Actin rings form plasma membrane diffusion barriers.

Abstract

In a fluid system like the plasma membrane, molecules will not enrich spontaneously. Since a high local concentration of specific molecules is vital for many membrane protein functions, a domain enriched in these molecules must be formed. This can be achieved by the generation of a physical diffusion barrier to the lateral movement of membrane components. One such barrier exists in the axon initial segment (AIS) of neurons, but the mechanism of the barrier so far remained elusive. Two candidates have emerged to be the cause of the membrane compartmentalization in the AIS: periodic actin rings (fence model) or large immobile transmembrane voltage-gated sodium channels (picket model). Here, we show that the diffusion barrier in the AIS is caused by the actin cytoskeleton. To do so, we combined in silico experiments with high-speed, high-density single-particle tracking and superresolution microscopy. In silico single-particle tracking (SPT) experiments clearly showed that the compartmentalization is compatible with the fence model but incompatible with the picket model. Furthermore, we show via SPT of membrane proteins in progenitor-derived neuronal cells that the diffusion barrier is also present in cells that do not exhibit an ion-channel enriched AIS, but do show actin rings along their processes. Further experiments using dual-colour live-cell stimulated emission depletion microscopy (STED) showed that periodic actin rings in cells are near (20 nm) the plasma membrane. Lastly, we show via correlative SPT/STORM that membrane molecules accumulate between actin rings. Taken together, we present here evidence for the compartmentalization of the plasma membrane by the actin cytoskeleton. Membrane proximal actin structures are a common feature of many cell types. Therefore, we speculate that the observed membrane compartmentalization is not specific to the neuronal cell lineage but rather a general mechanism of large-scale membrane organization.