Pathogenic Effect of Missense Mutations in ATP13A2 Figure 4. Co-localization of ATP13A2 with endosomal and lysosomal vesicular membranes in neurons. Confocal fluorescence microscopy reveals the co-localization of V5-tagged human ATP13A2 with lysosomal membranes (LAMP1-RFP), early (RFP-Rab5A) and late (GFP- Rab7A and GFP-Rab9A) endosomal compartments but fails to appreciably co-localize with recycling endosomes (GFP-Rab11A) or autophagosomes (GFP-LC3) within the soma of rat primary cortical neurons. Staining for the neuronal marker, bIII-tubulin, and nuclei (DAPI) are also indicated. Cytofluorograms and correlation coefficients (Rcoloc) indicate the extent of co-localization between ATP13A2 and vesicular markers. Confocal images are taken from a single z-plane at 0.15 mm thickness. Images are representative of at least two independent transfection experiments. Scale bar: 10 mm. doi:10.1371/journal.pone.0039942.g004 To assess ATPase activity, microsomal fractions were used for in vitro assays to monitor ATP13A2-mediated ATP hydrolysis by measuring the release of free c-phosphate produced by hydrolysis of ATP to ADP. ATPase assays were conducted in the presence of bafilomycin A1 (5 mM) to specifically inhibit the activity of the major vacuolar-type H+-ATPase (V-ATPase) that is present in vesicular membranes [22]. Microsomes expressing WT ATP13A2 markedly enhance ATP hydrolysis compared to control micro- somes, whereas microsomes containing SPCA1 exhibit substan- tially increased ATPase activity (Figure 7C), as previously PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e39942