High-speed single-particle tracking of phospholipids in the live cells plasma membrane uncovers dual-mobility and cholesterol-dependent anomalous subdiffusion at sub-millisecond.
Ching-Ya Cheng1*, Yu-Jo Chai1, Yi-Hung Liao1,2, Chih-Hsiang Lin1, Chia-Lung Hsieh1
1Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan
2Department of Physics, National Central University, Taoyuan, Taiwan
* Presenter:Ching-Ya Cheng, email:samu172004@gmail.com
Membrane compartmentalization and nanodomains are critical for the regulation of membrane functions. Cholesterol plays an important role in the formation of dynamic nanodomains in the liquid-ordered (Lo) phase, known as lipid rafts. Meanwhile, cytoskeleton meshwork and associated proteins act as diffusion barriers to the plasma membrane molecules. Although the biological significance of lipid rafts and cytoskeleton meshwork are well recognized, their governing mechanisms on the molecular diffusion at the nanoscale, i.e., the length scale in which the molecular interactions take place, remain elusive. To resolve the nanoscale membrane dynamics, in this work, we measured single-molecule diffusion by ultrahigh-speed single-particle tracking at 10,000 Hz. The synthetic biotinylated probe lipids were introduced into the plasma membrane of PtK2 cells through membrane fusion and then labeled by rhizavidin conjugated 30 nm AuNPs. Two probe lipids were investigated, the saturated lipid DSPE and the unsaturated lipid DOPE. Verified in the model membranes, the saturated lipid DSPE is prone to associate in the more ordered (e.g., Lo and gel) domains, while the unsaturated lipid DOPE prefers more disordered (liquid-disordered, Ld) domains. Anomalous subdiffusion was measured for both probe lipids on the PtK2 cell plasma membrane at the sub-millisecond timescale (0.1 – 1 ms) and the nanometer length scale (10 – 100 nm). The diffusion became free on the larger temporal and spatial scales. Dual mobility of nanoscopic lipid diffusion is discovered by statistical analysis. Using statistical analysis, we found the nanoscopic diffusion of the lipid exhibits dual mobilities, indicating the spatiotemporal heterogeneous motion.
The two mobilities are well presented by the model of hop diffusion which describes a free diffuser moving in the periodic semipermeable barriers created by the cytoskeleton meshwork. Through the analysis, we determined the confinement size and strength of the diffusion barrier. Consistent with our modeling, the confinement size was increased when the cortical actin was depolymerized by chemical drug treatment. Our data show that the two probe lipids experienced different levels of confinement; the diffusion of DSPE is more confined than DOPE. Depleting the membrane cholesterol by the chemical drug treatment enhanced the confinement of both lipids. The stronger confinement may originate from the formation of gel-like microdomains induced by cholesterol depletion. Our study shows that the membrane cholesterol modulates the interactions between the membrane nanodomains and cytoskeleton meshwork, which serves as a mechanism to regulate long-range molecular diffusion.

Keywords: plasma membrane nanodomain, cholesterol , ultrahigh-speed single-particle tracking, rhizavidin conjugated AuNPs, anomalous subdiffusion