DJ-1 in Mitochondrial Dynamics Figure 6. Assessing the induction and inhibition of autophagy by WIPI-1 puncta formation analyses in DJ-1 WT and DJ-1 deficient MEF. GFP-WIPI-1 puncta-formation analyses reveals that forced modulation of the autophagic activity is possible in DJ-1 deficient cells. WT (MEF DJ-1 +/+) (A) or DJ-1 KO MEF (MEF DJ-1 2/2) (B) were treated with standard inducers of autophagy, rapamycin and EBSS (medium lacking amino acids), a standard inhibitor of autophagy, wortmannin, or left untreated (mock treatment, control). Quantitative confocal microscopy was conducted. MEFs that displayed GFP-WIPI-1 puncta were counted as positive (puncta), and MEFs that did not displayed GFP-WIPI-1 were counted as negative (non- puncta). In total 200–300 MEFs were counted from 3 independent experiments (see supplemental figure S2A-H, supplemental table Table S1). P- values for puncta-positive DJ-1 WT cells upon rapamycin ( = 0,00067), wortmannin ( = 0,00026), EBSS ( = 0,00083), and p-values for puncta-positive for DJ-1 definient cells upon rapamycin ( = 0,00111), wortmannin ( = 0,00138), EBSS ( = 0,00117) are indicated (***). doi:10.1371/journal.pone.0009367.g006 standard treatments that modulate autophagy (EBSS and rapamycin as autophagy inducing agents, wortmannin as an autophagy inhibiting agent; supplemental figure S2A-H, supple- mental table Table S1). From this we conclude that loss of DJ-1 results in reduced basal autophagy (LC3 protein monitoring) but not in a complete block of the autophagic capacity (WIPI-1 puncta formation, LC3 protein monitoring). Reduced Colocalization of Mitochondria with Lysosomes in Cellular Models of Loss of DJ-1 Based on our finding of reduced levels of basal autophagy, we next investigated the clearance of dysfunctional mitochondria by autophagy using fluorescence microscopy on mitochondria and lysosomes. To determine the colocalization of mitochondria and lysosomes as a measure of lysosomal degradation of mitochondria we used specific dyes for differential staining of the respective organelles. Then we analyzed DJ-1 KO and DJ-1 WT MEF treated with EBSS Medium (Sigma-Aldrich, Germany) with live cell imaging (Fig. 7A). The scatter-plot analyses of colocalization pictures (Zeiss AxioVision 4.7 software, Zeiss, Germany) showed a significantly reduced co-staining of MitoTrackerH green and LysoTrackerH red in DJ-1 KO MEF, suggesting a reduced clearance of mitochondria by lysosomes (Figure 7A, 7B). This further supports our results showing impaired lysosomal degrada- tion in DJ-1 KO MEF. Next we assessed the mitochondrio-lysosomal colocalization in human fibroblasts from the E64D patient and controls. We found a significantly reduced number of mitochondria that overlap with lysosomes in EBSS-treated fibroblasts from the E64D patient compared to the healthy control (Fig. 7C). For the heterozygous PLoS ONE | www.plosone.org 9 February 2010 | Volume 5 | Issue 2 | e9367