Nano-Substructures of raft-Mimetic Liquid-Ordered Membrane Domains Revealed by High-Speed Single-Particle Tracking

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Biological membrane domains of distinct molecular compositions and organizations facilitate many important membrane functions. Lipid rafts are specialized membrane domains of liquid-ordered (Lo) phase that are found to be essential for various membrane processes. Despite of recent advances in identifying the biological significance of rafts, the dynamic nature and regulation mechanism of rafts are largely unknown due to the lack of technology to resolve the transient molecular interaction with rafts at the nanoscale. In this work, we investigate how rafts recruit and trap molecules by monitoring lateral diffusion of single lipids in raft-containing reconstituted membranes at ultrahigh spatiotemporal resolution. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, the motion of single lipids in the membrane is recorded via single-particle tracking (SPT) with 3 nm spatial precision and 20 μs temporal resolution. The processes of single molecules partitioning into and escaping from the raft-mimetic Lo domains are directly visualized in a continuous manner with unprecedented clarity. Surprisingly, we observe anomalous subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, that transiently trap the saturated lipids. Our data provide the first experimental evidence of non-uniform molecular organization of the Lo phase and show its critical role in determining molecular dynamics in the Lo phase. Our results also imply that the raft-associated molecules could be trapped between nano-subdomains of densely packed saturated lipids, surrounded by high density of cholesterol. The nanoscopic membrane environment created by the nano-subdomains of rafts is thus unique, which illustrates the essentials of lipid rafts of Lo phase in cell membrane.