Palmitoylation of Gephyrin Controls Receptor Clustering and Plasticity of GABAergic Synapses
Figure 4
Morphological analysis of palmitoylation-deficient gephyrin variants in primary hippocampal neurons.
(a) Representative examples are illustrated for WT gephyrin-GFP and 212,284Geph. Scale bar, 20 µm. (a′) Insets demonstrate postsynaptic clustering of gephyrin by colocalization with VGAT (red). 212,284Geph clusters show postsynaptic (arrowheads) and nonsynaptic (arrows) localization. (b, c) Quantitative analysis of (b) gephyrin cluster numbers and (c) VGAT puncta in gephyrin-GFP and 212,284Geph-transfected primary hippocampal neurons. (d) Colocalization of gephyrin-GFP and 212,284Geph clusters with GABAAR α2 immunofluorescence. Arrows show 212,284Geph clusters that are not localized at GABAergic synapses. Scale bar, 5 µm. (e) Quantitative analysis of cluster colocalized with α2-containing GABAARs. (f) Representative immunoblot of subcellular fractionation of 14 DIV cultured hippocampal neurons after incubation with 50 µM 2-BP or the solvent. (g–h) Quantitative line-scan analyses of fluorescence intensities ranging from individual clusters towards the cytosol as indicated by white arrows. Scale bar, 5 µm. (h′) Histogram shows quantification of cluster/cytoplasm intensity rations in gephyrin-GFP compared to 212,284Geph-transfected dendrites. All data are means ± SEM (***p<0.001 using Student's t test; NS, not significant). At least three independent cultures were used for the quantifications.