Figure 4. Lysosomal phenotype in DJ-1 WT and DJ-1 deficient MEF. (A) Lysosomal activity was determined by FACS analysis. Cells were stained with the specific lysosomal dye LysoTrackerH (Invitrogen, USA) 1 mM for 15 min at 37uC. The absorption of LysoTrackerH was measured by FACS. The uptake of LysoTrackerH is an indicator of the Lysosomal mass. DJ-1 KO cells revealed a significantly reduced uptake of LysoTrackerH compared to WT MEF (*p,0.0001, Student’s t-test). The experiments were performed in triplicate on three different days, the diagram represents 1 of 3 sets of experiments with similar results. (B) Ultrastructural analyzes of DJ- 1 deficient MEF and MEF from DJ-1 WT littermates were performed by electron microscopy. MEF lacking DJ-1 show multilamellar structures reminiscent of modified lysosomes. Bars 1 mm or 0,5 mm, respectively. (C) Quantification of lysosomal-like structures in DJ-1 WT and DJ-1 KO MEF. Multilamellar structured lysosomes were counted per cell and were significantly more frequent in DJ-1 KO cells compared to WT controls (p,0.001, Student’s t-test). A total of 150 individual cells were evaluated for the presence of multilamellar structures. doi:10.1371/journal.pone.0009367.g004 means by which cells degrade dysfunctional mitochondria. In this context, similar structures are believed to represent undigested cellular components that accumulate over time [31]. DJ-1 in Mitochondrial Dynamics Reduced Basal Autophagy in DJ-1 KO Cells Next, we asked whether impaired lysosomal degradation pathways in the absence of DJ-1 could be proven by upstream events that depend on functional lysosomes, such as autophagic clearance. Autophagy is a lysosomal degradation pathway responsible for the degradation of cytoplasmic material including long-lived proteins, misfolded proteins and cellular organelles such as mitochondria [32,33]. Interestingly, impaired autophagy has been correlated with the onset of neurodegeneration, such as the pathology of Alzheimer’s, Huntington’s and Parkinson’s disease [34]. However, molecular targets that confer autophagic dysfunc- tion in neurodegeneration are insufficiently identified. Here, we employed well established autophagy assays, such as LC3 protein monitoring [25] and WIPI-1 puncta formation quantification [22,23,24,34,35] to assess autophagy in DJ-1 KO MEF versus WT controls. Upon autophagic stimulation, LC3 becomes conjugated to phosphatidylethanolamine (LC3-II) and localizes at autopha- gosomal membranes. Unconjugated LC3 (LC3-I) is distributed throughout the cytoplasm, and in the nucleous. Hence the increase of endogenous LC3-II protein abundance monitors the autophagic activity. Thereby, basal autophagy as well as induced or inhibited autophagy can be compared using different cellular backgrounds. In addition, LC3 protein monitoring allows to analyze the autophagic flux, meaning the process from autophagosome formation to autophagolysosomal degradation. In experimental set-ups where autophagolysosomal degradation is blocked by the addition of lysosomal inhibitors, such as protease inhibitors, the further increase of LC3-II protein abundance reflects that autophagic flux occurs properly [36]. The WIPI-1 puncta formation analyses also monitors autoph- agic activity, because WIPI-1 specifically binds to Phosphatidyli- nositol 3-phosphate (PI(3)P) upon autophagic stimulation and localizes at autophagosomal membranes. Using GFP-WIPI-1, PI(3)P-bound GFP-WIPI-1 (puncta) can easily be visualized and quantified by fluorescence microscopy as a measure for the autophagic activity. Cells that display GFP-WIPI-1 fluorescent puncta undergo autophagic stimulation, and in cells without GFP- WIPI-1 puncta (non-puncta) autophagy is inactive. Of note, the usage of GFP-LC3 in such experiments leads to unwanted protein aggregations of GFP-LC3 that do not reflect autophagosomal membrane formation. In contrast, the usage of GFP-WIPI-1 does not lead to such protein aggregations [25]. Here, we first used the method of endogenous LC3 protein monitoring to ask whether or not basal autophagy is altered in models of loss of DJ-1 function. Strikingly, basal autophagy levels were reduced in DJ-1 KO MEF compared to WT control cells as reflected by a decrease LC3-II protein abundance, a measure for autophagosome-bound LC3 (Fig. 5A). We normalized LC3-II signal intensities over GAPDH in standard ECL analyses and show that the LC3-II signal intensity in DJ-1 KO cells (MEF DJ-1 2/2) was reduced to 0.297 when compared to WT cells (MEF DJ-1 +/+, set to 1) (Fig. 5A). However, the autophagic flux was not inhibited as shown by the addition of protease inhibitors (PI) during assay conditions that block final degradation (Fig. 5A). Comparing the LC3-II protein abundance during conditions with or without protease inhibitors, we found an increase of LC3-II signal intensity in WT cells from 1,000 to 3.013 (MEF DJ-1 +/+ PI) and also in DJ-1 KO cells from 0.297 (MEF DJ-1 2/2) to 2,539 (MEF DJ-1 2/2 PI). This result was further verified when cells were incubated in amino acid-free medium as a treatment that induces autophagy. Here, the further increase of LC3-II signal intensities when compared to basal LC3-II intensity levels in both, WT cells (MEF DJ-1 2/2 PI: 7.573) and DJ-1 KO cells (MEF DJ-1 +/+ PI: 3.392) confirm that the autophagic flux was PLoS ONE | www.plosone.org 7 February 2010 | Volume 5 | Issue 2 | e9367