Figure 8. KRS pathogenic ATP13A2 mutants fail to rescue defects of ATP13A2-null cells. (a and b) ATP13A2 pathogenic mutants do not rescue APS–LYS fusion. WT or ATP13A2-null (ATP13A2 KO) MEFs expressing GFP-mCherry-LC3 plus a control (Ctrl), ATP13A2WT, delC, F182L, or G504R plasmid. Fusion (red) or nonfusion (yellow) of APS–LYS is shown (a). Bar, 7.5 µm. Insets: expression of ATP13A2 variants (FLAG, white; bar, 7.5 µm) and magnified images (Zoom, bar, 1.5 µm). Fusion events were quantitatively analyzed (b). ***, P < 0.001; ****, P < 0.0001, WT: n = 31, KO/Ctrl: n = 37, KO/WT: n = 41, KO/delC: n = 33, KO/F182L: n = 29, KO/G504R: n = 27. (c and d) LC3 vesicle accumulation in cells expressing ATP13A2 pathogenic mutants. ATP13A2-null (ATP13A2 KO) HeLa cells expressing ATP13A2 (WT), delC, F182L, or G504R were detected for LC3 (green, upper panels) or ATP13A2 variants (V5, red, lower panels). Control (WT) and ATP13A2-null HeLa cells transfected with an empty plasmid (KO/Ctrl) served as controls (c). Bar, 7.5 µm. Quantitative analysis of LC3 puncta is shown (d). ****, P < 0.0001, n = 3. (e–g) ATP13A2 pathogenic mutants do not restore tubulin acetylation and LC3-II degradation in ATP13A2-null cells. ATP13A2-null (ATP13A2 KO) HEK293 cells expressing ATP13A2 variants (WT, delC, F182L, or G504R) were immunodetected for LC3 (LC3-I, LC3-II), ATP13A2 variants (FLAG), or α-tubulin (α-tub). WT and ATP13A2-null HEK293 cells transfected with an empty plasmid (KO/Ctrl) served as controls (e). Quantitation of ac-tub/α-tub and LC3-II/α-tub ratio is shown (f and g; n = 3). ***, P < 0.001; ****, P < 0.0001. (h) ATP13A2 pathogenic mutants do not promote protein aggregate degradation in ATP13A2-null cells. ATP13A2-null (KO) MEFs expressing ATP13A2 variants (WT, delC, F182L, or G504R) were treated with MG132 for 12 h to induce protein aggregation, followed by removal of MG132. Immunostaining with an anti-ubiquitin antibody and an anti-V5 tag antibody was used on insoluble protein aggregates (green, Ubi) and ATP13A2 (red, V5, lower panel), respectively. Cells transfected with control empty plasmid served as negative controls (KO/Ctrl). Dashed lines outline transfectants. Bar, 7.5 µm.
Figure 8.

KRS pathogenic ATP13A2 mutants fail to rescue defects of ATP13A2-null cells. (a and b) ATP13A2 pathogenic mutants do not rescue APS–LYS fusion. WT or ATP13A2-null (ATP13A2 KO) MEFs expressing GFP-mCherry-LC3 plus a control (Ctrl), ATP13A2WT, delC, F182L, or G504R plasmid. Fusion (red) or nonfusion (yellow) of APS–LYS is shown (a). Bar, 7.5 µm. Insets: expression of ATP13A2 variants (FLAG, white; bar, 7.5 µm) and magnified images (Zoom, bar, 1.5 µm). Fusion events were quantitatively analyzed (b). ***, P < 0.001; ****, P < 0.0001, WT: n = 31, KO/Ctrl: n = 37, KO/WT: n = 41, KO/delC: n = 33, KO/F182L: n = 29, KO/G504R: n = 27. (c and d) LC3 vesicle accumulation in cells expressing ATP13A2 pathogenic mutants. ATP13A2-null (ATP13A2 KO) HeLa cells expressing ATP13A2 (WT), delC, F182L, or G504R were detected for LC3 (green, upper panels) or ATP13A2 variants (V5, red, lower panels). Control (WT) and ATP13A2-null HeLa cells transfected with an empty plasmid (KO/Ctrl) served as controls (c). Bar, 7.5 µm. Quantitative analysis of LC3 puncta is shown (d). ****, P < 0.0001, n = 3. (e–g) ATP13A2 pathogenic mutants do not restore tubulin acetylation and LC3-II degradation in ATP13A2-null cells. ATP13A2-null (ATP13A2 KO) HEK293 cells expressing ATP13A2 variants (WT, delC, F182L, or G504R) were immunodetected for LC3 (LC3-I, LC3-II), ATP13A2 variants (FLAG), or α-tubulin (α-tub). WT and ATP13A2-null HEK293 cells transfected with an empty plasmid (KO/Ctrl) served as controls (e). Quantitation of ac-tub/α-tub and LC3-II/α-tub ratio is shown (f and g; n = 3). ***, P < 0.001; ****, P < 0.0001. (h) ATP13A2 pathogenic mutants do not promote protein aggregate degradation in ATP13A2-null cells. ATP13A2-null (KO) MEFs expressing ATP13A2 variants (WT, delC, F182L, or G504R) were treated with MG132 for 12 h to induce protein aggregation, followed by removal of MG132. Immunostaining with an anti-ubiquitin antibody and an anti-V5 tag antibody was used on insoluble protein aggregates (green, Ubi) and ATP13A2 (red, V5, lower panel), respectively. Cells transfected with control empty plasmid served as negative controls (KO/Ctrl). Dashed lines outline transfectants. Bar, 7.5 µm.

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