Simulations of SPT experiments reveal that actin rings act as a diffusion barrier. (a) Schematic representation of the cytoskeleton in neurons: the axon initial segment contains periodically organized actin rings (∼200 nm), which are interspersed by accumulations of channel proteins. (b) GPI-GFP forms membrane domains in DIV7 rat hippocampal neurons. Reconstruction of localizations of SPT experiment of GPI-GFP tagged with QDs (5,000 frames, 200 Hz). (c) Projection along the dotted line in (b). Scale bar is 100 nm. (d) Zoom in of box shown in (b). (e) Autocorrelation along the neuronal process shown in (c) reveals periodicity of GPI-GFP domains at ∼200 nm. (f) Schematic representation of the two competing hypotheses: Actin acts as a diffusion barrier or channel proteins act as a diffusion barrier. (g) Simulations of SPT experiments in Fluosim. Top: Model geometry mimicking the organization of actin rings. Middle top: Reconstruction of localizations of in silico SPT experiments over the geometry. Middle: In silico STORM reconstruction of actin rings. Middle bottom: Merge of SPT reconstruction and STORM of actin. Bottom: Crosscorrelation function (XCF) and individual autocorrelation functions (ACF) of SPT of GPI-GFP and STORM of actin reveals that GPI-GFP forms domains located between actin rings. (h) Same as (g) but with few channel proteins as a model geometry: No periodic pattern of GPI-GFP can be observed. (i) Same as (g) but with many channel proteins as model geometry: Periodic patterns of GPI-GFP can be observed but are localized on top of actin rings (in contrast to experimental data). If not otherwise indicated, scale bars are 500 nm.