Single-lipid dynamics in phase-separated supported lipid bilayers

Abstract

Phase separation is a fundamental organizing mechanism on cellular membranes. Lipid phases have complex dependencies on the membrane composition, curvature, tension, and temperature. Single-molecule diffusion measures a key characteristic of membrane behavior and relates to the effective membrane viscosity. Lipid diffusion rates vary by up to ten-fold between liquid-disordered (Ld) and liquid-ordered (Lo) phases depending on the membrane composition, measurement technique, and the surrounding environment. This manuscript reports the lipid diffusion on phase-separated supported lipid bilayers (SLBs) with varying temperature, composition, and lipid phase. Lipid diffusion is measured by single-particle tracking (SPT) and fluorescence correlation spectroscopy (FCS) via custom data acquisition and analysis protocols that apply to diverse membranes systems. We demonstrate agreement between FCS and SPT analyses with both the single-step length distribution and the mean squared displacement of lipids with significant immobile diffusers. Traditionally, SPT is sensitive to diffuser aggregation, whereas FCS largely excludes aggregates from the reported data. Protocols are reported for identifying and culling the aggregates prior to calculating diffusion rates via SPT. With aggregate culling, all diffusion measurement methods provide consistent results. With varying membrane composition and temperature, we demonstrate the importance of the tie-line length that separates the coexisting lipid phases in predicting the differences in diffusion between the Ld and Lo phases. HIGHLIGHTS• Lipid diffusion varies with the lipid phases, temperature, and aggregation • Aggregate culling yields consistent measurements from single-particle tracking and fluorescence correlation spectroscopy • Membrane with higher cholesterol content or at low temperature have more aggregates • A more variation in the diffusion rates occurred between the coexisting lipid phases at low temperatures and low cholesterol content

Nanoscale membrane curvature sorts lipid phases and alters lipid diffusion

“…All samples were equilibrated for >30 min prior to measurements of phase separation or lipid diffusion at a given temperature. The slow large-scale mixing of SLBs resulted in some of the largest domains (>10 μm diameter) remaining evident with blurred phase boundaries even when the sample temperature was greater than Tm, as described previously (24,48). The Tm for 2:2:1 SLBs was greater than 37 °C, as evident by the clear phase separation observed at this temperature.…”

“…DiPhyPC-containing SLBs yield aggregates of DPPE-Texas Red (24). Aggregates were apparent via a high fluorophore localization density in the absence of nanoparticles.…”

“…Comparisons between membrane curvature events and fluorophore aggregates were made via the size of the cluster of localizations, the local single-lipid diffusion rate, localization density, localization rate, and the proximity to the nanoparticles, as detailed in the Discussion. Aggregates were removed via localization density and long trajectory culling, as described previously (24). Briefly, all localizations that were not associated with membrane curvature were grouped based on their 2D location and acquisition time into 3D voxels.…”

“…The resulting diffusion coefficient through the XY-plane (Dxy) requires further correction to account for the 3D membrane shape. Monte Carlo simulations were performed for random walk on the surface that approximates the membrane bending over the 50-nm radius nanoparticle, as done previously (24,26,30,46). These simulations incorporated a varying lipid diffusion rate on the planar versus curved membrane and were analyzed to mimic the experimental conditions with an imposed localization uncertainty and image blur.…”

“…The single-lipid trajectories were observed within SLBs relative to the engineered curvature with a 50-nm radius. The application of Rayleigh distribution fitting rather than the conventional mean-square displacement analysis provided a precise determination of the diffusion coefficient across the nanoscale membrane curvature (24,26,46). The lipid trajectories were projected into the XY-plane and binned according to their distance from the curvature-generating nanoparticle (Fig.…”