DJ-1 in Mitochondrial Dynamics mutation carriers no significant alterations were observed (Fig. 7C). Our results support the concept of reduced mitochondrial clearance due to loss of DJ-1 in mammalian cells. Increased Mitochondrial Mass in DJ-1 KO Cells The regulation of mitochondrial turnover including clearance of dysfunctional mitochondria is essential for cellular homeostasis and survival [37]. Since our previous results gave evidence for disturbed mitochondrial degradation in DJ-1 KO MEF, we investigated, whether mitochondrial turnover may be affected and monitored the mitochondrial mass in cells with or without expression of physiological DJ-1. Using MitoTracker green (Molecular Probes, USA), which labels mitochondria irrespective of their functional status, we found that DJ-1 KO cells showed a significantly higher fluorescent signal than WT controls (Fig. 8A). This is indicative for an increased mitochondrial mass in DJ-1-KO cells compared to controls and correlates with our findings on lysosomal function and colocalization of mitochondria with lysosomal structures. In order to further validate our FACS-based method to quantify mitochondrial mass, we performed Western blot analyses of the mitochondrial complex IV subunit I, to quantify mitochondrial mass and to validate our FACS-based experiments for quantification by MitroTracker green staining (Invitrogen). These results showed increased levels of complex IV subunit I in DJ-1 KO cells compared to WT controls and provide additional evidence for reduced mitochondrial clearance due to loss of DJ-1 function Decreased MMP in DJ-1 WT Cells after Lysosomal Inhibition To investigate whether impaired lysosomal degradation in cells expressing physiological DJ-1 is sufficient to cause impaired mitochondrial function, we treated DJ-1 WT and DJ-1 KO cells with the lysosomal inhibitor bafilomycin A1 (BafA1). BafA1 is a Figure 8. Influence of lysosomal degradation of mitochondria on mitochondrial mass and its mechanistic backround. (A) To analyze mitochondrial mass in DJ-1 KO and DJ-1 WT MEF, cells were treated with 1 mM MitoTrackerH green FM (Invitrogen, USA) for 15 min at 37uC. Absorption of MitoTrackerH green FM was determined by FACS analysis. The uptake of MitoTrackerH was used as an indicator for the mitochondrial mass. We found evidence for increased mitochondrial mass in DJ-1 KO cells compared to controls (*p,0.001, Student’s t-test). (B) To further validate our observations on mitochondrial mass, we used an independent method quantifying complex IV subunit I as a mitochondrially encoded protein of the respiratory chain. We found increased protein levels of complex IV subunit I in KO cells compared to controls as indicative of an increased mitochondrial mass in the KO condition. (C) The influence of disturbed lysosomal activity on mitochondrial function was analyzed by measuring the MMP after treatment with BafA1 as an inhibitor of lysosomal function. Cells were treated overnight with 200 nM BafA1 (Calbiochem, Germany) and the integrity of MMP was determined by measuring the uptake of TMRE (Invitrogen, USA) by FACS analysis (CyAnADP, Beckman Coulter). (C) To analyze candidate pathways for DJ-1-mediated modulation of lysosomal degradation we investigated p42/p44 MAPK (ERK1/2) phosphorylation in the mitochondrial (MF) and cytosolic fraction (CF) of DJ-1 WT and DJ-1 KO MEF. Phosphorylation and expression of the ERK1/2 kinase was analyzed by WB using antibodies against phospho-ERK1/2 (Thr202/Tyr204). To define the fractions we used antibodies against prohibitin or GAPDH, as markers for the mitochondrial and the cytosolic fraction, respectively. Quantification of relative protein levels was performed by Image J software. The results indicate a significantly reduced level of phospho-ERK2 in the mitochondrial fraction (p,0.05; Student’s t test). doi:10.1371/journal.pone.0009367.g008 PLoS ONE | www.plosone.org 11 February 2010 | Volume 5 | Issue 2 | e9367