Accumulated positions of GPI-GFP molecules from SPT show a periodic pattern of ∼190 nm along the AIS that emerges as early as DIV3 during development. (A) Plot of SPT trajectories on the proximal axon of a DIV3 hippocampal neuron expressing GPI-GFP color-coded according to the instantaneous diffusion coefficient (D). Two areas are emphasized: one with a high (inset I) and one with a lower mean D (inset II). (B) Reconstructed image from all localizations acquired during SPT of QDs corresponding to the trajectories in A. (C) Trajectories color-coded for D on the proximal axon of a DIV 14 neuron. In a neurofascin-positive area (inset III), the lateral mobility is reduced compared with the distal axon that loops back to the neuron (arrow). The periodic pattern was observed directly on the AIS in DIV14 neurons. (D) Reconstructed image of all localizations from the SPT experiment in C. (E) Autocorrelations of localizations along the axon (dashed lines) reveal a periodicity of ∼190 nm emerging in areas of low D trajectories in insets II and III, but not in the area of high D in inset I. (F and G) A periodic pattern was observed along the entire proximal axon where the AIS is located but was usually more prominent in some regions (dashed line lines in IV and V). (H) Autocorrelations along segments IV and V show periodically arranged stripes of localizations. Bars, 1 µm.