Nanoscopic lipid diffusion in the live cell plasma membrane revealed by ultrafast single-molecule tracking.

Abstract

Membrane organization and dynamics underlie various molecular interactions required for membrane functions. Although the importance of investigating biological membranes at the nanoscale is well recognized, resolving nanoscopic structures and dynamics is technically challenging. In this work, we employ ultrafast single-particle tracking techniques to measure single phospholipid diffusion in the plasma membrane of live cells. We attach a monovalent gold nanoparticle to the headgroup of biotinylated phospholipid and record its diffusive motion on microsecond resolution and nanometer precision by advanced optical interference microscopy. We find that both saturated and unsaturated phospholipids (biotin-PEG-DSPE and biotin-PEG-DOPE, respectively) undergo anomalous subdiffusion in the length scale below 100 nm. The unsaturated lipid diffuses only slightly more freely than the saturated lipid. Moreover, the nanoscopic lipid diffusion is highly heterogeneous in space and time. With statistical analyses, we find that the apparent dual-mobility subdiffusion can describe measured diffusion. The subdiffusion agrees well with the hop diffusion model that describes a Brownian diffuser moving in periodic and partially permeable barriers created by the cytoskeleton. Inhibition of actin polymerization by the chemical drug CK-666 reduces confinement's effective size and strength. Furthermore, we examine the effect of cholesterol on lipid subdiffusion by manipulating the cholesterol concentration. Cholesterol depletion by methyl-b-cyclodextrin leads to a more restricted lipid diffusion. We attribute the more confined diffusion to the formation of solid-like gel-phase nanodomains that move slowly and act as diffusion obstacles. Lowing the system temperature shows similar effects on lipid diffusion, suggesting that the cholesterol-dependent lipid diffusion connects to the nanoscopic phase separation. Overall, this study provides experimental evidence that lipid diffusion in the cell membrane is heterogeneous at the nanoscale and regulated by the membrane cholesterol and actin cytoskeleton.