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.