Figure 2. Analysis of the integrity of mitochondria in DJ-1 deficient and DJ-1 WT MEF. (A) To determine intramitochondrial ROS production, DJ-1 WT and DJ-1 KO cells were treated with MitoSoxH (Invitrogen, USA) according to the manufacturer’s instruc- tions for 15 min at 37uC. Fluorescence emission was determined by FACS analysis. An increased intramitochondrial ROS production was observed in DJ-1 KO MEF compared to controls (p,0.05, Student’s t- test). Mitochondrial membrane integrity was assessed in DJ-1 WT and KO MEF and in DJ-1 KO cells stably transfected with either DJ-1 WT or the corresponding empty control vector, respectively. The MMP was determined after treatment with TMRM (Invitrogen, USA) for 15 min. at 37uC. The absorption of TMRM in the mitochondria was used as a marker for the integrity of MMP and determined by FACS analysis. (B) A significantly decreased MMP was found in DJ-1 KO cells compared to controls (*p,0.001, Student’s t-test). (C) Complementation of the DJ-1 KO phenotype with physiological DJ-1 significantly increased the MMP compared to empty vector control (*p,0.05, Student’s t- test). doi:10.1371/journal.pone.0009367.g002 DJ-1 in Mitochondrial Dynamics Therefore, we applied live cell imaging technique based on MitoTracker green (Molecular Probes, USA) and concomitant Hoechst 33342 (Molecular Probes, USA) staining. In DJ-1 KO cells, we found significant differences in the mitochondrial morphology compared to WT controls. Analysis of single mitochondria revealed that DJ-1 KO cells display disintegration of the mitochondrial network and reduced branching of mitochondria. Using Image J software to assess morphological parameters as the length (aspect ratio) and degree of branching (form factor) this was reflected by significant differences in the form factor between DJ-1 KO cells and WT controls (Fig. 3A and 3B). These results link the observed impairments of complex-I– dependent respiration, the increase in mitochondrial ROS production and the loss of MMP in DJ-1 KO cells with an unbalanced mitochondrial morphology. Our observations imply that the mitochondrial network, which is related to dynamic changes of mitochondrial morphology, may be modulated by DJ-1. In general, the number and morphology of mitochondria is critically regulated by mediators of mitochondrial fission, such as Drp1 and Fis1, and by mediators of mitochondrial fusion, such as OPA1 and mitofusins (Mfn1 and Mfn2) [27]. Therefore, we investigated if DJ-1 leads to changes in the steady state levels of Drp1, Mfn2, Fis1, and OPA1 proteins, respectively. No differences were observed in DJ-1 KO MEF compared to wild type controls (Fig. 3C). To control for species-specific effects, we next extended our morphological studies to human fibroblasts. Therefore we examined the mitochondrial morphology in fibroblasts from carriers of the E64D mutation in the DJ-1 gene [8]. We found that homozygosity for the mutant allele leads to a marked loss of the protein levels of DJ-1 in fibroblasts in vivo and thereby confirm previous in vitro data on the E64D and the L166P mutation that provided evidence for instability of the mutant protein (supple- mental figure S1) [8]. These results indicate that fibroblasts from homozygous carriers of the E64D mutation may provide a model to investigate the consequences of loss of DJ-1 function in humans. Our analyses of human fibroblasts from the homozygous index patient of the E64D family, two sibling carrying the E64D mutation in the heterozygous state and one unrelated healthy control revealed a similar mitochondrial phenotype as observed in DJ-1 KO cells with significantly reduced mitochondrial branching in the affected index patient compared to the heterozygous carriers and the healthy control (Fig. 3D, E). Our results indicate a conserved role of DJ-1 in the regulation of mitochondrial mophology among different species. Impaired Lysosomal Function in DJ-1 KO Cells Decreased MMP has been shown to interfere with mitochon- drial degradation that critically relies on lysosomal function [19]. Therefore, we analyzed a potential role of DJ-1 in terms of lysosomal activity using LysoTrackerH (Molecular Probes, USA) staining. We found a significant reduction in the fluorescent intensity of LysoTracker-treated DJ-1 KO MEF when compared to WT controls (Fig. 4A). These results are in line with impaired lysosomal activity and were subsequently followed by detailed ultrastructural analyzes. Electron microscopy of DJ-1 KO MEF revealed the presence of characteristic multilamellar structures containing electron-dense material and whorled membranous material, which was only rarely seen in the corresponding controls (Fig. 4B, 4C). Interestingly, these structures were reminiscent of cellular vacuoles containing pigments associated with neurological disorders, particularly caused by lysosomal dysfunction [28,29,30,31]. Indeed, autophagy-mediated lysosomal clearance is the only PLoS ONE | www.plosone.org 5 February 2010 | Volume 5 | Issue 2 | e9367