(A) Immunofluorescence of the proteins indicated at left in cortical neuronal cultures (DIV 18–24) from control, dynamin 1 KO, synaptojanin 1 KO and endophilin TKO mice. Clathrin, α–adaptin and amphiphysin 1 are predominantly diffuse in control neurons, but clustered in all three mutant genotypes. Dynamin 1 and auxilin are clustered in synaptojanin and endophilin mutant synapses, but auxilin is not clustered in dynamin 1 KO. Synaptojanin is slightly more diffuse in the absence of endophilin. The punctate Bassoon immunofluorescence is similar in all genotypes. Following treatment with TTX (1 μM, 14–18 h) to silence neuronal activity, clathrin was no longer clustered. Bar=15 μm.
(B) Quantification of the clustering of immunoreactivity. The y-axis represents the fold increase of fluorescence puncta in mutant synapses normalized to controls. *p<0.05, t-test. Bars=mean±SEM.
(C) Fluorescence analysis of EGFP-clathrin LC in WT and endophilin TKO neurons (DIV 18–24). Clathrin is predominantly diffuse in control neurons, but clustered in endophilin TKOs. Clustering was rescued by expression of endophilin 1 FL-Cherry, but not of the endophilin 1 BAR-Cherry construct.
(D) Quantification of the clustering of the clathrin fluorescence in the four conditions shown in (C). The y-axis represents the fold increase of fluorescence puncta in mutant synapses normalized to WT. Bars=mean±SEM, n.s. not significant, *p<.05, t-test.
(E) Left: Western blot analysis of starting lysates and CCV enriched fractions obtained from WT and endophilin TKO primary cultures (DIV 21). Right: Levels of proteins in the CCV enriched fractions from endophilin TKO cultures normalized to the WT values. Bars=mean±SEM, *p<0.05, t-test.