Figure 6. ATP13A2 regulates cortactin acetylation to promote APS–LYS fusion. (a and b) ATP13A2 regulates cortactin acetylation. Control (ATP13A2, WT) and ATP13A2-null (ATP13A2, KO) HEK293 cells were cotransfected with CBP (CBP, +) and either control empty plasmid (Cortactin, −) or a plasmid encoding FLAG-cortactin (Cortactin, +). Cortactin was immunoprecipitated followed by immunoblot analysis with an anti-acetylated lysine (Ac-K), an anti-FLAG-tag (FLAG-Cortactin), or anti-CBP antibodies (CBP; a). Relative levels of acetylated cortactin are shown (b). **, P < 0.01, n = 3. (c and d) Endogenous cortactin levels are not affected in ATP13A2. Expression of endogenous cortactin and actin in ATP13A2-null (KO) and WT control (WT) MEFs was detected (c). Quantitative analysis is shown (d; n = 3). (e and f) Cortactin rescues APS–LYS fusion in ATP13A2-null MEFs. ATP13A2-null MEFs (KO) were cotransfected with a plasmid encoding GFP-mCherry-LC3 with either an empty pCMV plasmid (KO/Ctrl) or a plasmid encoding FLAG-cortactin (KO/Cortactin). Fusion (red), nonfusion (yellow), exogenous cortactin (FLAG, white; bar, 7.5 µm), and high-magnification images (Zoom, bar, 1.5 µm) are shown (e). Quantitation of nonfused over total vesicles is shown (f). ****, P < 0.0001. KO/Ctrl: n = 37, KO/Cortactin: n = 46. (g and h) ATP13A2 and HDAC6 regulate cortactin/LYS colocalization. WT or ATP13A2-null (KO) MEFs transfected with control plasmid pcDNA3.1 (Ctrl), a plasmid encoding HDAC6 (HDAC6 WT), or a plasmid encoding HDAC6 mutant (HDAC6 H611A) were immunodetected for cortactin (green) and LAMP1 (red). Bar, 7.5 µm. Exogenous HDAC6 (FLAG, white; bar, 7.5 µm) and higher -magnification images (Zoom, bar, 1.5 µm) are also shown. Quantitative analysis of LAMP1-cortactin colocalization is shown (h). ***, P < 0.001; ****, P < 0.0001. WT: n = 39, KO/Ctrl: n = 41, KO/HDAC6 WT: n = 33, KO/HDAC6 H611A: n = 47. (i–l) Fractionation analysis of HDAC6 and cortactin in mouse liver tissues. Livers of ATP13A2-null mice (KO) and their WT control littermates (WT) were fractionated to isolate cytoplasm (Cyto), APS, autophagolysosome (APL), and LYS. The lysates of fractions and whole-cell lysates (WCL) were immunodetected for HDAC6, cortactin, actin, LAMP1, or LC3 (i). *, cortactin. The relative levels of HDAC6 (j), cortactin (k), and actin (l) in the lysosomal fraction of ATP13A2-null (KO) and their WT controls were calculated. ***, P < 0.001; ****, P < 0.0001, n = 3. (m) ATP13A2 regulates HDAC6 lysosomal localization. WT and ATP13A2-null (KO) HEK293 cells were immunodetected for LAMP1 (green) and HDAC6 (red). Bar, 5 µm. Arrows and dotted-line circles indicate LYS and HDAC6. The solid line in higher magnification images (Zoom; bar, 2.5 µm) indicates where colocalization analysis was performed. Histograms of LAMP1 and HDAC6 colocalization are shown (right panel). Bar, 5 µm. (n) Lysosomal HDAC6 activity is regulated by ATP13A2. HDAC6 activity of LYS and mitochondria (Mito) isolated from livers of WT or ATP13A2-null mice were assayed at 0, 15, 30, and 60 min. LYS fractions treated with trichostatin A were analyzed as a negative control. ***, P < 0.001; ****, P < 0.0001, n = 3.
