Fig. 1

Parkin over-expression in developing neurons causes severe climbing defects and premature death in Drosophila melanogaster. a Domain structure of Drosophila melanogaster Parkin, cloned with a N-terminal FLAG tag. The catalytic cysteine (C449), marked with an asterisk, was mutated to serine to generate a Ligase-Dead (LD) Parkin mutant. b L3 larva showing GFP over-expression pattern in developing neurons using the elav-GAL4 driver. c Anti-Parkin western blot showing Parkin levels in flies over-expressing one copy (Parkin^WT/+ and Parkin^LD/+) or two copies of FLAG-tagged WT (Parkin^WT) and LD Parkin (Parkin^LD), using elav-GAL4. GFP over-expression was used as a control. Four-fold less sample was loaded for Parkin^WT and Parkin^LD. d Climbing assay of stated genotypes at 5–8 and 17–20 days after eclosion. Parkin null (park ²⁵) flies were used as a negative control. Stars indicate 0% climbing ability and a skull represents complete death at the particular time point. Three technical replicates were performed with three biological replicates of each genotype and age. Columns represent average values and error bars show standard error of the mean (SEM). e Survival assay of stated genotypes. Survival graphs represent % of alive flies at each time point. Statistical significance was assessed by two-tailed paired Student’s-t test. Asterisk(s) indicate significance, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. Complete genotypes of flies used: elav-GAL4,UAS-GFP/CyO (GFP), elav-GAL4/CyO;UAS-Parkin ^WT /TM6 (Parkin^WT/+), elav-GAL4/CyO;UAS-Parkin ^LD /TM6 (Parkin^LD/+), elav-GAL4/CyO;UAS-Parkin ^WT (Parkin^WT), elav-GAL4/CyO;UAS-Parkin ^LD (Parkin^LD) and park ²⁵ (Parkin null)