Fig. 3

Endogenous DAT is clustered in nanodomains in the extensions and varicosities of cultured dopaminergic neurons. a–c, f–h Cross-section views through dopaminergic cell body showing the DAT distribution in the soma in an overall view, the soma edge, and in intracellular vesicular structures. DAT was visualized by immunolabeling with primary DAT antibody and Alexa405-Alexa647-conjugated secondary antibody. a, f Widefield image and corresponding reconstructed STORM image in the cell body. b, g Widefield image and corresponding STORM image at the soma edge; c, h Enlarged widefield or STORM image corresponding to the boxed regions in a, f, visualizing DAT in a putative endocytic vesicle. Yellow arrows highlight the circular distribution of the DAT signal. d, i Widefield image and reconstructed STORM image in a representative extension with varicosities. e, j Enlarged widefield image and corresponding STORM image of extension and varicosity. k Cross sectional profile along the boxed region in j showing the signal intensity as a function of the distance (µm). l, m STORM image and corresponding DBSCAN-based cluster map showing the degree of clustering of DAT in a representative varicosity. n, o STORM image and corresponding DBSCAN-based cluster map showing the degree of clustering of DAT in a representative extension. Clustered localizations are shown by color-coding with non-clustered localizations in gray. p DBSCAN-based quantification of the clustering of endogenous DAT in dopaminergic neurons in soma, varicosities and extensions. Data are % of localizations in cluster (means ± s.e.m., ***p < 0.001, one-way ANOVA and Bonferroni’s post-test). Data are from 16 to 32 images from six individual experiments. Red arrows mark DAT clustered in nanodomains. q Varicosity from image n, where the localizations are colored by the number of nearest neighbors within a 30 nm radius. r–t Example clusters of varied densities identified in image q, where the number of nearest neighbors within a 30 nm radius are identified by color and by position on the z axis. Scale bars a, f, l 5 μm, b, d, g, i 1 μm, c, h, m 200 nm, e, j, n–q, s 500 nm