Figure 6.

ATP13A2 regulates cortactin acetylation to promote APS–LYS fusion. (a and b) ATP13A2 regulates cortactin acetylation. Control (ATP13A2, WT) and ATP13A2-null (ATP13A2, KO) HEK293 cells were cotransfected with CBP (CBP, +) and either control empty plasmid (Cortactin, −) or a plasmid encoding FLAG-cortactin (Cortactin, +). Cortactin was immunoprecipitated followed by immunoblot analysis with an anti-acetylated lysine (Ac-K), an anti-FLAG-tag (FLAG-Cortactin), or anti-CBP antibodies (CBP; a). Relative levels of acetylated cortactin are shown (b). **, P < 0.01, n = 3. (c and d) Endogenous cortactin levels are not affected in ATP13A2. Expression of endogenous cortactin and actin in ATP13A2-null (KO) and WT control (WT) MEFs was detected (c). Quantitative analysis is shown (d; n = 3). (e and f) Cortactin rescues APS–LYS fusion in ATP13A2-null MEFs. ATP13A2-null MEFs (KO) were cotransfected with a plasmid encoding GFP-mCherry-LC3 with either an empty pCMV plasmid (KO/Ctrl) or a plasmid encoding FLAG-cortactin (KO/Cortactin). Fusion (red), nonfusion (yellow), exogenous cortactin (FLAG, white; bar, 7.5 µm), and high-magnification images (Zoom, bar, 1.5 µm) are shown (e). Quantitation of nonfused over total vesicles is shown (f). ****, P < 0.0001. KO/Ctrl: n = 37, KO/Cortactin: n = 46. (g and h) ATP13A2 and HDAC6 regulate cortactin/LYS colocalization. WT or ATP13A2-null (KO) MEFs transfected with control plasmid pcDNA3.1 (Ctrl), a plasmid encoding HDAC6 (HDAC6 WT), or a plasmid encoding HDAC6 mutant (HDAC6 H611A) were immunodetected for cortactin (green) and LAMP1 (red). Bar, 7.5 µm. Exogenous HDAC6 (FLAG, white; bar, 7.5 µm) and higher -magnification images (Zoom, bar, 1.5 µm) are also shown. Quantitative analysis of LAMP1-cortactin colocalization is shown (h). ***, P < 0.001; ****, P < 0.0001. WT: n = 39, KO/Ctrl: n = 41, KO/HDAC6 WT: n = 33, KO/HDAC6 H611A: n = 47. (i–l) Fractionation analysis of HDAC6 and cortactin in mouse liver tissues. Livers of ATP13A2-null mice (KO) and their WT control littermates (WT) were fractionated to isolate cytoplasm (Cyto), APS, autophagolysosome (APL), and LYS. The lysates of fractions and whole-cell lysates (WCL) were immunodetected for HDAC6, cortactin, actin, LAMP1, or LC3 (i). *, cortactin. The relative levels of HDAC6 (j), cortactin (k), and actin (l) in the lysosomal fraction of ATP13A2-null (KO) and their WT controls were calculated. ***, P < 0.001; ****, P < 0.0001, n = 3. (m) ATP13A2 regulates HDAC6 lysosomal localization. WT and ATP13A2-null (KO) HEK293 cells were immunodetected for LAMP1 (green) and HDAC6 (red). Bar, 5 µm. Arrows and dotted-line circles indicate LYS and HDAC6. The solid line in higher magnification images (Zoom; bar, 2.5 µm) indicates where colocalization analysis was performed. Histograms of LAMP1 and HDAC6 colocalization are shown (right panel). Bar, 5 µm. (n) Lysosomal HDAC6 activity is regulated by ATP13A2. HDAC6 activity of LYS and mitochondria (Mito) isolated from livers of WT or ATP13A2-null mice were assayed at 0, 15, 30, and 60 min. LYS fractions treated with trichostatin A were analyzed as a negative control. ***, P < 0.001; ****, P < 0.0001, n = 3.

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