispersed and allowed to settle in assay buffer containing an oxygen-scavenging system and 1 mM GTP. After imaging was started, dynamin-1 was added to the solution with a pipette to a final concentration of 0.5 μM. The movie is composed of frames captured via 100 ms exposures at 1-s intervals and is played at 7 frames/s speed. (MOV 2429 kb)
Preparation of membrane tethers. Demonstration of how the Lab-Tek chamber is tilted to achieve formation of membrane tethers. (MOV 4728 kb)
Fission of membrane tethers. SUPER templates were dispersed and allowed to settle in assay buffer containing an oxygen-scavenging system and 1 mM GTP. Plain silica beads (d = 20 μm) were added to the solution onto one side of the chamber and allowed to settle. Tethers were pulled by tilting the chamber and rolling the large beads over the layer of SUPER templates. After imaging was started, dynamin-1 was added to the solution with a pipette to a final concentration of 0.5 μM. The movie is composed frames captured via 100 ms exposures at 1-s intervals and is played at 1 frame/s speed. (MOV 233 kb)
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Neumann, S., Pucadyil, T. & Schmid, S. Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir. Nat Protoc 8, 213–222 (2013). https://doi.org/10.1038/nprot.2012.152
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