Polarization is a decisive step in neuronal development and once axonal and somatodendritic domains are generated, they generally persist over the lifetime of an organism. The axonal initial segment (AIS), a stretch of 50 – 100 µm length close to the cell body, bears a specialized cytoskeleton and maintains the polarized distribution of neuronal molecules by blocking the exchange of molecules between axon and soma. The adaptor protein ankyrinG is essential for the assembly of this diffusion barrier and crosslinks ion channels and adhesion molecules to the cytoskeleton. However, the molecular mechanism for the obstruction of molecular diffusion across the AIS remains unclear. Recent insights revealed an ordered arrangement of the cytoskeleton not only in the AIS but also further down the axon, raising the question, how the nanoscopic organization of cytoskeletal molecules in the AIS may directly influence the lateral motion of membrane molecules. Here we aim to use novel single-molecule localization-based superresolution microscopy in primary hippocampal neurons to investigate the molecular architecture of the AIS and to understand the mechanism by which membrane protein diffusion is locally restricted. To do so, we will correlate membrane protein diffusion during neuronal polarization in single cells over time with the nanoscopic organization of the AIS. We hypothesize that a regularly spaced structure based on AnkyrinG specifically reduces membrane mobility in the AIS. Our results will clarify the molecular mechanism that controls the diffusion barrier at the AIS and contribute to our understanding of how neuronal polarity is achieved and more generally, how the membrane cytoskeleton organizes the plasma membrane.