Fig. 4.

Cholesterol depletion impairs DAT nanodomain clustering in extensions and varicosities of dopaminergic neurons. a, c, e, g, i, k Representative STORM images showing DAT distribution in extensions and varicosities of dopaminergic neurons under control conditions (a, c), after cholesterol removal with 5 mM mβCD (e, g), or after actin depolymerization with 5 μg ml⁻¹ CytD (i, k). DAT was visualized by immunolabeling with primary DAT antibody and Alexa405-Alexa647-conjugated secondary antibody. b, d, f, h, j, l DBSCAN-based cluster maps of the DAT signal in a, c, e, g, i, k. Clustered localizations are shown by color-coding with non-clustered localizations in gray. m, n DBSCAN quantifications DAT nanodomain localization in dopaminergic neurons after cholesterol removal or actin depolymerization, showing m clustering in varicosities and n clustering in extensions. Data are fraction of localizations in clusters in % (means ± s.e.m., ***p < 0.001, one-way ANOVA and Bonferroni’s post-test). o, p Probability distribution of the cluster sizes in varicosities (o) and extensions (p) at control conditions (black) or after treatment with mβCD (red) or CytD (blue). Data are based on from 9 to 39 images from three individual experiments. Red arrows mark DAT clustered in nanodomains. Scale bars 500 nm