STED Nanoscopy Reveals the Ubiquity of Subcortical Cytoskeleton Periodicity in Living Neurons

In the axons of cultured hippocampal neurons, actin forms various structures, including bundles, patches (involved in the preservation of neuronal polarity), and a recently reported periodic ring-like structure. Nevertheless, the overlaying organization of actin in neurons and in the axon initial segment (AIS) is still unclear, due mainly to a lack of adequate imaging methods. By harnessing live-cell stimulated emission depletion (STED) nanoscopy and the fluorescent probe SiR-Actin, we show that the periodic subcortical actin structure is in fact present in both axons and dendrites. The periodic cytoskeleton organization is also found in the peripheral nervous system, specifically at the nodes of Ranvier. The actin patches in the AIS co-localize with pre-synaptic markers. Cytosolic actin organization strongly depends on the developmental stage and subcellular localization. Altogether, the results of this study reveal unique neuronal cytoskeletal features. [Display omitted] •Dendrites exhibit periodic actin organization•Cytoskeletal proteins show the same periodicity at nodes of Ranvier•Cytosolic actin organization is developmentally and spatially regulated•Actin patches in the axon initial segment co-localize with synaptic markers Harnessing STED nanoscopy, D’Este et al. reveal novel patterns of actin organization at the nanoscale in living hippocampal neurons.