Biomolecules, such as proteins and nucleic acids, spontaneously condense into functional clusters in cells in response to external environments through phase separation. Recent studies have suggested that these clusters, called biomolecular condensates, participate in the assembly of pivotal structures at biological membrane surfaces, including immunological synapses, focal adhesions and endocytic vesicles. Two existing in vitro model membranes, supported lipid bilayers and giant vesicles, have been used to study the phase separation of biomolecules on the membrane. However, the presence of the support (often solid) in supported lipid bilayers may reduce the mobility of lipids and membrane-bound biomolecules, possibly affecting phase separation dynamics. Also, giant vesicles often require challenging time-resolved visualization due to their curved three-dimensional geometry. Moreover, biomolecules can access only one side of the membrane surface in these model membranes. Collectively, these restrictions have hampered extensive investigations of membrane-associated phase separation and the accompanying dynamic membrane reorganization, which frequently occurs on both sides of the membrane simultaneously.
To circumvent these technical limitations, we developed an array of free-standing planar lipid membranes based on the self-assembly of lipids at the oil–water interface. To this end, an electron microscopy grid with hexagonal holes was placed onto the lipid-containing oil layer and subsequently immersed into the aqueous buffer, forming two lipid monolayers at the oil–water interfaces. Then, spontaneous adhesion between two lipid monolayers occurred due to the oil removal between monolayers, resulting in free-standing lipid bilayers. We created dozens of membranes within the hexagonal holes of electron microscopy grids, each with a diameter of approximately 100 μm. Importantly, biomolecules could access both sides of the membrane surface by introducing thin spacers between the EM grid and the glass coverslip while being compatible with high-resolution fluorescence microscopy.
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