Supported Bilayers with Excess Membrane Reservoir (SUPER): Novel Templates for Vesicular and Non-Vesicular Transport Studies

Abstract

Our understanding of membrane-localized processes has largely been gained from the use of liposome-based systems. However reactions involving budding and fission of membranes are difficult to analyze using liposome-based systems since their buoyancy imposes a fundamental limitation on separating end-products of such reactions. Supported bilayers formed by liposome fusion on glass represent an attractive solution. Conventional supported bilayers are however deficient in membrane reservoir necessary for membrane budding and fission reactions. We report a novel system of supported bilayers with excess membrane reservoir (SUPER) and have analyzed factors that contribute to their formation. The excess reservoir in this system originates from higher binding affinity of liposomes to glass and depends on the presence of anionic lipids in the membrane and high salt content in the buffer. This template formed on silica beads allows the seamless application of microscopy-based assays to analyze membrane-localized processes as well as sedimentation-based assays to isolate vesicular and non-vesicular products released from the membrane. We demonstrate the utility of SUPER templates by the direct visualization of amphiphile-induced membrane tubulation prior to solubilization and dynamin-catalyzed membrane fission prior to vesiculation. Our results highlight the general applicability of SUPER templates in analyzing several forms of vesicular and non-vesicular transport processes.