Ultra‐Stable Freestanding Lipid Membrane Array: Direct Visualization of Dynamic Membrane Remodeling with Cholesterol Transport and Enzymatic Reactions

Abstract Cell membranes actively change their local compositions, serving essential biological processes such as cellular signaling and endocytosis. Although membrane dynamics is vital in the cellular functions, the complexity of natural membranes has made its fundamental understanding and systematic assessment difficult. Here, a powerful artificial membrane system is developed for real‐time visualization of the spatiotemporal dynamics of membrane remodeling. Through well‐defined air/oil/water interfaces on grid holes, tens of planar lipid bilayer membranes are easily created, and their reproducibility, controllability, and generality are highlighted. The freestanding membranes are large but also highly stable, facilitating direct long‐term monitoring of dynamic membrane reconstitution caused by external stimuli. As an example to demonstrate the superiority of this membrane system, the effect of cholesterol trafficking, which significantly affects biophysical properties of cell membranes, is investigated at different membrane compositions. Cholesterol transport into and out of the membranes at different rates causes anomalous lipid arrangements through cholesterol‐mediated phase transitions and decomposition, which have never been witnessed before. Furthermore, enzyme‐induced membrane dynamics is successfully shown in this platform; sphingomyelinases locally generate asymmetry between two membrane leaflets. This technique is broadly applicable for exploring the membrane heterogeneity under various membrane‐based reactions, providing valuable insight into the membrane dynamics.