Single-Lipid Diffusion Behaviors in Cell Membranes Modulated by Cholesterol-Based Heterogeneity

Over a century after the proposal of Fluid Mosaic Model, the relationship between functionally related multiple-scale spatial heterogeneity of the cell membrane and mobility of component molecules, both inherent features of cell membrane, remains elusive. Single-lipid tracking enables the analysis of structural heterogeneity at different spatial scales within the cell membrane from a lipid diffusion perspective. Herein, specifically designed cholesterol (Chol)-based membrane systems were utilized to investigate the distinct impacts of molecular-level interactions between diverse membrane components and micrometer-scale spatial confinement on lipid diffusion. The results demonstrate that the incorporation of Chol into 1,2-dioleoyl-<i>sn-glycero</i>-3-phosphocholine (DOPC) membranes decelerates lipid diffusion, with a positive correlation observed between the degree of deceleration and the mole ratio of Chol molecules. Across all these systems, lipid diffusion consistently adheres to the continuous time random walk (CTRW) model, indicating lipid entrapment resulting from specific molecular interactions. Conversely, micrometer-scale spatial confinement induced by phase separation not only reduces the diffusion rate of DOPC molecules but also triggers a transition from CTRW to fractional Brownian motion (fBM) or random walk on a fractal (RWF) mode within a confinement width range of 6.3-5.4 μm, suggesting a crowded microenvironment. In living cell membranes, this transformation in lipid diffusion is observed following Chol depletion, implying that lipid raft disruption leads to increased crowding within the lipid microenvironment. This study enhances our understanding of the relationship between lipid diffusion and membrane microenvironment across different spatial scales while providing insights into characterizing spatially heterogeneous structures within cell membranes from the perspective of lipid diffusion.