Long-Lived Planar Asymmetric Suspended Membranes for Investigating Protein Dynamics and Membrane Fusion

Abstract Membrane fusion is pivotal in cellular signaling and trafficking, requiring an intricate understanding of the interactions between proteins and lipids. Addressing the limitations of studying complex protein dynamics in cellular environments, a reductionist in vitro membrane model system is essential. Such a method begins with minimal components and incrementally adds complexity, allowing for the isolation and detailed examination of individual molecular species, an effort unattainable with traditional cellular techniques. Suspended lipid membranes accurately mimic cellular architecture by providing a unique environment with aqueous compartments on both sides, exceptional lateral diffusion properties, a solvent-free environment, and excellent stability for time-lapse imaging. Here, we present a detailed protocol for generating pore-spanning based planar suspended lipid membranes from native and reconstituted membranes that are ideal for time-lapse imaging using total internal reflection and confocal microscopy techniques. Our approach enables precise single-molecule protein counting, comprehensive analysis of protein dynamics, and the conduct of single-vesicle fusion assays. This method stands out for its exceptional temporal resolution, essential for accurately monitoring vesicle docking and fusion kinetics. The natural biological composition and lipid asymmetry of these membranes, combined with their excellent diffusion capabilities, greatly enhance the ability of studying the dynamics of membrane proteins. This protocol marks a significant advancement in membrane fusion research and offers deep insights into the nuances of cellular signaling and mechanisms.