Figure 7.

σ₂-adaptin is required specifically for AP2 complex and clathrin stability. (A–E) Representative images of third instar larval boutons from control, heteroallelic σ₂-adaptin mutant (angur⁷/AP2σ^KG02457), and transgene-rescued (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457) NMJ synapses co-labeled with HRP (red) and other synaptic proteins (green): (A) α-adaptin; (B) β₂-adaptin; (C); EYFP-Clc (D) Endophilin 1, and (E) Dynamin 1. The histograms on the right show quantification of these synaptic proteins in the boutons, expressed as percentage of control levels. The fluorescence intensity of at least 50 individual boutons was measured and the background subtracted for the quantification. ***P < 0.0001. Error bars represent standard error of the mean. Statistical analysis based on one-way ANOVA followed by post-hoc Tukey’s multiple-comparison test. (F) Western blot analysis of various synaptic proteins in larval brain of control, heteroallelic σ₂-adaptin mutant (angur⁷/AP2σ^KG02457 and angur⁷/angur⁵), and rescued animals (actin5C/+; angur⁷/UAS-AP2σ, AP2σ^KG02457). Level of α-actin was used as loading control. The level of clathrin was assessed by probing the blots with anti-GFP antibody. For the EYFP-Clc experiment, the control genotype was elav^C155/+; EYFP-Clc/+; the heteroallelic mutant genotype was elav^C155/+; EYFP-Clc/+; angur⁷/AP2σ^KG02457; and the rescue genotype was elav^C155/+; EYFP-Clc/+; angur⁷/UAS-AP2σ, AP2σ^KG02457.