Nanodisc-Mediated Visualization of Crowding-Induced Condensation and Membrane Reorganization in Two-Dimensional Membrane Environments

Although biological membranes exhibit complex and dynamic organization, the mechanistic role of molecular crowding in governing lateral membrane heterogeneity remains poorly characterized experimentally. Here, we provide direct experimental visualization showing that crowding-induced condensation of membrane-anchored macromolecules above the bilayer interface reorganizes membrane dynamics and gives rise to spatially heterogeneous lipid mobility. Both PEGylated lipids and membrane-anchored proteins undergo surface-density-driven condensation on supported lipid bilayers, forming immobile regions that constrain lipid diffusion. Notably, the condensation threshold varies inversely with PEG chain length and surface density, defining a quantitative relationship between molecular size and crowding strength. To directly visualize these crowding-induced structures, we employed nanodelivery using lipid-loaded nanodiscs, revealing a clear correspondence between condensed crowder regions and diffusion barriers reminiscent of the picket-fence model of live-cell membranes. Similar condensation behavior observed for protein-crowded SLBs demonstrates the generality of this crowding-driven mechanism. Together, these findings establish surface-density-driven crowding and condensation of membrane-anchored macromolecules as a key physical mechanism underlying lateral membrane inhomogeneity and position nanodelivery as a general approach for interrogating membrane organization across synthetic and biological systems.