The periodic confinement areas overlap with βII-spectrin immunostaining and are excluded from actin rings. (A) Proximal axon of a DIV5 hippocampal neuron with trajectories from SPT of GPI-GFP with QDs overlaid with a superresolution micrograph of βII-spectrin. (B) Reconstructed image of all localizations from SPT in the region of interest (white box, A) shows a regular pattern along the axis of the axon. (C) Superresolution micrograph of βII-spectrin confirms periodicity of the submembrane cytoskeleton in the region of interest. (D) Overlay of trajectories (magenta) and βII-spectrin (white). (E) Overlay of localizations from SPT (magenta) and cytoskeletal marker βII-spectrin (cyan). Regions where both overlap are black. (F) Auto- and cross-correlation along the axon (dashed line, D) confirms that both SPT localizations and βII-spectrin are arranged periodically at ∼190 nm. The cross-correlation places clusters of localizations from SPT on top of βII-spectrin with an offset of ∼25 nm. (G) Proximal axon of a DIV6 hippocampal neuron with trajectories from SPT of GPI-GFP with QDs overlaid with a superresolution micrograph of actin. (H) Reconstructed image of all localizations from SPT in region of interest (white box, G) shows a regular pattern along the axis of the axon. (I) Superresolution micrograph of actin confirms periodicity of the submembrane cytoskeleton in the region of interest. (J) Overlay of trajectories (magenta) and cytoskeletal marker actin (white). (K) Overlay of localizations from SPT (magenta) and cytoskeletal marker actin (cyan). Regions where both overlap are black. (L) Auto- and cross-correlation along the axon (J, dashed line) confirms that both SPT localizations and actin are arranged periodically at ∼190-nm spacing. The cross-correlation places clusters of localizations from SPT in between the actin rings with an offset of ∼20 nm. Bars, 500 nm.