Nanoscopic compartmentalization of membrane protein motion at the axon initial segment

The axon initial segment (AIS) is enriched in specific adaptor, cytoskeletal and transmembrane molecules. During AIS establishment, a membrane diffusion barrier is formed between the axon and the somatodendritic domain. Recently, an axonal periodic pattern of actin, spectrin and ankyrin forming 190 nm distanced, ring-like structures has been discovered. However, whether this structure is related to the diffusion barrier function is unclear.Here, we performed single particle tracking timecourse experiments on hippocampal neurons during AIS development. We analyzed the mobility of lipid-anchored molecules by high-speed single particle tracking and correlated positions of membrane molecules with the nanoscopic organization of the AIS cytoskeleton.We observe a strong reduction in mobility early in AIS development. Membrane protein motion in the AIS plasma membrane is confined to a repetitive pattern of ~190 nm spaced segments along the AIS axis as early as DIV4 and this pattern alternates with actin rings. Our data provide a new model for the mechanism of the AIS diffusion barrier.