Crowding-driven condensation and membrane reorganization

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 (SLBs), forming immobile regions that constrain lipid diffusion. Notably, the condensation threshold varies inversely with poly(ethylene glycol) chain length and surface density, defining a quantitative relationship between molecular size and crowding strength. To directly visualize these crowding-induced structures, we employed a nanodisc-mediated lipid delivery ("nanodelivery") approach, revealing a clear correspondence between condensed crowder regions and diffusion barriers reminiscent of the picket-fence model of live-cell membranes. Similar behavior in protein-crowded SLBs demonstrates the generality of this crowding-driven mechanism. Using minimal two-layer and three-layer reconstituted architectures, we further demonstrate that surface density and macromolecular crowding above the membrane function as tunable control parameters that drive phase transitions within membrane-tethered macromolecular layers, independent of anchoring chemistry, while the underlying lipid bilayer remains laterally continuous. Extension of this framework to living cells reveals analogous suppression of membrane mobility under high surface-density conditions, indicating that this crowding-induced reorganization is not restricted to SLBs model systems. Together, these findings establish surface-density-driven phase transitions of membrane-anchored macromolecules as a general physical mechanism underlying lateral membrane inhomogeneity and position nanodelivery as a versatile approach for interrogating membrane organization across synthetic and biological systems.