High-Speed Single-Particle Tracking of GM1 in Model Membranes Reveals Anomalous Diffusion due to Interleaflet Coupling and Molecular Pinning

Abstract

The biological functions of the cell membrane are influenced by the mobility of its constituents, which are thought to be strongly affected by nanoscale structure and organization. Interactions with the actin cytoskeleton have been proposed as a potential mechanism with the control of mobility imparted through transmembrane “pickets” or GPI-anchored lipid nanodomains. This hypothesis is based on observations of molecular mobility using various methods, although many of these lack the spatiotemporal resolution required to fully capture all the details of the interaction dynamics. In addition, the validity of certain experimental approaches, particularly single-particle tracking, has been questioned due to a number of potential experimental artifacts. Here, we use interferometric scattering microscopy to track molecules labeled with 20–40 nm scattering gold beads with simultaneous <2 nm spatial and 20 μs temporal precision to investigate the existence and mechanistic origin of anomalous diffusion in bilayer membranes. We use supported lipid bilayers as a model system and demonstrate that the label does not influence time-dependent diffusion in the small particle limit (≤40 nm). By tracking the motion of the ganglioside lipid GM1 bound to the cholera toxin B subunit for different substrates and lipid tail properties, we show that molecular pinning and interleaflet coupling between lipid tail domains on a nanoscopic scale suffice to induce transient immobilization and thereby anomalous subdiffusion on the millisecond time scale.

Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation

“…A link between GM1, pinning and interdigitation has recently been found in both simulations and model membrane studies (Spillane et al, 2014; Sun et al, 2015). It seems likely that the lo-domain formation observed in the plasma membrane after cross-linking outer leaflet components, described above, causes the pinned molecules to stretch and hence make contact with the inner leaflet lipids, both nucleating and stabilizing the domains.…”

Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterol nanoclusters

“…Moreover, a recent study of the SPM dynamic in the cellular PM revealed that instead of clearly partitioning into nanodomains, SPM was mainly transiently trapped51. In the case of the ganglioside GM1, another lipid described as partitioning into L o phases, its transient trapping was shown to depend on molecular pinning and interleaflet coupling between lipid tail domains52. This suggest that Gag could not only generates his own lipid nano-domains at the inner leaflet of the cellular PM but also induce the formation of domains in the outer leaflet of the cellular PM by transiently trapping SPM or other components.…”

Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

“…Diffusion of single nanoparticles on supported bilayer membranes has been studied by high-speed iSCAT microscopy before2223. In the previous work, anomalous diffusion, multimobility and also nano-confinements of the particles were observed due to the multiple lipid binding sites on the particle, clustering of the lipids, and the substrate effects, which complicate the investigation of single-molecule dynamics in the membrane.…”