Highly stable planar asymmetric suspended membranes for investigating protein dynamics and membrane fusion.

Membrane fusion is central to cellular signaling and trafficking, requiring a detailed understanding of protein-lipid interactions. Studying these dynamic events in live cells presents challenges due to their complexity and heterogeneity. To address this, we developed a reductionist in vitro membrane model system that enables the controlled investigation of individual molecular components. This approach begins with a minimal membrane environment, with the opportunity for the stepwise addition of specific components to incrementally increase complexity achieving a level of experimental precision often unattainable in cellular studies. We developed suspended lipid membranes, a platform that uses pore-spanning lipid bilayers formed on microfabricated silicon chips with micrometer-sized holes. These membranes closely mimic native cellular architecture by maintaining aqueous compartments on both sides, providing a solvent-free, near-native environment with exceptional lateral diffusion properties. Their high stability makes them ideal for time-lapse imaging and dynamic process analysis using total internal reflection fluorescence and confocal microscopy. Here we present a detailed protocol for generating pore-spanning, planar suspended lipid membranes from native and synthetic reconstituted lipids using our silicon chip platform. Using SNARE proteins and molecular chaperones, we demonstrate the system's ability to capture ultrafast membrane fusion events. Additionally, we demonstrate single-molecule protein counting, protein dynamics analysis and single-vesicle fusion assays using fluorescently labeled proteins and vesicles. The ability to preserve native lipid asymmetry, biological composition and lateral diffusion makes this method a powerful tool for dissecting membrane fusion mechanisms and other membrane biological processes with unparalleled precision.