Surface-expressed DHHC2 is essential for generation of postsynaptic nanodomains. (A) DHHC2 constructs used. EKKNR, an ER-retention signal; SEP, super-ecliptic pHluorin. Blue boxes, DHHC catalytic domain. extra, extracellular space; cyto, cytoplasm; lum, lumen. (B) Surface-expressed and total DHHC-HA are shown in green and red, respectively. Bars: (top) 10 µm; (bottom) 2 µm. (C) Endogenous DHHC2 (green) exists as small discrete clusters (arrows) at the center of hPF11-stained postsynaptic regions (red) in a spine. Bar, 1 µm. (D) Endosomal (arrowheads) and synaptic membrane localization (arrows) of DHHC2. GFP-DHHC2 or DHHC2-SEP was coexpressed with TfR-mCherry or PSD-95-mCherry. Bar, 5 µm. (E and F) Hippocampal neurons were transfected with PF11-GFP-miR-DHHC2 (for co-cistronic expression) and miRNA-resistant DHHC2 (resDHHC2-WT, CS, or ER), and live-imaged using STED microscopy. PF11-GFP–labeled nanodomains (gray boxes) and multi-nanodomain synapses (E, arrowheads; white boxes) were counted (F). In total, 35 (WT), 4 (CS), and 11 (ER) dendrites from two or three independent experiments were analyzed. Bar: (E) 1 µm. (G and H) miRNA against DHHC2 (marked by EmGFP) was expressed with resDHHC2 WT or –ER. Neurons were stained using a conventional PSD-95 antibody (red) and imaged by confocal microscopy. miLacZ indicates a control miRNA. Arrows denote PSD-95-negative dendritic spines/filopodia (G). Approximately 50 dendrites from 2 independent experiments were analyzed (H). resDH2, resDHHC2. ***, P < 0.001, by one-way ANOVA with post-hoc Tukey’s test (F and H). Bar: (G) 5 µm.