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Literature DB >> 23288321

Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir.

Sylvia Neumann¹, Thomas J Pucadyil, Sandra L Schmid.

Abstract

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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Year: 2013 PMID： 23288321 PMCID： PMC4753780 DOI： 10.1038/nprot.2012.152

Source DB: PubMed Journal: Nat Protoc ISSN： 1750-2799 Impact factor: 13.491

28 in total

Analyzing membrane remodeling and fission using supported bilayers with excess membrane reservoir.

Review 1. Regulated portals of entry into the cell.

2. Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin.

3. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission.

4. Clathrin-coated vesicle formation from isolated plasma membranes.

5. Robust colorimetric assays for dynamin's basal and stimulated GTPase activities.

6. Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes.

7. Dynamin undergoes a GTP-dependent conformational change causing vesiculation.

8. SNAREpins: minimal machinery for membrane fusion.

9. Curvature of clathrin-coated pits driven by epsin.

10. Stage-specific assays for coated pit formation and coated vesicle budding in vitro.

1. Identification and function of conformational dynamics in the multidomain GTPase dynamin.

2. Membrane fission by protein crowding.

3. Topography design in model membranes: Where biology meets physics.

Review 4. A novel fluorescence microscopic approach to quantitatively analyse protein-induced membrane remodelling.

5. A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission.

6. In Vitro Reconstitution Platforms of Mammalian Cell-Free Expressed Membrane Proteins.

7. Dual role of BAR domain-containing proteins in regulating vesicle release catalyzed by the GTPase, dynamin-2.

8. A simple supported tubulated bilayer system for evaluating protein-mediated membrane remodeling.

9. Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions.

10. A high-throughput platform for real-time analysis of membrane fission reactions reveals dynamin function.